Genetics, Medicine, and the Plain People ANRV386-GG10-23 ARI 29 July 2009 15:38 Genetics, Medicine, and the Plain People Kevin A. Strauss1,2,3,∗ and Erik G. Puffenberger1,2,∗ 1 Clinic for Special Children, Strasburg, Pennsylvania 17579; email: kstrauss@clinicforspecialchildren.org, epuffenberger@clinicforspecialchildren.org 2 Department of Biology, Franklin and Marshall College, Lancaster, Pennsylvania 17603 3 Lancaster General Hospital, Lancaster, Pennsylvania 17604 Annu. Rev. Genomics Hum. Genet. 2009. 10:513–36 First published online as a Review in Advance on July 16, 2009 The Annual Review of Genomics and Human Genetics is online at genom.annualreviews.org This article’s doi: 10.1146/annurev-genom-082908-150040 Copyright c© 2009 by Annual Reviews. All rights reserved 1527-8204/09/0922-0513$20.00 ∗Equal contributors. Key Words Anabaptist, deme, haplotype, identity-by-descent, Mendelian, single-nucleotide polymorphism Abstract The Old Order Amish and Old Order Mennonite populations of Pennsylvania are descended from Swiss Anabaptist immigrants who came to the New World in the early eighteenth century. Today they live in many small endogamous demes across North America. Geneti- cally, these demes have dissimilar allele frequencies and disease spectra owing to unique founders. Biological and social aspects of Old Order communities make them ideal for studies in population genetics and genomic medicine, and over the last 40 years, advances in genomic sci- ence coincided with investigational studies in Plain populations. Newer molecular genetic technologies are sufficiently informative, rapid, and flexible to use in a clinical setting, and we have successfully integrated these tools into a rural pediatric practice. Our studies with the Pennsyl- vania Plain communities show that population-specific genetic knowl- edge provides a powerful framework in which to prevent disease, reduce medical costs, and create new insights into human biology. 513 A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 Knowledge of genetic anatomy is having a com- parably strong and pervasive influence on all of medicine. . . . Genomic anatomy permits medicine to become more predictive and preventive. At the same time, diagnosis and treatment are rendered more sensitive, specific, effective, and safe. –Victor A. McKusick, 2001 (50) Be not conformed to this world, but be ye trans- formed by the renewing of your mind. Romans 12:2 INTRODUCTION The Old Order Amish and Old Order Mennonite (Plain) people of North America have contributed much to our understanding of human genetics. Although wary of technol- ogy, they have cooperated with clinical inves- tigators for more than 40 years, believing the knowledge gained could help alleviate suffer- ing in their own communities and elsewhere. The history of genetic discovery among Plain sects has broad relevance: It charts the trans- formation of human genetics from an esoteric discipline into a foundation of clinical medicine (50). Beginning in 1962, inherited disorders among Old Order communities were the sub- ject of exceptional observational studies, col- lected in the 1978 landmark Medical Genetic Studies in the Amish (1). Between 1975 and 1988, the advent of Southern blotting (80), genetic markers (17), linkage analysis, DNA sequenc- ing (48, 76), and the polymerase chain reaction (PCR) (12) enabled investigators to describe these disorders in more precise molecular terms (31, 43, 66, 71, 74, 75, 81). Recently, single- nucleotide polymorphism (SNP) microarray technology has accelerated this process (67, 68, 89, 90) and provided fascinating details about the genetic structure of inbred populations. Over the past two decades, the focus has shifted to treatment. Genetic studies within Old Order populations have revealed how per- sonal genetic information can be integrated into everyday clinical practice (23, 37, 50). Here, we present original as well as previously published data that inform the larger field of genomics and fulfill a central promise of the Human Genome Project—to harness genetic knowledge to heal the sick (88), prevent disability (87), and reduce medical costs (59, 87–89). THE PLAIN POPULATIONS Contemporary Old Order groups fall under the collective term Plain people, which refers to Christian groups that live simply, dress plainly, and live in the modern world but remain sep- arate from it. They include the Amish, Old Order and Conservative Mennonites, Old Order Brethren, and Hutterites (D. Kraybill, personal communication). Our clinical practice serves Old Order Amish and Mennonite peo- ple of Pennsylvania and Maryland, who are the focus of this review. The Anabaptist movement began in 1525 amid the Protestant Reformation. It was based on the idea that baptism should be an inten- tional act of adults who wanted to join an austere Christian community (41, 65, 69). For this and other beliefs, early Anabaptists were tortured and killed as heretics for more than 200 years as they moved throughout Europe in exile. By the 1670s, most had settled in Switzerland (65). Jakob Amman (1656–1730), a Mennonite bishop living in the canton of Berne, believed that many Anabaptists had compro- mised their religious discipline to avoid per- secution (41). He insisted upon the practice of shunning (Meidung), the social exclusion of noncompliant church members, and in 1693 forced a division among Mennonite preachers that marked the formation of the Amish church (41). William Penn offered Anabaptists asylum in the New World, and several thousand ar- rived in the port of Philadelphia between 1683 and 1770. The Mennonites generally appeared earlier in the records, whereas the Amish ar- rived in the latter half of this migration (1727– 1770). Several hundred Amish and Mennonites 514 Strauss · Puffenberger A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 eventually settled in Lancaster County (27, 65, 69). The present-day Lancaster County Amish population of approximately 50,000 individu- als is derived from perhaps 50 to 200 of these original founders (54). Most early Anabaptist settlements in Pennsylvania failed, and resid- ual members joined later Anabaptist immi- grants who moved west to Ohio, Indiana, and Illinois (54), where they remain concentrated. About 70% of contemporary Amish occupy just four counties: Lancaster County, Pennylvania; Holmes and Geauga Counties, Ohio; and LaGrange County, Indiana (54). The Old Order Amish in America have doubled in the last 16 years to approximately 226,395 individ- uals (D. Kraybill, personal communication). Following the migration to North America, Amish culture became extinct in Europe (41), while forces of assimilation and change reached Anabaptist immigrants. Some regional Amish and Mennonite churches were further divided by ideological conflicts (65), and by the 1860s groups that held fast to tradition were des- ignated the Old Order by their more pro- gressive counterparts (41, 65). Philosophical differences, reinforced by geographical separa- tion resulted in many endogamous subdivisions called demes. MEDICAL GENETIC STUDIES In 1962, Dr. Victor A. McKusick reviewed John Hostetler’s Amish Society for Johns Hopkins University Press and recognized the potential for using the Amish to study medical genetics (Table 1) (54). That same year, he read about David Krusen, a Lancaster County doctor who reported a high prevalence of achondroplasia among the Amish. These events sparked a productive collaboration between McKusick and Hostetler (54), and McKusick made his first field trips to Lancaster County with Dr. Krusen (31). He soon recognized that dwarfism among the Amish was not achondroplasia, but rather two distinct recessive conditions: Ellis-van Creveld syndrome and cartilage-hair hypoplasia (Figure 1) (52, 53). Figure 1 This Amish boy with cartilage-hair hypoplasia has the characteristic physical features of short-limbed dwarfism, sparse hair, and small nails. He suffered from neuronal intestinal dysplasia and combined immune deficiency, rare complications of the disorder, and had a bone marrow transplant at 28 months of age. He died of intractable autoimmune hemolytic anemia one year later (photo used with parent permission). Over the next 16 years, the Amish were subjects of diverse genetic studies. Many of these were detailed clinical descriptions of re- cessive disorders, but there were also studies about blood groups (16), HLA antigens (42), immunoglobulin levels (56), chromosomal vari- ation (35), and the heritability of complex traits (e.g., blood pressure, glucose tolerance, and cancer risk) (26, 44, 72). Seminal works from this era are collected in the 1978 publication Medical Genetic Studies in the Amish (1), edited by McKusick, which describes 18 previously rec- ognized and 16 newly diagnosed Mendelian dis- orders among the Amish of Pennsylvania, Ohio, and Midwestern states. Medical Genetic Studies in the Amish established many known principles of population genetics that remain pertinent today (Table 1). www.annualreviews.org • Genetics, Medicine, and the Plain People 515 A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . 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ANRV386-GG10-23 ARI 29 July 2009 15:38 Table 1 Characteristics of Plain populations conducive to genetic studies McKusick et al. 1964 (53) • A self-defined, closed population (little gene inflow) • Western European origin • Small number of founding members • Concentrated population settlements (relative immobility) • Extensive geneological records • High standards of living (i.e., nutrition, hygiene) and physical health • Strong interest in inherited illness and the healing arts • Relatively uniform social, economic, and occupational circumstances • Large sibships (e.g., average 5–8 per family) • Low illegitimacy rate • Children with birth defects or genetic disorders cared for at home • Multiple discrete subpopulations (demes) allow comparative analysis Molecular update, 2008 • Large haplotype blocks (small population of recent origin) from identity-by-descent around disease loci • Mutation homogeneity (founder effect) • Population-specific allele frequencies Before the advent of molecular genetic tech- nology, McKusick used genealogical records to infer identity-by-descent for pathogenic alleles. He traced the mutation for pyruvate kinase deficiency to “Strong Jacob” Yoder and his wife, who immigrated in 1742, and traced the Ellis-van Creveld allele through 40 sibships, 73 affected individuals, and 80 parents to Samuel King and his wife, who also immigrated in the 1740s (49). Although complex pedigree analysis is seldom necessary today, these studies highlighted the advantages of genetic linkage analysis and disease gene mapping in Plain populations. MOLECULAR GENETIC STUDIES Population Genetics Early investigators recognized the distinct na- ture of Amish demes based on their immi- gration history and surname distribution (49). Surnames are inherited through the pater- nal line, largely independent of selection or inbreeding. Deviations in their frequency within a population over time simulate ran- dom genetic drift. Presently, the name Stoltzfus accounts for 26% of Amish households in Lancaster County. Three other surnames (King, Fisher, and Beiler) each account for 10%–12% of the total. Similarly, among Weaverland and Groffdale Mennonites, the surname Martin accounts for 20% of house- holds. Mitochondrial DNA haplotypes, inher- ited through the maternal line, demonstrate similar drift over the last 12–14 generations (Table 2). Within each Anabaptist subpopula- tion, pathogenic alleles may also increase (or de- crease) in frequency by random chance. When the founding population is small, random allele frequency shifts from one generation to the next can be profound; some rare mutations become quite common, while others become extinct. This underlying genetic structure made Plain populations ideal for early genetic map- ping studies, which relied on large collections of patients and traditional linkage analysis. Formal linkage analysis was necessary because DNA markers were sparse and genotyping was time- consuming and expensive. Typically, 300–400 microsatellite markers were genotyped in af- fected individuals, their parents, and siblings in an attempt to link a phenotype to a genomic region. Many mapping studies in Plain popula- tions have identified disease genes in this way (6, 13, 19, 20, 47, 62, 63, 66, 74, 75, 81, 92, 95). It has long been known that isolated pop- ulations have a high incidence of rare genetic 516 Strauss · Puffenberger A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 Table 2 A frequency distribution of surnames and mitochondrial hypervariable region (HVR1) haplotypes among four Pennsylvania Amish and Mennonite demes1 Groffdale Weaverland Lancaster Juniata and Mifflin Mennonites Mennonites Amish Amish Surname Martin 18% 21% 0% 0% Zimmerman 18% 9% 0% 0% Hoover 8% 6% 0% 0% Nolt 7% 5% 0% 0% Weaver 5% 7% 0% 0% Stoltzfus/Stoltzfoos 0% 0% 26% 0% King 0% 0% 12% 0% Fisher 0% 0% 11% 0% Beiler/Byler 0% 0% 11% 7% Peachey 0% 0% 0% 29% Swarey 0% 0% 0% 13% Kanagy 0% 0% 0% 11% Yoder 0% 0% 1% 11% mtDNA haplotype 16298C 16311C 20% 5% 0% 0% 1669T 16126C 16261T 13% 19% 0% 0% Cambridge reference 8% 0% 0% 38% 16304C 7% 5% 0% 8% 1693C 16304C 6% 11% 0% 0% 16294T 16304C 2% 11% 3% 4% 16126C 16294T 16296T 4% 8% 0% 0% 16129A 16311C 16316G 2% 8% 0% 0% 16209C 16304C 1% 8% 0% 0% 16260T 16356C 0% 0% 36% 0% 16051G 16092C 16129C 16183C 16189C 16362C 4% 0% 26% 0% 16129A 16264T 16311C 0% 0% 15% 4% 16124C 16189C 16356A 0% 0% 5% 15% 16189C 2% 0% 0% 12% 1 These data demonstrate significant genetic drift and distinct paternal and maternal lineages within Plain demes. In our local Amish population, the 16260T 16356C haplotype accounts for 36% of HVR1 haplotypes. By genealogical analysis, it can be shown that all copies of this haplotype were derived from a single ancestral female who migrated to Pennsylvania in the eighteenth century. In the Groffdale Mennonites, HVR1 16298C 16311C predominates (20%), whereas 1669T 16126C 16261T is the most common haplotype among the Weaverland Mennonites (19%). Thirty-eight percent of the Juniata and Mifflin County Amish harbor the HVR1 Cambridge reference sequence. disorders. This, along with inbreeding and a restricted founder pool, has been taken as evidence of low genetic diversity. However, microsatellite marker (50) and SNP data challenge this view. Using Affymetrix 10K GeneChip arrays, we found that calculated heterozygosity across 9833 autosomal SNPs was 33.7% and 34.8% for Amish (N = 80) and Mennonite (N = 80) individuals, respectively. These values are similar to the 35.5% measured in an European control population (these data refer to the Affymetrix European control set for the 10K arrays; from Affymetrix, Santa Clara, CA). The comparisons may be affected by drift, www.annualreviews.org • Genetics, Medicine, and the Plain People 517 A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 which can lead to allele fixation. Among Men- nonites, 351 SNPs are monoallelic; 233 of these are also monoallelic in the European control population. Thus, 118 (i.e., 351–233) SNP al- leles have become fixed in the Mennonite pop- ulation, whereas 204 different SNP alleles have reached fixation in the Amish. Further inspection of SNP allele frequen- cies shows that Amish and Mennonite popu- lations are genetically dissimilar. We compared 10095 SNPs for significant allele frequency dif- 70 60 50 40 30 20 10 60 50 40 30 20 10 60 50 40 30 20 10 a b c Mennonite vs. European Amish vs. European Amish vs. Mennonite PercentageNumber of SNPs 190 750 1493 1.9 7.4 14.8 Chromosome number SN P X 2 va lu e SN P X 2 va lu e SN P X 2 va lu e 2 4 6 8 10 0 0 0 12 14 16 18 20 22 ferences (chi-square > 10.828, alpha = 0.001) among Lancaster County Amish, Lancaster County Mennonites, and the Affymetrix Eu- ropean control set. Amish and Mennonite sam- ples were more similar to European controls than to each other (Figure 2). This signif- icant finding underscores the importance of choosing proper control groups for association and linkage mapping studies (Figure 3) (65). It is also relevant to clinical work; we have long appreciated that genetic diseases of Amish and Mennonite populations are quite distinct (Table 3) (59, 65). Of the 67 disorders we de- lineated among the Pennsylvania Amish and Menonnite people, only five are found in both populations and only two share the same molec- ular defect. Thus, for any particular child who presents with a medical problem, the genetic differential diagnosis depends critically on pop- ulation of origin (59). Random genetic drift is a compelling and parsimonious explanation for allele frequency ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 2 Single-nucleotide polymorphism (SNP) allele frequencies are highly dissimilar between Old Order Amish and Mennonites. We performed a χ 2 analysis of allele frequencies for 10095 SNPs from the Affymetrix GeneChip Human Mapping 10K Arrays. We used 80 Amish and 80 Mennonite females and performed the analysis with high cut-off values (χ 2 > 10.828, alpha = 0.001). We also compared the two Plain populations with Affymetrix European controls. Genome-wide SNP χ 2 values are plotted in panels (a), (b), and (c) to highlight overall differences. (a) The Mennonite population was moderately dissimilar to the European controls. One hundred ninety SNPs (1.9%) demonstrated statistically significant differences between these two groups. (b) The Amish demonstrated greater allele frequency differences; roughly 7.4% of SNPs exhibited significant differences with European controls. (c) The Amish and Mennonite comparison was most striking as nearly 1 in 7 SNPs showed significant allele frequency differences. We surmise that these large differences are due to genetic drift within each population. Since the Amish founding population was smaller, we expect larger shifts due to drift over time. This is evident by the comparison with European controls, where the Amish show a nearly fourfold increase in allele frequency differences as compared to the Mennonites. 518 Strauss · Puffenberger A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 shifts within a small, isolated population. How- ever, allele frequencies might have been shaped by selection as well. For example, Anabaptist settlers were exposed to new pathogens that likely exerted significant selective pressures. Although difficult to measure, allele frequency shifts point to interesting gene candidates. From our GeneChip 10K genotype data, the SNP rs1986710 demonstrates the largest allele frequency differences between Plain popula- tions and the Affymetrix European controls and HapMap data sets. The A allele frequency is 94% in Amish and 96% in Mennonites, compared to 54% in Affymetrix European controls. This SNP resides within an intron of LPAR1, a gene associated with immunity and inflammation. When compared to control data (Americans of European descent), Amish and Mennonite samples are skewed toward larger blocks of homozygous SNPs (Figure 4). Within any isolated population, homozygous markers that occur in “runs” or blocks are likely autozygous (identical-by-descent); i.e., both the maternal and paternal alleles derive from a common ancestor. Now, we can use microarray data to quantify this—the genome-wide estimate of au- tozygosity is 4.1% and 2.5% in Amish and Mennonite individuals, respectively. Similar es- timates based on homozygous megabases of ge- nomic DNA yield comparable results (3.9% and 2.4%). This implies that the average Amish individual harbors ∼120 Mb of autozygous DNA. The probability that any two random Amish individuals would be autozygous for the same genomic region and the same haplotype is very remote. Relatively high genetic diversity and low allele sharing help facilitate successful autozygosity mapping studies, even with very small sample sizes. Modern Disease Gene Mapping Large-scale SNP genotyping methodologies are a major technological breakthrough because they allow simultaneous and rapid genotyping of many markers randomly distributed across the entire genome. This reduces analytical time Chromosome number 2 4 6 8 10 12 14 16 18 20 22 X SN P X 2 va lu e SN P X 2 va lu e GCDH 0 60 50 40 30 20 10 0 60 50 40 30 20 10 a b Figure 3 Population-specific SNP allele frequencies are critical for association mapping within Plain populations. As a test case, we analyzed SNP profiles of 30 Amish children with glutaric aciduria type 1 (GA1), who were all homozygous for GCDH c.1262C>T. The GCDH gene maps to a region of chromosome 19 that has poor SNP coverage on GeneChip 10K arrays, making homozygosity mapping impossible. As an alternative, we attempted to map the disease using X2 analysis of SNP allele frequency differences between affected children and controls. This analysis depends on accurate control allele frequencies as well as extended linkage disequilibrium surrounding the disease gene. When we compared affected GA1 children to Old Order Amish controls, we easily identified the location of GCDH. However, when we used Old Order Mennonite allele frequency data, background noise increased and the mapping signal on chromosome 19 was obscured. and cost, and allows autozygosity analysis to be done with fewer affected patients. With suf- ficiently dense SNP coverage, it is no longer necessary to infer genotype and haplotype shar- ing between distant markers; increased marker density provides enough information to reveal sharing directly. Higher marker density also simplifies analysis. Large shared blocks of ho- mozygous SNPs can be detected with simple counting and graphing algorithms using stan- dard spreadsheet software (67, 90). We have www.annualreviews.org • Genetics, Medicine, and the Plain People 519 A nn u. R ev . G en om . 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ANRV386-GG10-23 ARI 29 July 2009 15:38 Table 3 Thirty-seven Amish and 29 Mennonite disorders understood at the molecular level among demes of Pennsylvania, Ohio, and Maryland Disorder Gene DNA Variant Protein Variant Population Deme∗ 3-methylcrotonylglycinuria MCCC2 295G>C Glu99Gln Amish A 3-ß-OH-steroid dehydrogenase deficiency HSD3B2 35G>A Gly11Glu Amish A Aldosterone deficiency CYP11B2 5 bp deletion Amish A Amish microcephaly SLC25A19 530G>C Gly177Ala Amish A Bardet-Biedl syndrome BBS1 1169T>G Met390Arg Amish A Bartter syndrome CLCNKB gene deletion Amish A, B Cardiomyopathy, dilated, with AV block LMNA 568C>T Arg190Trp Amish A Cartilage-hair hypoplasia RMRP 70A>G Amish A, O Cortical dysplasia and focal epilepsy CNTNAP2 3709delG Amish A, B, O Crigler-Najjar syndrome UGT1A1 222C>A Tyr74Ter Amish A Ellis-van Creveld syndrome EVC IVS13+5G>T Amish A Familial hypercholanemia BAAT 226A>G Met76Val Amish A Familial hypercholanemia TJP2 143T>C Val48Ala Amish A Galactosemia GALT 563A>G Gln188Arg Amish A, E Gittelman syndrome SLC12A3 1924C>A Arg642Gly Amish A, B Gittelman syndrome SLC12A3 8627 bp deletion Amish A, B Gutaric aciduria, type 1 GCDH 1262C>T Ala421Val Amish A Gutaric aciduria, type 3 C7orf10 895C>T Arg299Trp Amish A, B McKusick-Kaufman syndrome MKKS [250C>T + 724G>T] His84Tyr/Ala242Ser Amish A Nemaline rod myopathy TNNT1 505G>T Glu180Ter Amish A Nephrotic syndrome NPHS2 413G>A Arg138Gln Amish A Osteogenesis imperfecta COL1A2 2098G>T Gly610Cys Amish A Pelizaeus-Merzbacher-like syndrome GJA12 203A>G Tyr68Cys Amish A Phenylketonuria PAH 280-282delATC Amish A Phenylketonuria PAH 782G>A Arg261Gln Amish A Propionic acidemia PCCB 1606A>G Asn536Asp Amish A, B Severe combined immune deficiency RAG1 2974A>G Lys992Glu Amish A Sitosterolemia ABCG8 1720G>A Gly574Arg Amish A Weil-Marchesani syndrome ADAMTS10 15 exon del Amish A Chronic granulomatous disease CYBB 1335C>A Cys445Ter Amish E Troyer syndrome SPG20 1110delA Amish O 11-hydroxylase deficiency CYP11B1 1343G>A Arg448His Amish B Adenosine deaminase deficiency ADA 646G>A Gly216Arg Amish B Byler disease ATP8B1 923G>T Gly308Val Amish B Cockayne syndrome ERCC6 IVS14+1G>T Amish B GM3 synthase deficiency ST3GAL5 694C>T Arg232Ter Amish B, O Primary ciliary dyskinesia DNAH5 4348C>T Gln1450Ter Amish B Pyruvate kinase deficiency PKLR 1436G>A Arg479His Amish B Sudden infant death with dysgenesis of the testes TSPYL1 457 458insG Amish B Homocystinuria MTHFR 1129C>T Arg377Cys Amish J (Continued ) 520 Strauss · Puffenberger A nn u. 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ANRV386-GG10-23 ARI 29 July 2009 15:38 Table 3 (Continued ) Disorder Gene DNA Variant Protein Variant Population Deme∗ 3-methylcrotonylglycinuria MCCC2 518insT Mennonite M, X Alpha-1 antitrypsin deficiency SERPINA1 1096G>A Glu342Lys Mennonite M Biotinidase deficiency BTD 1459T>C Trp487Arg Mennonite M Congenital nephrotic syndrome NPHS1 1481delC Mennonite M Congenital nephrotic syndrome NPHS1 3250delG Mennonite M Crigler-Najjar syndrome UGT1A1 222C>A Tyr74Ter Mennonite M Cystinuria SLC3A1 IVS6+2T>C Mennonite M Cystinuria SLC3A1 1354C>T Arg452Trp Mennonite M Cystinuria SLC7A9 201C>T Ile67Ile Mennonite M Cystinuria SLC7A9 1166C>T Thr389Met Mennonite M Deafness, nonsyndromic GJB2 35delG Mennonite M Factor 11 deficiency F11 1327C>T Arg443Cys Mennonite M Fragile X syndrome FMR1 (CGG)n expansion Mennonite M Glycogen storage disease, type 6 PYGL IVS13+1G>A Mennonite M Hirschsprung disease EDNRB 828G>T Trp276Cys Mennonite M, S LYK5 deficiency LYK5 7304 bp deletion Mennonite M Maple syrup urine disease BCKDHA 1312T>A Tyr438Asn Mennonite M, S Medium-chain acyl-CoA dehydrogenase deficiency ACADM 985A>G Lys329Glu Mennonite M Medium-chain acyl-CoA dehydrogenase deficiency ACADM IVS4-30A>G Mennonite M Mevalonate kinase deficiency MVK 803T>C Ile268Thr Mennonite M Mevalonate kinase deficiency MVK 1174G>A Ala392Thr Mennonite M Phenylketonuria PAH IVS12+1GA Mennonite M Phenylketonuria PAH 782G>A Arg261Gln Mennonite M Propionic acidemia PCCB 1606A>G Asn536Asp Mennonite M, S Restrictive dermopathy ZMPSTE24 54 55insT Mennonite M Severe combined immune deficiency IL7R 2T>G Mennonite M Spinal muscular atrophy SMN1 exon 7 deletion Mennonite M Tyrosine hydroxylase deficiency TH 698G>A Arg233His Mennonite M Tyrosinemia, type 3 HPD 85G>A Ala29Thr Mennonite M Vitamin B12 deficiency AMN 44 bp deletion Mennonite M Properdin deficiency PFC 379T>G Cys127Gly Mennonite X Osteoporosis-pseudoglioma syndrome LRP5 1225A>G Thr409Ala Mennonite S Osteoporosis-pseudoglioma syndrome LRP5 1275G>A Trp425Ter Mennonite S Phenylketonuria PAH IVS10-11G>A Mennonite S Cardiomyopathy, hypertrophic SLC25A4 523delC Mennonite W Periodic fever (TRAPS) TNFRSF1A 362G>A Arg121Gln Mennonite W Salla disease SLC17A5 115C>T Arg39Cys Mennonite W Tyrosinemia, type 3 HPD 479A>G Tyr160Cys Mennonite W Tyrosinemia, type 3 HPD 1005C>G Ile335Met Mennonite W ∗A, Lancaster County (PA) Amish; B, Juniata and Mifflin County (PA) Amish; O, Holmes and Geauga County (OH) Amish; J, Somerset County (PA) Amish; M, Weaverland and Groffdale Conference Mennonites (PA and elsewhere); S, Stauffer Mennonites (PA/MD); W, Western MD; X, other unspecified Mennonites; E, eastern shore of Maryland Amish. www.annualreviews.org • Genetics, Medicine, and the Plain People 521 A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 a b A ve ra g e n u m b er o f b lo ck s D ev ia ti o n fr o m c o n tr o l 20 15 10 –5 –10 –15 –20 –25 –30 –35 –40 Amish Mennonite Control Block size 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100 >100 Amish Mennonite Block size 222222 42 62 82 102 122 142 162 182 202 6 5 4 3 2 1 0 5 0 Figure 4 Plain populations demonstrate increases in homozygous block size and number compared to Americans of European descent. Blocks of homozygosity were quantified by their SNP content and physical size. (a) The average number of homozygous SNP blocks per person is shown. Amish and Mennonite individuals have more (and larger) runs of homozygosity than do controls. (b) The figure plots deviations from the average control block data by size (i.e., number of SNPs contained in each homozygous block). The Plain populations are relatively deficient in small homozygous runs, which are replaced by larger blocks. The excess runs of homozygosity range in size from about 20 to over 200 adjacent SNPs (highlighted area). For each Plain population, excess homozygosity relative to European controls is found in a relatively small number of large blocks, comprising roughly 116.7 and 72.5 Mb of homozygous DNA in Amish and Mennonite genomes, respectively. used these techniques to map disorders with as few as two affected children (89). We have applied various methods to iden- tify 75 different mutations segregating in the Plain populations of southeastern Pennsylva- nia and elsewhere. The most straightforward method is candidate gene analysis—sequencing a candidate gene known to cause a pheno- type. Phenylketonuria mutations in several unrelated Old Order Amish patients from Lan- caster County were identified in this way, and the same strategy was applied to disorders such as 5,10-methylenetetrahydrofolate reduc- tase (MTHFR) deficiency, factor XI deficiency, and Crigler-Najjar syndrome. A related approach, candidate gene local- ization, uses SNP data to narrow a list of candidate genes that are associated with a particular phenotype and scattered across the genome, provided the child is autozygous sur- rounding the disease gene locus. By correlating homozygous blocks to the location of can- didate genes, we can often identify a single gene for targeted sequencing. This method has been used successfully to map autosomal reces- sive deafness, Cockayne syndrome, megaloblas- tic anemia, and hereditary spastic paraplegia (Figure 5). Finally, disease gene mapping uses genetic marker data to map unknown recessive pheno- types by comparing homozygous blocks among multiple affected individuals (67, 68, 89, 90). This strategy exploits genetic properties com- mon to young populations with a small number of founders, and also assumes mutation homo- geneity. We often delineate locus boundaries rapidly and efficiently, but the shared homozy- gous blocks tend to be quite large. The average shared block is 6.3 Mb (mean 3.7 patients per study) and contains dozens and sometimes hun- dreds of genes. Thus, although finding the rele- vant block can be simple, identifying the disease gene within it can be daunting. We have initiated over 40 separate ge- netic mapping studies in Plain populations of Pennsylvania, Maryland, Indiana, and Ohio. For 14 of these, we unequivocally mapped the disease gene and identified the pathogenic se- quence variant. For 10 others, we delineated a chromosomal region but have yet to identify the causative gene (Table 4). For the remainder, mapping results are inconclusive due to insuf- ficient sample size or the presence of multiple shared homozygous blocks. 522 Strauss · Puffenberger A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 Locus Location Number of homozygous SNPs Location score 25 15 10 5 0 35 30 20 SPG20 1q24 2q37 3q27 6q23 8p12 10q22 13q13 13q14 14q 14q21 14q23 15q14 15q21 16q24 SPG23 SPG30 SPG14 SPG25 SPG5A SPG27 SPG20 SPG24 SPG32 SPG28 SPG15 SPG11 SPG21 SPG7 SPG5B SPG35 Chromosome number H o m o zy g o u s b lo ck s iz e 2 4 6 8 10 12 14 16 18 20 22 Figure 5 Two brothers from a Midwestern Amish deme presented to our clinic with developmental delay, short stature, and symmetric spastic diplegia. Recessive hereditary spastic paraplegia (SPG) was considered in the genetic diagnosis, but a review of Online Mendelian Inheritance in Man revealed multiple SPG loci scattered throughout the genome. This daunting gene list would likely preclude further genetic testing. However, assuming identity-by-descent for the disease gene, we used 10K SNP data to exclude the large majority of SPG loci and show that both boys were homozygous for a large block of DNA surrounding the locus for Troyer syndrome (SPG20); sequencing confirmed homozygosity for the known single-base-pair deletion (1110delA) in SPG20. MENDELIAN DISORDERS IN PLAIN POPULATIONS The Evolution of Clinical Methods Amish genetic studies between 1964 and 1988 predated the era of molecular mapping and did not deal explicitly with the problem of providing care. Many research subjects died or were crippled because they lacked appro- priate medical services (57). In 1989, Holmes and Caroline Morton established the small nonprofit Clinic for Special Children to serve Amish and Mennonite children who suffered from genetic disorders (7, 8). The Clinic was sited in rural Lancaster County (Figure 6), which gave families nearby access to affordable care (60) and provided a framework for study- ing genetic disorders in a natural setting (41, 45). It is a paradigm of community genetics (46), designed to serve particular people living in a specific place and time (15, 57, 59, 65). The Old Order communities support the work as a way to care for their own (8, 59). Today, Clinic staff manage 1029 active patients representing over 105 different Mendelian disorders. From the Clinic’s inception, on-site labo- ratory studies were continually shaped by the needs of patients; research and clinical care were inseparable. Between 1989 and 1993, a gas chromatograph-mass spectrometer donated by Hewlett-Packard was used to screen 1232 Amish children for glutaric aciduria type 1 (GA1) (58). In 1990, two Mennonite churches donated a high-performance liquid chromato- graph that has run more than 17,000 amino acid samples for the diagnosis and management of maple syrup urine disease (MSUD) (59). In the early years of the Clinic, operating these in- struments on the front line of clinical practice meant that GA1 could be diagnosed within a few hours of life (i.e., from amniotic fluid ob- tained at delivery) (58), and MSUD could be detected in asymptomatic newborns less than 1 day old (60). These methods were a crucial www.annualreviews.org • Genetics, Medicine, and the Plain People 523 A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 Table 4 Twenty-three genetic disorders mapped using SNP microarrays at the Clinic for Special Children since 2003. The causative gene was identified for 14 conditions, whereas 9 other disorders are mapped to a region but the gene remains unknown Disease Chromosome Gene Nephrotic syndrome, Amish 1 NPHS2 Pelizaeus-Merzbacher-like syndrome 1 GJA12 Bartter syndrome 1 CLCNKB Hypertrophic cardiomyopathy 4 SLC25A4 Primary ciliary dyskinesia 5 DNAH5 Sudden infant death with dysgenesis of the testes 6 TSPYL1 Salla disease 6 SLC17A5 Cortical dysplasia and focal epilepsy 7 CNTNAP2 Glutaric aciduria, type 3 7 C7orf10 Severe combined immune deficiency 11 RAG1 Deafness, Mennonite 13 GJB2 Gitelman syndrome 16 SLC12A3 Polyhydramnios, megalencephaly, and symptomatic epilepsy 17 STRADA Weil-Marchesani syndrome 19 ADAMTS10 Posterior column ataxia with retinitis pigmentosa 1 Congenital nystagmus 1 Usher-like syndrome 5 Hydrocephalus 6 Lethal seizure syndrome 7 Mental retardation, nonsyndromic 12 Junctional ectopic tachycardia 15 Spastic paraplegia 15 “Yoder” dystonia 15 “Schwartz” syndrome 20 Microcephaly 22 first step toward disease prevention, allowing affected newborns to start therapy days before state screening results were complete. In 1999 we began direct DNA sequencing, which led quickly to mutation identification for numerous disorders and carrier testing for GA1, MSUD, and several other conditions (58, 60, 83, 91). Affymetrix donated a GeneChip scanner in 2005. It proved a powerful tool for mapping (67, 88, 89) and also revealed important details about allele dynamics within Plain populations. In 2007, we acquired a Roche real-time PCR system (LightCycler) through charitable contributions from the A.J. Stamps Foundation and CSL Behring. This instrument uses melting curve analysis to detect mutations in about an hour (94). It is ideal for community-based genetic testing [see sidebar 1, 5,10-Methylenetetrahydrofolate Reductase (MTHFR) Deficiency] and can be adapted to practical clinical problems such as low-resolution HLA matching (88). Spectrum of Mendelian Disease Table 3 lists 29 Mennonite and 37 Amish disorders understood at the molecular level, restricted for the purpose of this review to populations of Pennsylvania and Maryland. Al- though there is some crossover, demes of Ohio, 524 Strauss · Puffenberger A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 Indiana, and elsewhere suffer from different genetic disorders. Even within Pennsylvania, there are several genetically distinct groups, and these distinctions are clinically important (see sidebar 2, Genetic Epidemiology). Ninety percent of the conditions listed in Table 3 are recessive, and 90% of these result from a single allele restricted to a particular deme (54, 55). However, there are several exceptions. Among Old Order Mennonites, who stem from a larger founding popula- tion, we have identified multiple mutations for phenylketonuria, cystinuria, medium- chain acyl-CoA dehydrogenase deficiency, mevalonate kinase deficiency, osteoporosis- pseudoglioma syndrome, and tyrosinemia type 3. Among the Amish, biotinidase de- ficiency, galactosemia, Gitelman syndrome, and phenylketonuria demonstrate mutation heterogeneity, and familial hypercholanemia exhibits locus heterogeneity. Although most of the disorders we manage are recessive, we have also identified several X- linked (e.g., fragile X syndrome, chronic granu- lomatous disease, and properdin deficiency) and dominant conditions (e.g., LMNA cardiomy- opathy and polycystic kidney disease), and recognize others yet to be mapped (sympha- langism, multiple exostoses, arteriovenous mal- formation). Although fewer in number, these disorders nevertheless exact a heavy toll when they occur in large extended families. We have also found de novo mutations in a child with lissencephaly (PAFAH1B1 deletion), a child with Apert syndrome (FGFR2), an adult with idiopathic torsion dystonia (DYT1), and sev- eral children with Prader-Willi or Angelman syndrome (chromosome 15 deletions). Finally, using 10K SNP arrays we have identified chro- mosomal abnormalities at the resolution of a standard karyotype in numerous children with syndromic developmental delay (Figure 7). Although certain mutations are concen- trated in Amish and Mennonite demes, they are seldom unique to these groups. About 20% of individuals leave the Old Order each generation, allowing mutations to flow into Figure 6 The Clinic for Special Children is sited on an untillable corner of farmland in rural Lancaster County, Pennsylvania. The land was the gift of an Amishman who has two granddaughters with glutaric aciduria type 1. It was built with the donated labor of Amish and Mennonite workers. The Clinic is readily accessible to most of its patients and has on-site biochemical and molecular testing. SIDEBAR 1. 5,10-METHYLENETETRAHYDROFOLATE REDUCTASE (MTHFR) DEFICIENCY An Amish boy, born in Somerset County in 1998, had psychomo- tor delay and slow head growth by 3 months of age, and an MRI showed global cerebral atrophy and hypomyelination. He was seen by numerous pediatric subspecialists but remained without a diagnosis after more than $10,000 of diagnostic testing. We saw him in October 2003, and based on a biochemical workup identified a pathogenic 1129C>T change in MTHFR (87). De- spite treatment with high-dose betaine, he remained severely re- tarded, autistic, and mute. We made several field trips to Somer- set County where we identified 68 MTHFR c.1129C>T carriers among 230 healthy members of the deme (estimated population carrier frequency 30%). In collaboration with Dr. Naylor and his colleagues, we developed a LightCycler method for detecting MTHFR c.1129C>T in dried filter paper blood spots (28). Mid- wives offered Amish parents the option of getting a “fifth blood spot” to test for the MTHFR mutation. The first newborn diag- nosed by LightCycler screening in 2004 was sister to the proband (87). She started therapy during her second week of life and has no signs of brain damage after four years of follow-up. www.annualreviews.org • Genetics, Medicine, and the Plain People 525 A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 SIDEBAR 2. GENETIC EPIDEMIOLOGY Most Amish babies are delivered at home, breastfeed, and do not receive parenteral vitamin K. In Lancaster County, two recessive alleles in the Amish (TJP2 c.143T>C and BAAT c.226A>G) dis- rupt bile salt metabolism and result in vitamin K malabsorption (20). Thus genetic factors interact with cultural practice to deter- mine actual risk: over the past 10 years, three Amish infants with bile salt disorders presented with massive intracerebral and retinal hemorrhages from vitamin K deficiency. One child survived with dense spastic hemiparesis and unilateral blindness. The other two died of acute brain injuries. In one case, the parents were wrong- fully accused of child abuse and temporarily lost custody of their other children. Screening for bile salt disorders and neonatal vita- min K administration are pressing public health concerns against this genetic background. In Juniata and Mifflin Counties, a mu- tation in TSPYL (457 458insG) among Amish children causes brain disease and a dysautonomia syndrome (SIDDT) that ac- counts for 20% of overall infant mortality and perhaps half of all sudden infant deaths in the region (67). To prevent sudden infant death in Mifflin County, efforts to understand and treat SIDDT syndrome are at least as important as the “back to sleep” campaign (http://nichd.nih.gov/sids/). the outbred population (59). Moreover, most pathogenic alleles probably existed in Europe before the Anabaptist migration. For example, all four Plain mutations of the PAH gene were first identified in Europeans with phenylke- tonuria (Table 3), and the only GCDH variant known to cause glutaric aciduria type 1 in the Amish (c.1262C>T) is among the more com- mon mutations found throughout Europe and the United States. Curiously, several founder mutations from Finland are also distributed in North American Plain sects (i.e., RMRP c.70A>G and SLC17A5 c.115C>T). There are no known genealogical ties between Swiss An- abaptists and Finns, suggesting that these mu- tations are either ancient or recurrent (51). For 21 (31%) conditions listed in Table 3, an experienced clinician can make a correct diagnosis based on the clinical presentation and a single confirmatory test. A few oth- ers are distinctive and easily recognized (e.g., Amish lethal microcephaly, Ellis-van Creveld syndrome, Weil-Marchesani syndrome, etc.). Only 8 (12%) are reliably detected by state newborn screening. Importantly, about 40% of the disorders cannot be diagnosed by rou- tine clinical methods; they present to physi- cians as nonspecific problems such as failure to thrive, jaundice, bruising, itching, develop- mental delay, cerebral palsy, mental retardation, autism, seizures, short stature, or weakness. An understanding of genetic risks within a popu- lation makes it clear that these general terms apply to many discrete causes (Table 5) (see sidebar 3, Glutaryl-CoA Dehydrogenase Defi- ciency). The same is likely true in outbred pop- ulations, but it is much harder to investigate. When we encounter a novel phenotype, in- vestigational studies are needed to determine the molecular cause and then translate this in- formation into useful clinical testing (67, 68, 90). Many core genetic laboratories offer DNA isolation and storage as well as biochemical ge- netics testing. However, few offer the flexible genotyping and sequencing services needed to map a new disease. Fewer still achieve these aims within a clinically relevant time frame or at an acceptable cost to self-pay families. Our laboratory functions as a specialized core fa- cility with the sole purpose of solving patient- and community-centered problems quickly and economically (88). We have moved the tools of genetic research to the clinical laboratory and invested heavily in their continued use and ef- ficacy. Consequently, when evaluating a new patient we are as likely to sequence a gene or generate SNP genotypes as we are to order an electrolyte profile. TRANSLATIONAL MEDICINE AND THE TREATMENT OF GENETIC DISEASE Treatment of Genetic Disease Just over 20 years ago, half of known genetic disorders were considered untreatable and only about 10% could be treated in a highly effective 526 Strauss · Puffenberger A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 manner (93). We have seen extraordinary progress in Plain populations over the past two decades. For about 40% of the disorders we manage, simple dietary intervention has a deci- sive effect. These include conditions such as bi- otinidase deficiency, galactosemia, and vitamin B12 malabsorption. Protocols for the treatment of maple syrup urine disease (MSUD), Crigler- Najjar syndrome, and severe combined immune deficiency (SCID) are more complex but still quite effective (2, 57, 60, 91). These latter con- ditions can now be cured by tissue transplant, currently the only reliable form of gene therapy (85, 91). Problems such as pyruvate kinase de- ficiency, cystinuria, and deafness are treatable, but not perfectly so. Nevertheless, like diabetes, depression, and cancer, skillful medical care al- lows people to live longer, suffer less, and enjoy more independence. For 13 (21%) genetic conditions listed in Table 3, eventual outcome is not affected by treatment; five of these disorders are lethal dur- ing infancy. They are among the most common genetic disorders we encounter; among the −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 7 Affymetrix GeneChip Human Mapping 10K Arrays are useful for copy-number assessment. Although the 10K arrays lack fine resolution for this application, our data show that they perform at least as well as a standard karyotype. We readily identify copy-number abnormalities in the 2–4 Mb range, validated by standard metaphase karyotype (http://www.mayomedicallaboratories.com/). Our analyses use the copy-number data generated by the Affymetrix Genotype software. We calculate mean and standard deviations for the fluorescence at each SNP marker and then generate Z scores for the patient at each SNP marker. This partially corrects for polymorphic copy-number variation within the genome. Panels (a) and (b) show two small deletions of ∼4.1 and ∼4.2 Mb, respectively. The first patient had multiple congenital anomalies, and the second patient was diagnosed with Angelman syndrome after SNP analysis detected monosomy on chromosome 15. Panels (c) and (d ) depict trisomy of chromosome 2q markers and a complex rearrangement involving partial deletion and duplication of chromosome 8. Data in panels (b) and (d ) are from males, and hemizygosity for X chromosome markers is apparent. Amish, for example, we have a record of 85 chil- dren with lethal microcephaly born between 1960 and 2008 and 88 children born with ne- maline rod myopathy (TNNT1 ) since 1989 9 6 3 0 –3 –6 9 6 3 0 –3 –6 9 6 3 0 –3 –6 9 6 3 0 –3 –6 c a d b Chromosome number 2 4 6 8 10 12 14 16 18 20 22X SN P c o p y n u m b er Z s co re SN P c o p y n u m b er Z s co re SN P c o p y n u m b er Z s co re SN P c o p y n u m b er Z s co re www.annualreviews.org • Genetics, Medicine, and the Plain People 527 A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 SIDEBAR 3. GLUTARYL-CoA DEHYDROGENASE DEFICIENCY Before 1989, Amish children with GA1 were commonly misdi- agnosed with cerebral palsy or acute viral encephalitis. The first genetic study at the Clinic for Special Children identified GA1 as the cause of “Amish cerebral palsy” in 16 disabled children who traced to a single founding couple (58). Beginning in 1989, the Clinic offered amniotic fluid and urine organic acid screening for newborns, comprehensive pediatric follow-up care, and inpa- tient management for illnesses. Dr. Naylor introduced statewide supplemental screening for GA1 in 1994. Between 1989 and the present, the rate of brain injury in GA1 decreased from 94% to 36% (83). Thus the efficacy of treatment in GA1, as with many genetic conditions, is relative. Although GA1 is the most com- mon mimic of cerebral palsy in Lancaster County, other hered- itary conditions can produce a similar phenotype and, equally importantly, not every child with dystonia has a Mendelian dis- order. Acquired causes of brain injury are intermingled with, and difficult to distinguish from, genetic disease. (43, 59). Even for such “untreatable” prob- lems, medical care is important (57, 59). Before the Clinic was established, Amish children with nemaline rod myopathy were repeatedly sub- jected to invasive and costly interventions (e.g., muscle biopsies, nerve conduction testing, elec- tromyography, magnetic resonance imaging, echocardiograms, etc.) that collectively cost the community millions of dollars. A thoughtful physician informed with the right genetic di- agnosis can help a family determine appropri- ate limits of medical care while also protecting the child from futile interventions and the com- mon miseries of hunger, thirst, dyspnea, and pain. Presymptomatic Diagnosis and Disease Prevention Children born today with conditions such as GA1, MTHFR deficiency, or MSUD— once thought untreatable (39)—now grow up healthy if diagnosed early in life (see sidebars 1, 3, and 4) (60, 83, 87). Presymptomatic diagnosis creates a window of opportunity (22, 33, 40, 83). This was the idea behind Guthrie & Susi’s test for phenylketonuria in newborns, published in 1963 (36). Thirty years later, shortly after Dr. Morton began his work in Lancaster County, Edwin Naylor and colleagues were using tan- dem mass spectrometry to screen newborns for a much broader spectrum of genetic conditions (21). Screening does not, however, assure good outcome. Early diagnosis is of no value with- out a pragmatic follow-up plan designed to deal with the dynamic interplay among genes, envi- ronment, and disease as it unfolds over time. Genes encode proteins embedded in complex biological networks. These networks are con- tained within cells, grouped into tissues, and able to interact within physiological systems. Understanding disturbances at all of these lev- els, as experienced by intact humans in their natural setting, is the basis for designing effec- tive therapies (38, 70). This is vividly illustrated by the history of MSUD in Lancaster County (59). Before 1989, one-third of Mennonite chil- dren with MSUD died during childhood. Most who survived were severely disabled [see side- bar 4, Changing the Natural History of Maple Syrup Urine Disease (MSUD)] (57, 59, 60). Since the Clinic started offering local services, outcomes for MSUD have improved (60). No child died while under our care for metabolic crisis, and all Mennonite children with MSUD born after 1988 attained normal developmen- tal milestones. Knowledge of pathophysiology, not genetics, fueled this progress. Through careful study of many individuals in diverse clinical circumstances, we learned how defi- ciency of branched-chain ketoacid dehydrogen- ase disrupts critical biological processes such as metabolic adaptation to fasting and illness (86), cerebral amino acid transport (86, 87), and cell volume control (60). These processes change with age and are influenced by many nutri- tional and environmental variables. Our treat- ment protocol evolved in lockstep with our un- derstanding of this complex physiology (60) and allowed us to prevent many serious neurologi- cal consequences of MSUD. 528 Strauss · Puffenberger A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 Table 5 Gene mutations result in specific medical conditions that are often described with nonspecific terms: mental retardation, cerebral palsy, autism, sudden infant death, sepsis, and epilepsy1 Clinical diagnosis Disorder Gene Cerebral palsy Glutaric aciduria type 1 GCDH Propionic acidemia PCCB Gap junction deficiency GJA12 Crigler-Najjar syndrome UGT1A1 Hereditary spastic paraplegia SPG20 Dopamine-responsive dystonia TH Idiopathic torsion dystonia DYT1 Mental retardation Phenylketonuria PAH MTHFR deficiency MTHFR Maple syrup urine disease BCKDHA Salla disease SLC17A5 Bardet-Biedl syndrome BBS1 Fragile X syndrome FMR1 Epilepsy Biotinidase deficiency BTD CDFE syndrome (CASPR2) CNTNAP2 GM3 synthase deficiency ST3GAL5 PMSE syndrome STRADA Stroke/hemorrhage Hypercholanemia,TJP2 type TJP2 Hypercholanemia, BAAT type BAAT Factor V Leiden F5 Sitosterolemia ABCG8 Alpha-1-antitrypsin deficiency SERPINA1 Sudden death SIDDT syndrome TSPYL 3-beta-HSD deficiency HSD3B2 Hypertrophic cardiomyopathy SLC25A4 Lethal infection Galactosemia GALT Properdin deficiency PFC SCID, IL7 receptor type IL7R Adenosine deaminase deficiency ADA RAG1 deficiency, Omenn RAG1 1 This table lists a selection of genetic syndromes that cause developmental disorders in Amish and Mennonite children. Recognizing genetic predispositions that underlie such disorders is a key step toward planning effective prevention strategies. Disease prevention has substantial eco- nomic repercussions (57). Based on our expe- rience with MSUD and several other treatable genetic conditions that affect the nervous system (Table 3), we have prevented severe mo- tor disability or mental retardation in an es- timated 150 children over a 20-year period. This saved the Plain communities about $180 million in direct and indirect costs (3). Thus annually, our operating budget of $1.4 million reduces the community economic burden by over $8 million. To help uninsured patients, preventative therapies must be affordable. For example, early www.annualreviews.org • Genetics, Medicine, and the Plain People 529 A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 SIDEBAR 4. CHANGING THE NATURAL HISTORY OF MAPLE SYRUP URINE DISEASE (MSUD) Between 1966 and 1988, 36 Mennonite children were born with MSUD in Lancaster County. They all became encephalopathic within the first week of life and required weeks or months of intensive hospital treatment that cost tens of thousands of dollars. Follow-up care was expensive, far away, and disorganized (59, 73). MSUD patients were often sick at home for days before arriving comatose in Philadelphia with critical cerebral edema. No aspect of therapy dealt specifically with this complication, and 14 (36%) of these children died of brain herniation within 24 h of hospitalization (59, 73). Since 1989, we have managed 59 Mennonite infants with MSUD. Thirty-two (59%) were diagnosed between 12 and 24 h of life by on-site amino acid analysis or, more recently, di- rect mutation detection from umbilical cord blood using real- time PCR. Among infants diagnosed before 3 days of age, 94% were treated safely at home. To manage crises locally, we made MSUD hyperalimentation solution available 24 h a day at Lancaster General Hospital and had on-site amino acid analysis with a 30-min turnaround time. We have successfully managed over 200 metabolic crises at Lancaster General with an average hospital stay of just 4 days. By combining general pediatric care and specialized services, a much larger number of metabolic crises were averted out of hospital; the overall hospitalization rate for our MSUD cohort is currently only 0.4 hospital days per patient per year. studies of vitamin B12 deficiency led to reli- able and inexpensive forms of replacement ther- apy (61). Now, for Mennonite children with hereditary B12 malabsorption (AMN deletion), once daily sublingual methyl-B12 prevents life- threatening anemia and spinal cord injury for about $30 per year. However, if sublingual methyl-B12 were to be developed as a new thera- peutic today, it would cost between $500 million and $2 billion to bring to the U.S. market, and its cost would be prohibitive for many self-pay families (4). Although we can expect exciting new advances in the treatment of genetic dis- ease, attendant costs will put effective therapies beyond the reach of some families. PLAIN PEOPLE AT THE FRONTIER OF GENOMIC MEDICINE When McKusick and colleagues began their studies of the Amish in 1962 (31), medical ge- netics was just a fledging discipline and molec- ular studies were limited to descriptions of ab- normal chromosome number and structure (9). Polymerase chain reaction was not introduced until 1986 (12), and polymorphic microsatel- lite markers were not widely used until the early 1990s (50). The Human Genome Project started in October 1990, and the complete draft of the human genome was published in Febru- ary 2001 (25, 96). Thus, genetic discovery in the Plain communities developed in parallel with larger events taking place in the field of ge- nomics. Genetic science has become a cornerstone of medicine, but the practice of medicine has been equally vital to progress in genetic science. Bench scientists and clinicians have different interests and responsibilities. Basic scientists investigate physiologic systems, tis- sues, membranes, pathways, and molecules. They are primarily concerned with patterns and mechanisms. Physicians, in contrast, are prin- cipally concerned with the welfare of individ- uals, “for the patient is no mere collection of symptoms, signs, disordered functions, dam- aged organs, and disturbed emotions. [The pa- tient] is human, fearful, and hopeful, seeking relief, help, and reassurance” (29). These differing perspectives influence how one views progress in genomic science (82). Consider three decades of progress in cellu- lar immunology. Over the past 30 years, im- munogenetics research has expanded at an ex- plosive pace; we now understand many cellular immune responses in exquisite molecular de- tail (18). Yet among 41 Amish and Mennonite children born with SCID during the same time period, only half received a bone marrow trans- plant and 63% died by 2 years of age (88). There was little funding for patient-centered research (34), and hospitals with expertise in SCID were far away and cost too much (57). 530 Strauss · Puffenberger A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 We came face to face with this problem in January 2007, when an Amish newborn pre- sented to our Clinic with Omenn syndrome, a variant of SCID (Figure 8) (88). She was sick and uninsured, and needed timely, affordable care. We were able to use 10K SNP genotypes from the child and her siblings to delineate the RAG1/RAG2 locus, and used these same data to quickly and inexpensively find an HLA- matched stem cell donor among her siblings. This entire process—from clinical presentation to genetic diagnosis to donor identification— took less than two weeks and saved the fam- ily between $20,000 and $80,000. Applied ge- nomics enabled access to life-saving therapy. In a medical-industrial research environ- ment that views case reports and small cohorts as poor evidence (98), this one Amish child gave us a glimpse of McKusick’s vision for ge- nomic medicine (Figure 9) when he wrote: “[G]enomics is likely to render medicine more predictive and, therefore, more preventive. . . on the traditional turf of clinical medicine, di- agnosis will become more specific and pre- cise, and treatment also more specific and safer. Genomics-based individualization of medical care aims to achieve the right treatment for the right patient” (50) (italics added). Thus, the fu- ture of translational genomics is about patient discovery, not gene discovery (14, 34, 77). The idea has a modern emphasis but is not new; Hippocrates believed that diseases as such did not exist in nature. To him, what existed natu- rally were sick people, and no two people were sick in exactly the same way (77). The ethical imperative to care for individ- uals with genetic disease is apparent, but the scientific value of such work should not be over- looked. The connection of a gene to a human phenotype is the only way to fully appreciate a gene’s function in vivo (24, 49, 55, 59, 83). In this respect, mice, fish, and flies will always fall short of modeling human disease (see sidebar 5, New Disease Pathways in Human Brain Development). The astute physician, working daily to care for one or a few patients with a rare and complex disorder, can develop key insights that deepen our understanding (68, 90), RAG1/RAG2 C h ro m o so m e 11 HLA gene cluster C h ro m o so m e 6 2 4 6 8 10 12 14 16 18 20 22 a b c Chromosome number Figure 8 An Amish girl was born in 1993 with erythroderma, hepatosplenomegaly, hypereosinophilia, hypogammaglobulinemia, and B cell deficiency. She grew poorly and had recurrent infections. She was provisionally diagnosed with Omenn syndrome, a variant of severe combined immune deficiency (SCID), but died at 3 months of age before the diagnosis could be confirmed or a transplant attempted. In January 2007, her sister presented as a newborn with alopecia and erythroderma, developed a systemic Staphylococcus aureus infection at three weeks of age, and had immunologic studies consistent with SCID. (a) A homozygosity plot was generated for the proband ( gray peaks); identity- by-state (IBS) plots for each pairwise comparison between the proband and her unaffected siblings were superimposed on this plot. Genotype-identical regions (colored lines) between the proband and her siblings (i.e., IBS = 2) denote regions where the disease gene cannot reside. The red line at the top is a summary of the exclusion mapping. This analysis demonstrated that only a single, homozygous genomic region on chromosome 11 was consistent with linkage within the pedigree. (b) The RAG1 and RAG2 loci mapped to the homozygous interval on chromosome 11. RAG1 sequencing revealed homozygosity for a pathogenic c.2974A>G variant in the patient. (c) The homozygosity plot of chromosome 6 in the proband and her seven unaffected siblings. Compared to the proband, only one sibling is haplo-identical for the major HLA loci on chromosome 6. Subsequently, HLA typing of the patient and her potential sibling donor was performed using standard serotyping and molecular probes; these data corroborated the SNP matching. The patient had a successful unmodified bone marrow transplant at 62 days of age. She is now 2 years old, fully engrafted, and healthy. DNA extracted from paraffinized bone marrow showed that her sister, who died 14 years earlier, was also homozygous for RAG1 c.2974A>G. www.annualreviews.org • Genetics, Medicine, and the Plain People 531 A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 SIDEBAR 5. NEW DISEASE PATHWAYS IN HUMAN BRAIN DEVELOPMENT In 2006, we discovered a homozygous frameshift mutation of the CNTNAP2 gene in a group of Amish children with cortical dys- plasia, focal epilepsy, and developmental regression (90). Remark- ably, mice with homozygous Cntnap2 mutations have no central nervous system disease. Nevertheless, the findings prompted new studies about the role of CNTNAP2 in human brain development. Within two years this gene was linked to idiopathic autism (5, 10, 11), schizophrenia (32), and mutism (97) in outbred populations across the country and throughout the world. Thus, the study of rare Mendelian disorders links specific gene families or protein networks—i.e., “interactomes”—to relevant processes in human biology. In the words of William Harvey (ca. 1657): “Nature is nowhere accustomed more openly to display her secret mysteries than in cases where she shows traces of her workings apart from the beaten path; nor is there any better way to advance the proper practice of medicine than to give our minds to the discovery of the usual law of nature by careful investigation of cases of rarer forms of disease.” expand our diagnostic capabilities (88, 89), and create new opportunities for prevention (34, 58, 60, 87). In the era of genomic medicine, physicians and molecular biologists who work closely together can translate these insights into real clinical benefits. Such collaborations will help us tackle big problems at the frontier of genomic medicine, including multigenic dis- ease (e.g., atherosclerosis, hypertension, men- tal illness, etc.), modifier genes, and epigenetics (24, 30, 64, 78, 79). DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review. ACKNOWLEDGMENTS We thank Donna Robinson and Christine Hendrickson for assistance in data collection and for their exceptional contributions to patient care. Drs. Nicholas Rider and Terry Sharrer provided important insights and helpful suggestions. Don Kraybill and his colleagues at the Young Center for Anabaptist Studies, Elizabethtown College, provided demographic data. Holmes and Caroline Morton envisioned the Clinic for Special Children and had the tenacity to make it real; we are Figure 9 Victor A. McKusick (left), D. Holmes Morton (right), and Bowie sitting on the front porch at the Clinic for Special Children, spring 2007. Finally, it is important to remember that ad- vances in molecular science only reveal what can be done with genetic information. Perhaps the Plain people can teach us something impor- tant about what should be done with it (24, 57). The Old Order communities measure the value of medical research not in grant awards, publi- cations, or academic promotions, but in human terms: alleviation of pain, prevention of disabil- ity, equitable delivery, and fair cost. They un- derstand that to translate genetic information into better public health, one must first com- mit to caring for individuals. 532 Strauss · Puffenberger A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . ANRV386-GG10-23 ARI 29 July 2009 15:38 deeply indebted to them for their extraordinary example and ongoing contributions to people of the Plain communities. Finally, we thank the children and families whom we serve; they continue to teach us much about science, medicine, and meaningful work (57). LITERATURE CITED 1. McKusick VA, ed. 1978. Medical Genetic Studies of the Amish. 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The Cult of Statistical Significance. Ann Arbor: Univ. Mich. Press 536 Strauss · Puffenberger A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . AR386-GG10-FM ARI 6 August 2009 7:15 Annual Review of Genomics and Human Genetics Volume 10, 2009 Contents Chromosomes in Leukemia and Beyond: From Irrelevant to Central Players Janet D. Rowley � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1 Unraveling a Multifactorial Late-Onset Disease: From Genetic Susceptibility to Disease Mechanisms for Age-Related Macular Degeneration Anand Swaroop, Emily Y. Chew, Catherine Bowes Rickman, and Gonçalo R. Abecasis � � �19 Syndromes of Telomere Shortening Mary Armanios � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �45 Chronic Pancreatitis: Genetics and Pathogenesis Jian-Min Chen and Claude Férec � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �63 The Genetics of Crohn’s Disease Johan Van Limbergen, David C. Wilson, and Jack Satsangi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �89 Genotyping Technologies for Genetic Research Jiannis Ragoussis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 117 Applications of New Sequencing Technologies for Transcriptome Analysis Olena Morozova, Martin Hirst, and Marco A. Marra � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 135 The Posttranslational Processing of Prelamin A and Disease Brandon S.J. Davies, Loren G. Fong, Shao H. Yang, Catherine Coffinier, and Stephen G. Young � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 153 Genetic Testing in Israel: An Overview Guy Rosner, Serena Rosner, and Avi Orr-Urtreger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 175 Stewardship of Human Biospecimens, DNA, Genotype, and Clinical Data in the GWAS Era Stephen J. O’Brien � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 193 Schistosoma Genetics: New Perspectives on Schistosome Biology and Host-Parasite Interaction Ze-Guang Han, Paul J. Brindley, Sheng-Yue Wang, and Zhu Chen � � � � � � � � � � � � � � � � � � � � 211 Evolution of Genomic Imprinting: Insights from Marsupials and Monotremes Marilyn B. Renfree, Timothy A. Hore, Geoffrey Shaw, Jennifer A. Marshall Graves, and Andrew J. Pask � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 241 v A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . AR386-GG10-FM ARI 6 August 2009 7:15 Methods for Genomic Partitioning Emily H. Turner, Sarah B. Ng, Deborah A. Nickerson, and Jay Shendure � � � � � � � � � � � � � 263 Biased Gene Conversion and the Evolution of Mammalian Genomic Landscapes Laurent Duret and Nicolas Galtier � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 285 Inherited Variation in Gene Expression Daniel A. Skelly, James Ronald, and Joshua M. Akey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 313 Genomic Analyses of Sex Chromosome Evolution Melissa A. Wilson and Kateryna D. Makova � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 333 Sequencing Primate Genomes: What Have We Learned? Tomas Marques-Bonet, Oliver A. Ryder, and Evan E. Eichler � � � � � � � � � � � � � � � � � � � � � � � � � � � 355 Genotype Imputation Yun Li, Cristen Willer, Serena Sanna, and Gonçalo Abecasis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387 Genetics of Athletic Performance Elaine A. Ostrander, Heather J. Huson, and Gary K. Ostrander � � � � � � � � � � � � � � � � � � � � � � � � 407 Genetic Screening for Low-Penetrance Variants in Protein-Coding Diseases Jill Waalen and Ernest Beutler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 431 Copy Number Variation in Human Health, Disease, and Evolution Feng Zhang, Wenli Gu, Matthew E. Hurles, and James R. Lupski � � � � � � � � � � � � � � � � � � � � � 451 The Toxicogenomic Multiverse: Convergent Recruitment of Proteins Into Animal Venoms Bryan G. Fry, Kim Roelants, Donald E. Champagne, Holger Scheib, Joel D.A. Tyndall, Glenn F. King, Timo J. Nevalainen, Janette A. Norman, Richard J. Lewis, Raymond S. Norton, Camila Renjifo, and Ricardo C. Rodríguez de la Vega � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 483 Genetics, Medicine, and the Plain People Kevin A. Strauss and Erik G. Puffenberger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 513 Indexes Cumulative Index of Contributing Authors, Volumes 1–10 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 537 Cumulative Index of Chapter Titles, Volumes 1–10 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 541 Errata An online log of corrections to Annual Review of Genomics and Human Genetics articles may be found at http://genom.annualreviews.org/ vi Contents A nn u. R ev . G en om . H um an G en et . 2 00 9. 10 :5 13 -5 36 . D ow nl oa de d fr om a rj ou rn al s. an nu al re vi ew s. or g by D r. K ev in S tr au ss o n 10 /0 8/ 09 . F or p er so na l us e on ly . Search Annual Reviews Annual Reviews Online Annual Review of Genomics and Human Genetics Online Most Downloaded Genomics and Human Genetics Reviews Most Cited Genomics and Human Genetics Reviews Annual Review of Genomics and Human Genetics Errata View Current Editorial Committee All Articles in the Annual Review of Genomics and Human Genetics, Vol. 10 Chromosomes in Leukemia and Beyond: From Irrelevantto Central Players Unraveling a Multifactorial Late-Onset Disease: From Genetic Susceptibility to Disease Mechanisms for Age-Related MacularDegeneration Syndromes of Telomere Shortening Chronic Pancreatitis: Genetics and Pathogenesis The Genetics of Crohn’s Disease Genotyping Technologies for Genetic Research Applications of New Sequencing Technologies for Transcriptome Analysis The Posttranslational Processing of Prelamin A and Disease Genetic Testing in Israel: An Overview Stewardship of Human Biospecimens, DNA, Genotype, and Clinical Data in the GWAS Era Schistosoma Genetics: New Perspectives on Schistosome Biology and Host-Parasite Interaction Evolution of Genomic Imprinting: Insights from Marsupials and Monotremes Methods for Genomic Partitioning Biased Gene Conversion and the Evolution of Mammalian Genomic Landscapes Inherited Variation in Gene Expression Genomic Analyses of Sex Chromosome Evolution Sequencing Primate Genomes: What Have We Learned? Genotype Imputation Genetics of Athletic Performance Genetic Screening for Low-Penetrance Variants in Protein-Coding Diseases Copy Number Variation in Human Health, Disease, and Evolution The Toxicogenomic Multiverse: Convergent Recruitment of ProteinsI nto Animal Venoms Genetics, Medicine, and the Plain People