Developmental and degenerative features in a complicated spastic paraplegia Developmental and Degenerative Features in a Complicated Spastic Paraplegia M. Chiara Manzini, PhD,1 Anna Rajab, PhD,2 Thomas M. Maynard, PhD,3 Ganeshwaran H. Mochida, MD, MMSc,1,4,5 Wen-Hann Tan, BMBS,5 Ramzi Nasir, MD, MPH,6 R. Sean Hill, PhD,1 Danielle Gleason, BA,5 Muna Al Saffar, MBChB, CCGC1,5 Jennifer N. Partlow, MS, CGC,1,5 Brenda J. Barry, MS, CGC,1,5 Mike Vernon, MSc,3 Anthony-Samuel LaMantia, PhD,3 and Christopher A. Walsh, MD, PhD1,5 Objective: We sought to explore the genetic and molecular causes of Troyer syndrome, one of several compli- cated hereditary spastic paraplegias (HSPs). Troyer syndrome had been thought to be restricted to the Amish; however, we identified 2 Omani families with HSP, short stature, dysarthria and developmental delay— core fea- tures of Troyer syndrome—and a novel mutation in the SPG20 gene, which is also mutated in the Amish. In addition, we analyzed SPG20 expression throughout development to infer how disruption of this gene might generate the constellation of developmental and degenerative Troyer syndrome phenotypes. Methods: Clinical characterization of 2 non-Amish families with Troyer syndrome was followed by linkage and sequencing analysis. Quantitative polymerase chain reaction and in situ hybridization analysis of SPG20 expression were carried out in embryonic and adult human and mouse tissue. Results: Two Omani families carrying a novel SPG20 mutation displayed clinical features remarkably similar to the Amish patients with Troyer syndrome. SPG20 mRNA is expressed broadly but at low relative levels in the adult brain; however, it is robustly and specifically expressed in the limbs, face, and brain during early morphogenesis. Interpretation: Null mutations in SPG20 cause Troyer syndrome, a specific clinical entity with developmental and degenerative features. Maximal expression of SPG20 in the limb buds and forebrain during embryogenesis may explain the developmental origin of the skeletal and cognitive defects observed in this disorder. ANN NEUROL 2010;67:516 –525 Hereditary spastic paraplegias (HSPs) comprise severaldisorders commonly divided into 2 subgroups: “pure” HSPs characterized by progressive spasticity in the lower limbs due to pyramidal tract degeneration and “complicated” HSPs, where lower limb spasticity is asso- ciated with a variety of other neurological signs and clin- ical features. Complicated HSPs are clinically heteroge- neous, mainly autosomal recessive syndromes, frequently described and mapped in sporadic families within inbred populations.1–3 Because of this heterogeneity, diagnosis Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.21923 Received Jun 25, 2009, and in revised form Oct 26. Accepted for publication Nov 6, 2009. Address correspondence to Dr Walsh, Chief, Division of Genetics, Children’s Hospital Boston, Howard Hughes Medical Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Room 14047.1, Center for Life Sciences Building, 300 Longwood Ave CLS 14047.1, Boston, MA 02115. E-mail: christopher.walsh@childrens.harvard.edu From the 1Department of Neurology and Howard Hughes Medical Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 2Genetic Unit, Directorate General of Health Affairs, Ministry of Health, Muscat, Sultanate of Oman; 3Department of Cell and Molecular Physiology and UNC Neuroscience Center, University of North Carolina School of Medicine, Chapel Hill, NC; 4Pediatric Neurology Unit, Massachusetts General Hospital, Boston, MA; 5Division of Genetics and Manton Center for Orphan Disease Research, Children’s Hospital Boston, Boston, MA; and 6Division of Developmental Medicine, Children’s Hospital Boston, Boston, MA. Additional Supporting Information can be found in the online version of this article. ORIGINAL ARTICLE 516 © 2010 American Neurological Association and recommendations for genetic testing in these disor- ders have been daunting tasks. Troyer syndrome (Online Mendelian Inheritance in Man #275900) is a complicated HSP associated with short stature, skeletal abnormalities, dysarthria, and developmen- tal delay, first described in the Old Order Amish.4,5 Since the original description in 1967, several Troyer-like syn- dromes have been reported,6 –9 but they often differed from classical Troyer syndrome in their neurological or skeletal features. The Amish founder mutation is a single nucleo- tide deletion in the SPG20 gene,10 leading to the loss of the spartin protein11; however, no additional SPG20 mu- tations were subsequently identified,5 and it was suspected that Troyer syndrome may be restricted to the Amish. We identified an Omani kindred presenting with clinical features resembling Troyer syndrome. All affected Omani individuals had a novel homozygous null mutation in SPG20, and their clinical descriptions matched closely to those of Amish Troyer syndrome individuals of comparable ages. Because Troyer syndrome is associated with develop- mental features, such as short stature, skeletal abnormali- ties, and global developmental delay, we investigated the sites of action of SPG20 during development in humans and mice. SPG20/Spg20 expression in the adult is relatively modest and widespread in the nervous system; however, maximal and focal expression is observed in the embryonic limb buds, face, and forebrain during early morphogenesis, which might explain the developmental phenotypic changes involving the extremities, face, and brain. Subjects and Methods Enrollment and Clinical Studies The subjects belonged to 2 families residing in a remote region of Oman and were originally ascertained by a local clinical geneticist. Detailed family and medical histories were obtained by a genetic counselor, who is a native Arabic speaker, and a developmental pediatrician, also a native Arabic speaker, conducted a develop- mental assessment. A pediatric neurologist performed a standard neurological examination, and a second clinical geneticist obtained anthropometric measurements. The height of all subjects and head circumference of subjects under the age of 36 months were plotted on the Centers for Disease Control and Prevention 2000 growth charts,12 and head circumference of subjects above 36 months and all other anthropometric measurements were plotted on standard charts.13 Written, informed consent was obtained from the subjects or their legal guardians. The Ministry of Health in Oman and the institutional review board of Children’s Hospi- tal Boston approved this study. Linkage Analysis Genomic DNA was purified from lymphocytes separated from peripheral blood using commercial kits (Qiagen, Valencia, CA). Six hundred nanograms of genomic DNA was used to hybridize Affymetrix Human SNP Array 6.0 at the Genomic Analysis Core of the University of North Carolina (UNC) Neuroscience Center of the UNC School of Medicine, Chapel Hill, North Carolina. After removal of low-quality calls and of Mendelian and non-Mendelian errors using Merlin software,14 the single nucleotide polymorphism (SNP) data were analyzed using Alle- gro software15 under a fully penetrant autosomal recessive model. For microsatellite analysis, highly polymorphic microsat- ellite markers were chosen from the Marshfield database in the University of California at Santa Cruz Genome Browser. Fluo- rescently labeled polymerase chain reaction (PCR) primers (Ap- plied Biosystems, Foster City, CA) were used to amplify DNA samples using standard conditions, and PCR products were re- solved on an Applied Biosystems 3130xl Genetic analyzer. SPG20 Sequencing Analysis Sequencing of SPG20 (NM_015087; NM_001142294-6) cod- ing region was performed by SeqWright (Houston, TX) on PCR products after amplification of genomic DNA. PCR prim- ers were designed for each exon including at least 50bp of flank- ing intronic sequences. Primer sequences are available on re- quest. Once a putative change was identified, at least 96 control DNAs (192 alleles) were tested to exclude the possibility of a benign polymorphic change. Patient Cell Lines and Mutated Protein/mRNA Detection Transformed lymphoblastoid cell lines were established from pe- ripheral blood of affected and unaffected individuals at the Part- ners Center for Personalized Genetic Medicine (Cambridge, MA). Cell lines were maintained in RPMI medium supplemented with 10% fetal bovine serum, glutamine (2mM) and penicillin/strep- tomycin (100U/100�g) (all from Gibco, Grand Island, NY). Pro- tein lysates were obtained by boiling a cell pellet in Laemmli sam- ple buffer (Bio-Rad Laboratories, Hercules, CA). Western blotting was performed using standard protocols on Bio-Rad equipment. Presence of spartin was assessed using a rabbit anti-SPG20 anti- body (Proteintech Group, Chicago, IL) and a goat anti-rabbit IRDye-800CW secondary antibody (LI-COR Biosciences, Lin- coln, NE) detected on an Odyssey Imager (LI-COR Biosciences). Total RNA was purified from patient cell lines using the mirVana RNA Isolation Kit (Ambion Applied Biosystems, Foster City, CA). Random primed cDNA was generated using reverse transcriptase (Promega, Madison, WI) and analyzed by quantita- tive PCR (qPCR) using SYBR Green reagents (Applied Biosys- tems) on an ABI 7500 qPCR platform as previously described.16 Primer sequences are available in Supplementary Table. SPG20/Spg20 Expression Analysis cDNA samples from human fetal and adult postmortem brain tissue were obtained commercially (BioChain Institute, Hayward, CA and Clontech, Mountain View, CA). cDNA from embryonic and postnatal mouse brains was generated as described above. qPCR was performed as described above (see Supplementary Ta- ble for primer sequences). DNA templates for in situ hybridiza- tion probes were cloned using a 550bp fragment of the Spg20 Manzini et al: Novel SPG20 Mutation April, 2010 517 coding sequence using the following primers: Spg20-BamHI 5�- ctacatggatccgatatcaaccggaggagcagccaaagtcagc-3� and Spg20-SalI 5�-gtccttgtcgactgcccctggcttctcttcctccacctg-3�. Digoxigenin-labeled riboprobes were synthesized and in situ hybridization was per- formed as previously described.17 Video images of the hybridized sections were obtained on a Wild Leitz (Heerbrugg, Switzerland) photomicroscope or Leica (Nussloch, Germany) DMR micro- scope under consistent illumination conditions. Results Identification of a Novel SPG20 Mutation in an Omani Kindred We examined 2 related Omani families presenting with short stature, dysarthria, motor and cognitive develop- mental delay, and increased muscle tone (families 1 and 2 in Fig 1A). The fathers (individuals 1-2 and 2-1) were brothers, and the mothers (1-1 and 2-2) were aunt and niece. Family 1 had 9 children: 4 were reportedly unaf- fected females, who were married and lived elsewhere, 1 was a male (1-4), who was unaffected by clinical exami- nation, 3 were affected males (1-3, 1-6, and 1-7), and 1 was an affected female (1-5). Family 2 had 8 children: 4 unaffected girls and 1 unaffected boy, 3 of whom were present at the time of our evaluation (2-4, 2-5 and 2-6), 1 affected male (2-3), 1 affected female (2-7). The young- est child (2-8) was small for her age and could neither talk nor walk, but did not display overt neurological symptoms; we were not able to obtain DNA from her for genotyping or sequencing. FIGURE 1: Affected individuals carry a homozygous null mutation in the SPG20 gene. (A) Pedigree of 2 related Omani families affected with short stature, spasticity, dysarthria, and developmental delay. Affected individuals are in black and unaffected in white. The status of individual 2-8 (in gray) could not be determined, because she is too young to properly assess neurological symptoms. All numbered individuals were examined, but genomic DNA was only collected from indi- viduals for whom microsatellite analysis is shown. Microsatellite analysis revealed common maternal (in yellow) and paternal (in blue) haplotypes in all affected. Microsatellite markers in the linkage region identified by the single nucleotide poly- morphism analysis are in bold. One homozygous marker (D13S219) is common to all affected individuals (highlighted in orange). (B) The homozygous region contains 6 genes, including SPG20 (in orange). (C) The affected individuals carried a homozygous 2bp deletion in SPG20, which was present in heterozygosity in the carriers. (D) Western blot analysis of patient cell lines showed that full-length SPG20 protein is missing in the affected individuals (A) compared with a nonaffected noncarrier sibling (NA). (E) Quantitative polymerase chain reaction analysis of cDNA from the patient cell lines indicated that SPG20 mRNA is not present in the affected individual. deltaRN � signal magnitude expressed by the difference in the normalized reporter (RN) values. ANNALS of Neurology 518 Volume 67, No. 4 Genomic DNA was obtained from most individuals in the pedigree and hybridized to Affymetrix Human SNP Array 6.0 chips for genome-wide genotyping. We suspected the presence of a homozygous ancestral mutation because the families resided in an isolated mountain village and large stretches of homozygous DNA identified in all indi- viduals suggested some degree of consanguinity; however, we could not ascertain any shared ancestry as far as 5 gen- erations removed from the probands by pedigree analysis. Thus, the SNP data was first analyzed following an auto- somal recessive model assuming no consanguinity. Linkage analysis identified 1 region of 7.0Mb flanked by SNPs rs11619306 and rs9576104 on chromosome 13q12.3 (log of odds � 3.6). Microsatellite analysis confirmed a shared maternal and paternal haplotype in all affected individuals (see Fig 1A). Individual 1-4, who was unaffected, was ho- mozygous for the maternal haplotype throughout most of the linkage region, excluding marker D13S219, which was homozygous on all affected individuals. At this locus, the SNP data indicated an 844kb region of homozygosity be- tween markers rs1418987 and rs9315443, shared only by affected but not unaffected individuals. Among the 6 genes in this region (see Fig 1B), SPG20 was the strongest can- didate gene, because individuals carrying an SPG20 muta- tion were affected with a remarkably similar phenotype, a complicated form of hereditary spastic paraplegia associated with short stature, dysarthria, and developmental delay, called Troyer syndrome.4,10 Sequencing of the SPG20 coding regions revealed a homozygous 2bp deletion (c.364_365delAT), which re- sulted in an amino acid substitution followed by a stop codon in the first coding exon (p.M122VfsX1; see Fig 1C). This mutation was confirmed in all affected individ- uals, and the inheritance pattern was confirmed in the rest of the families. This mutation was not identified in con- trol individuals, indicating that this is not a common polymorphism. Since the SPG20 mutation in the Amish (c.1110delA) was reported to be a null mutation,11 we generated lympho- blastoid cell lines from 2 affected (1-3 and 1-5) and 1 unaf- fected individual (2-5). We found that the full-length spartin protein was absent in the affected individuals (see Fig 1D). We also carried out qPCR for SPG20 on cDNA samples from 2 of the lymphoblastoid lines and could not detect any mRNA in the affected individual (see Fig 1E), indicating that this mutation is a null allele. Clinical Characterization of Patients Carrying a Novel SPG20 Mutation To ascertain phenotypic similarities or differences among individuals carrying SPG20 mutations, we compared the clinical presentations of the Omani individuals with the clinical description of 21 Amish patients by Proukakis et al5 (Table). All affected individuals in the Omani cohort presented with short stature and dysarthria, and were de- layed in reaching motor and cognitive developmental milestones. They all had some difficulties walking, with clumsy, mildly spastic gait, which their parents reported to be worsening over time. The most common physical features were relative hypertelorism and overgrowth of the maxilla leading to overbite, as well as hand and feet anomalies such as brachydactyly (5/6 patients), hammer toes, and pes cavus (Fig 2). Additional nonspecific skeletal malformations observed in the hands were clinodactyly, camptodactyly, and hypoplastic 5th middle phalanges (see Fig 2E, F). Most affected individuals had persistent cog- nitive deficits and poor performance in school, but no emotional lability was reported. Detailed neuropsycholog- ical testing was not available. The neurological examination revealed distal amyot- rophy (4/6 patients), dysmetria in the upper extremities (3/6 patients), hyperreflexia, which was more severe in the lower limbs (4/6 patients), and ankle clonus (3/6 pa- tients). Heel cords were tight in the 4 older affected in- dividuals. Brain magnetic resonance imaging (MRI) was performed in 2 individuals (1-7 at 14 years and 2-3 at 19 years). Both individuals had mild atrophy of the cerebellar vermis, mild white matter volume loss, and revealed periventricular white matter hyperintensity in T2- weighted images, consistent with gliosis (see Fig 2I, J). The mean age of our cohort (16.6 years) was younger than the Amish cohort, which might explain the milder and more variable phenotype observed in the Omani individuals; therefore, we subdivided the Amish cohort into a younger subset (Amish I) comparable in age to our cohort (�27 years; mean age, 15 years) and an older subset (Amish II) (�28 years; mean age, 43.5 years). Our cohort closely matched the description of the Amish I group, whereas the Amish II group was more severely affected, consistent with the progressive, degener- ative aspects of Troyer syndrome (see Table). Expression of Spg20 in the Developing and Adult Nervous System There appear to be 2 major components in Troyer syn- drome: an early developmental aspect and a neurodegen- erative process. The combination of phenotypes, includ- ing limb, craniofacial, and behavioral anomalies, is common in complex genetic disorders affecting early mor- phogenetic events,18 and the relevant genes are often spe- cifically expressed in the embryonic rudiments of the af- fected adult structures. We first analyzed SPG20 expression in the developing and adult human brain using qPCR. SPG20 was expressed at modest levels in the fetal Manzini et al: Novel SPG20 Mutation April, 2010 519 and adult human brain compared to a highly expressed neurally specific gene, synaptophysin (SYP; Fig 3A, B). Levels of SPG20 were highest in the amygdala, cortex, and thalamus, and lowest in the hippocampus and cere- bellum. Although clearly detectable, SPG20 was expressed at substantially lower levels than SYP. Expression levels and distribution of the mouse orthologue of SPG20, Spg20, in the adult mouse brain parallel those of the hu- man, with some divergence; particularly, relative expres- sion of Spg20 in the hippocampus was slightly elevated compared with humans. In addition, we analyzed Spg20 expression in mouse spinal cord and brainstem and found that it was highest in the spinal cord of all brain regions measured. As in the human, Spg20 was expressed at com- paratively lower levels than Syp. Spg20 is not a brain- specific gene, as it is also expressed in several other or- gans.10,19 It is, however, developmentally regulated, as expression was maximal at midgestation and embryonic day (E)10, and declined precipitously thereafter (see Fig 3C–F). We then used in situ hybridization to localize Spg20 in adult, fetal, and embryonic mouse brain (Fig 4). Spg20 was expressed at relatively low levels in neurons and glia throughout the adult brain, including glia in fiber tracts. Modestly elevated expression was seen throughout hip- pocampal stratum pyramidale of the CA fields and den- tate gyrus and the transitional parahippocampal/entorhi- nal cortex. Low expression was seen in the cerebral cortex with no apparent laminar or cell class specificity. The only additional site of elevated expression in the forebrain was the habenular complex, including the habenular re- cess appended to the corpus callosum. Spg20 is expressed throughout the cerebellum in Purkinje cells, granule cells, and scattered cells in the molecular layer. In the brain- TABLE: Clinical Features of the Omani Troyer Syndrome Individuals and Comparison with the Amish Cohort Characteristics Individuals Omani Amish I Amish II 1–3 1–5 2–3 1– 6 1–7 2–7 Age (yr) 27 19 19 17 14 4 Mean 16.6 Mean 15 Mean 43.5 Sex M F M M M F Height (cm) 149 133 145 146 NA 83 Short Short Short % �3rd �3rd �3rd �3rd �3rd OFC (cm) 52.4 51.6 52.8 54.8 53.0 48.4 Normal Normal Normal % 3rd–10th �3rd 10th 25th–50th 10th–25th 3rd–10th Developmental milestones and cognition Talked at 2 years 2 years 3 years Late Late Late Late (100%) Late (80%) Late (87%) Walked at 2 years 3 years 1.5 years 2.5 years 3 years 1.5 years Late (67%) Late (80%) Late (87%) Poor school performance � � � � � NA �(80%) �(100%) �(80%) Emotional lability � � � � � NA No �(70%) �(91%) Neurological exam Dysarthria � � � � � NA �(100%) �(100%) �(100%) Tongue dyspraxia � � � � � NA �(80%) Often Often Distal amyotrophy � � � � � � �(67%) �(70%) �(100%) Hyperreflexia Upper � � � � � � �(33%) �(30%) �(90%) Lower � � � � � � �(67%) �(100%) �(100%) Ankle clonus � � � � � � �(50%) �(30%) �(36%) Clumsy, spastic gait � � � � � � �(100%) �(100%) �(100%) Dysmetria � � � � � � �(50%) �(70%) �(100%) Skeletal abnormalities Overbite � � � � � ? �(100%) No No Hypertelorism � � � � � � �(83%) No No Hand/foot abnormalities � � � � � � �(100%) �(100%) �(100%) OFC � occipitofrontal circumference; NA � not available. ANNALS of Neurology 520 Volume 67, No. 4 stem, there is robust expression in large neurons, probably motor neurons, and in the facial nucleus. Spg20 is also expressed in cells distributed throughout the spinal cord with no noticeable discontinuities. As predicted from the qPCR data, expression in the fetal brain is relatively low, with no apparent regional distinctions. Aside from ele- vated expression in the lens placode and pigment epithe- lium, cochlear epithelium, and condensing mesenchyme at sites of myogenic or cartilage formation, there is little focal expression at fetal stages (see Fig 4L–N; E12.5 and E14.5 are shown). In contrast to the later stages, and, as predicted from the qPCR analysis, Spg20 expression in the midgestation embryo is elevated and highly patterned. In the E10.5 embryos, shortly after neural tube closure, Spg20 is spe- cifically expressed in the initial frontonasal mass/forebrain, craniofacial structures, aortic arch/heart primordium, and limb buds during morphogenesis, with lowest expression in the heart (Fig 4O, P). qPCR analysis confirms this dis- tribution; expression levels in limb buds, branchial arches, and frontonasal mass/forebrain are substantially elevated compared with the heart as well as with whole E10.5 em- bryo (Fig 4R). Our observations suggest that Spg20 has its most specific and maximal activity in the limbs, face and forebrain during early morphogenesis. This is remarkable, because the sites of phenotypic manifestations in the af- fected members of the Omani Troyer syndrome kindred described here include anomalies of the limb extremities, face, and brain. Discussion Our identification of a novel, disease-associated SPG20 mutation in an Omani kindred indicates for the first time that Troyer syndrome is not restricted to the Amish, as previously proposed.2 Complicated autosomal recessive HSPs are heterogeneous disorders often lacking clear clin- ical and molecular diagnostic guidelines. Of the 17 dis- tinct loci for complicated HSP identified to date, more FIGURE 2: Morphological and radiological features of Omani individuals with Troyer syndrome. Comparison between (A, C) an unaffected and (B, D) an affected individual revealed (A, B) relative hypertelorism and (C, D) pronounced overbite. Examples of skeletal anomalies in the extremities: brachydactyly (short digits; E, F), camptodactyly (flexed digit; asterisks in E, F), hammer toes (asterisks in G, H), and clinodactyly (curved digit; arrowheads in G, H). Brain magnetic resonance imaging shows atrophy of the cerebellar vermis (arrowhead in I) and white matter hyperintensity in T2-weighted images, which is more prominent posteriorly (arrowheads in J). Manzini et al: Novel SPG20 Mutation April, 2010 521 than half have been described in single families or isolated populations.1,2 In the past 4 decades, several HSP syn- dromes with Troyer-like features have been reported, but none matched the clinical presentation in the Amish cases.5 Some of these disorders were later mapped to dif- ferent loci such as SPG26,6,20 SPG39,21 and ARSACS.22,23 Even within the isolated region where these Omani families reside, we had described a similar extended pedigree with dysarthria, mental retardation, cerebral palsy, and micro- cephaly.24 Linkage analysis ruled out the SPG20 locus in the second kindred, confirming that the 2 disorders are separate clinical and genetic entities (A.R., R.S.H., C.A.W., unpublished data). Four individuals in this ex- tended family (N � 45 individuals) also were heterozy- gous carriers for the SPG20 mutation, highlighting its presence in this isolated population. Thus, this novel mu- tation in SPG20 is geographically and genetically distinct from that in the Amish population; nevertheless, both mutations result in the phenotypic spectrum associated with Troyer syndrome. FIGURE 3: Quantitative polymerase chain reaction analysis of SPG20/Spg20 expression in human and mouse tissues. (A) There is modest variation in SPG20 local expression in the adult human brain when distinct regions are compared to whole brain samples. (B) Human SPG20 expression is substantially lower than that of the brain-specific gene synaptophysin (SYP) across all brain regions with the exception of the amygdala. (C) A similar profile of regionally variable Spg20 expression, with some modest differences, is seen in the mouse brain. (D) Lower Spg20 expression is observed when compared with Syp. (E) Spg20 is expressed in several murine tissues beside the brain. (F) Spg20 is developmentally regulated in the mouse embryo with highest expression levels in the whole embryo at E10.5. �CT � ; Bas Gang � basal ganglia; DeltaCT � signal magnitude expressed by the difference in CT values. Hippoc � hippocampus; Mesen � mesenchyme; Cerebel � cerebellum; Sp Cord � spinal cord; Sk Muscle � skeletal muscle; Rel. � relative. ANNALS of Neurology 522 Volume 67, No. 4 Although a clear Troyer syndrome diagnosis is dif- ficult to reach in young subjects due to the variability and mildness of their symptoms, direct comparison of clinical features showed that the Omani cohort closely resembled the age-matched Amish Troyer syndrome group (Amish I), suggesting that SPG20 null mutations cause a well- defined phenotype. As in the Amish, we observed short stature, skeletal abnormalities in the extremities, develop- mental delay, and dysarthria of possible cerebellar origin from an early age. Therefore, a diagnosis of Troyer syn- drome must be considered when this constellation of phe- notypes is present in young children. Facial dysmorphism FIGURE 4: In situ analysis of Spg20 expression in mice. (A–J) Spg20 expression in the adult brain is modest and focal. (E) Cortical expression is moderate and not layer-specific. Relatively higher expression is observed in (B) the hippocampus, (C) glial cells in the corpus callosum, (D) entorhinal cortex, (F, G) habenular complex, (H) cerebellum, (I) facial nucleus (fN), and (J) spinal cord. (K) Fetal expression is also modest and distributed in the brain, with higher expression in (L) the lens placode (le), (M) the cochlear epithelium, and (N) condensing mesenchyme at sites of myogenic and cartilage formation (asterisks). (O) Early embryonic expression (embryonic day 10.5) is highly patterned and enhanced in the forebrain (fb), frontonasal mass (fnm), maxilla (mx), branchial arches (ba1, ba2), heart (h), and limb buds (flb). (P) The antisense control is completely unlabeled. (Q) Selective, focally elevated expression in the fnm/fb, flb, and ba1/ba2 is confirmed by qPCR after microdissection compared to the heart (h) and whole embryo (Wh). CA1 � ; CA2 � ; CA3 � ; CC � corpus callosum; m � molecular layer; P � Purkinje cells layer; g � granule cell layer; dh � dorsal horn; vh � ventral horn; Ctx � cortex; LGE � lateral ganglionic eminence; MGE � medial ganglionic eminence; r � retina. Manzini et al: Novel SPG20 Mutation April, 2010 523 and skeletal features are subtle, and in single cases in non- consanguineous populations this disorder could be ini- tially diagnosed simply as cerebral palsy. Spasticity and distal amyotrophy appeared in the teenage years and worsened slowly over time. In the Amish, brain MRI re- vealed white matter abnormalities, which were less severe in the youngest patient examined (age 15 years), suggest- ing a progressive worsening of the condition.5 MRI anal- ysis indicates that the Omani Troyer syndrome individu- als of comparable ages also have mild white matter abnormalities, supporting the hypothesis that white mat- ter degeneration accompanies disease progression, al- though direct neuronal degeneration cannot be ruled out. In addition, we identified mild cerebellar atrophy, which is consistent with the cerebellar signs. Serial MRI scans of the Omani individuals might resolve whether progressive neurological symptoms are correlated with increased white or gray matter degeneration. Despite many similarities to the Amish cohort, a few specific differences were observed in the affected Omani individuals, including the presence of hypertelorism and a pronounced overbite, and the ab- sence of inappropriate emotional responses. As new cases are identified it will be interesting to assess whether these traits have variable penetrance or are population specific. Functional studies on spartin, the protein encoded by SPG20, have identified multiple roles in protein ubiq- uitination25–27 and lipid droplet formation,19,25,26 and a possible link to endothelial growth factor receptor signal- ing.27 Previous expression analyses by Northern blot or qPCR identified widespread SPG20/spartin localization in the brain and in other tissues10,19; however, it was unclear whether regional and cellular dynamism in SPG20 expres- sion could explain the phenotypes observed in the affected individuals. Our study shows that whereas SPG20/Spg20 expression is modest and virtually ubiquitous in the adult and developing brain, early stages of embryonic develop- ment show maximal levels of Spg20 in the limbs, face, and forebrain primordia. This localized expression sug- gests a parallel role for SPG20/Spg20 in morphogenesis and differentiation at these phenotypic sites; accordingly, a loss of function mutation may contribute to the pheno- typic spectrum of Troyer syndrome. Acknowledgment This work was supported by the NINDS (grant RO1 NS 35129, C.A.W.), NICHD (grant RO1 HD029178, A.-S.L.), the Dubai Harvard Foundation for Medical Re- search (C.A.W.), the Manton Center for Orphan Disease Research (C.A.W.), the Muscular Dystrophy Association (Development Grant, M.C.M.), and the University of North Carolina (Reynolds Faculty Fellowship, A.-S.L.). C.A.W. is an Investigator of the Howard Hughes Medical Institute. We thank the families for their cooperation and their hospitality; the local nurses and doctors for their as- sistance; Megumi Aita at the University of North Caro- lina In Situ Core for help with the in situ analysis; Daniel Rakiec at Children’s Hospital, Boston for help with the sample preparation and analysis; and the following col- leagues at Children’s Hospital Boston for helpful discus- sion on the clinical features of the patients: Hope E. Dickinson, a speech pathologist from the Department of Otolaryngology and Communication Enhancement, and Janice Ware, a child psychologist and associate director of the Developmental Medicine Center. Authorship M. Chiara Manzini and Anna Rajab contributed equally and are co-first authors. Potential Conflicts of Interest Nothing to report. References 1. Harding AE. Classification of the hereditary ataxias and paraple- gias. Lancet 1983;1:1151–1155. 2. Depienne C, Stevanin G, Brice A, Durr A. Hereditary spastic paraplegias: an update. 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