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1.  MYBPC1 mutations impair skeletal muscle function in zebrafish models of arthrogryposis 
Human Molecular Genetics  2013;22(24):4967-4977.
Myosin-binding protein C1 (MYBPC1) is an abundant skeletal muscle protein that is expressed predominantly in slow-twitch muscle fibers. Human MYBPC1 mutations are associated with distal arthrogryposis type 1 and lethal congenital contracture syndrome type 4. As MYBPC1 function is incompletely understood, the mechanism by which human mutations result in contractures is unknown. Here, we demonstrate using antisense morpholino knockdown, that mybpc1 is required for embryonic motor activity and survival in a zebrafish model of arthrogryposis. Mybpc1 morphant embryos have severe body curvature, cardiac edema, impaired motor excitation and are delayed in hatching. Myofibril organization is selectively impaired in slow skeletal muscle and sarcomere numbers are greatly reduced in mybpc1 knockdown embryos, although electron microscopy reveals normal sarcomere structure. To evaluate the effects of human distal arthrogryposis mutations, mybpc1 mRNAs containing the corresponding human W236R and Y856H MYBPC1 mutations were injected into embryos. Dominant-negative effects of these mutations were suggested by the resultant mild bent body curvature, decreased motor activity, as well as impaired overall survival compared with overexpression of wild-type RNA. These results demonstrate a critical role for mybpc1 in slow skeletal muscle development and establish zebrafish as a tractable model of human distal arthrogryposis.
PMCID: PMC3836476  PMID: 23873045
2.  Exome Sequencing Identifies a Rare HSPG2 Variant Associated with Familial Idiopathic Scoliosis 
G3: Genes|Genomes|Genetics  2014;5(2):167-174.
Idiopathic scoliosis occurs in 3% of individuals and has an unknown etiology. The objective of this study was to identify rare variants that contribute to the etiology of idiopathic scoliosis by using exome sequencing in a multigenerational family with idiopathic scoliosis. Exome sequencing was completed for three members of this multigenerational family with idiopathic scoliosis, resulting in the identification of a variant in the HSPG2 gene as a potential contributor to the phenotype. The HSPG2 gene was sequenced in a separate cohort of 100 unrelated individuals affected with idiopathic scoliosis and also was examined in an independent idiopathic scoliosis population. The exome sequencing and subsequent bioinformatics filtering resulted in 16 potentially damaging and rare coding variants. One of these variants, p.Asn786Ser, is located in the HSPG2 gene. The variant p.Asn786Ser also is overrepresented in a larger cohort of idiopathic scoliosis cases compared with a control population (P = 0.024). Furthermore, we identified additional rare HSPG2 variants that are predicted to be damaging in two independent cohorts of individuals with idiopathic scoliosis. The HSPG2 gene encodes for a ubiquitous multifunctional protein within the extracellular matrix in which loss of function mutation are known to result in a musculoskeletal phenotype in both mouse and humans. Based on these results, we conclude that rare variants in the HSPG2 gene potentially contribute to the idiopathic scoliosis phenotype in a subset of patients with idiopathic scoliosis. Further studies must be completed to confirm the effect of the HSPG2 gene on the idiopathic scoliosis phenotype.
PMCID: PMC4321025  PMID: 25504735
HSPG2; perlecan; idiopathic scoliosis; exome sequencing
3.  Copy number analysis of 413 isolated talipes equinovarus patients suggests role for transcriptional regulators of early limb development 
Talipes equinovarus is one of the most common congenital musculoskeletal anomalies and has a worldwide incidence of 1 in 1000 births. A genetic predisposition to talipes equinovarus is evidenced by the high concordance rate in twin studies and the increased risk to first-degree relatives. Despite the frequency of isolated talipes equinovarus and the strong evidence of a genetic basis for the disorder, few causative genes have been identified. To identify rare and/or recurrent copy number variants, we performed a genome-wide screen for deletions and duplications in 413 isolated talipes equinovarus patients using the Affymetrix 6.0 array. Segregation analysis within families and gene expression in mouse E12.5 limb buds were used to determine the significance of copy number variants. We identified 74 rare, gene-containing copy number variants that were present in talipes equinovarus probands and not present in 759 controls or in the Database of Genomic Variants. The overall frequency of copy number variants was similar between talipes equinovarus patients compared with controls. Twelve rare copy number variants segregate with talipes equinovarus in multiplex pedigrees, and contain the developmentally expressed transcription factors and transcriptional regulators PITX1, TBX4, HOXC13, UTX, CHD (chromodomain protein)1, and RIPPLY2. Although our results do not support a major role for recurrent copy number variations in the etiology of isolated talipes equinovarus, they do suggest a role for genes involved in early embryonic patterning in some families that can now be tested with large-scale sequencing methods.
PMCID: PMC3598331  PMID: 22892537
talipes equinovarus; microduplication; microdeletion; transcription
4.  Multiplexed direct genomic selection (MDiGS): a pooled BAC capture approach for highly accurate CNV and SNP/INDEL detection 
Nucleic Acids Research  2014;42(10):e82.
Despite declining sequencing costs, few methods are available for cost-effective single-nucleotide polymorphism (SNP), insertion/deletion (INDEL) and copy number variation (CNV) discovery in a single assay. Commercially available methods require a high investment to a specific region and are only cost-effective for large samples. Here, we introduce a novel, flexible approach for multiplexed targeted sequencing and CNV analysis of large genomic regions called multiplexed direct genomic selection (MDiGS). MDiGS combines biotinylated bacterial artificial chromosome (BAC) capture and multiplexed pooled capture for SNP/INDEL and CNV detection of 96 multiplexed samples on a single MiSeq run. MDiGS is advantageous over other methods for CNV detection because pooled sample capture and hybridization to large contiguous BAC baits reduces sample and probe hybridization variability inherent in other methods. We performed MDiGS capture for three chromosomal regions consisting of ∼550 kb of coding and non-coding sequence with DNA from 253 patients with congenital lower limb disorders. PITX1 nonsense and HOXC11 S191F missense mutations were identified that segregate in clubfoot families. Using a novel pooled-capture reference strategy, we identified recurrent chromosome chr17q23.1q23.2 duplications and small HOXC 5′ cluster deletions (51 kb and 12 kb). Given the current interest in coding and non-coding variants in human disease, MDiGS fulfills a niche for comprehensive and low-cost evaluation of CNVs, coding, and non-coding variants across candidate regions of interest.
PMCID: PMC4041413  PMID: 24682816
5.  Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene 
Human Molecular Genetics  2012;22(6):1097-1111.
Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development.
PMCID: PMC3578410  PMID: 23223018
6.  Genetics of Clubfoot 
Modern advances in genetics have allowed investigators to begin to identify the complex etiology of clubfoot. It has become increasingly apparent that clubfoot is a heterogeneous disorder with a polygenetic threshold model explaining its inheritance patterns. Several recent genetics studies have identified a key developmental pathway, the PITX1-TBX4 transriptional pathway, as being important in clubfoot etiology. Both PITX1 and TBX4 are uniquely expressed in the hindlimb which helps explain the foot phenotype seen with mutations in these transcription factors. Future studies are needed to develop animal models to determine the exact mechanisms by which these genetic abnormalities cause clubfoot and to test other hypotheses of clubfoot pathogenesis.
PMCID: PMC3229717  PMID: 21817922
7.  Copy number variants and infantile spasms: evidence for abnormalities in ventral forebrain development and pathways of synaptic function 
European Journal of Human Genetics  2011;19(12):1238-1245.
Infantile spasms (ISS) are an epilepsy disorder frequently associated with severe developmental outcome and have diverse genetic etiologies. We ascertained 11 subjects with ISS and novel copy number variants (CNVs) and combined these with a new cohort with deletion 1p36 and ISS, and additional published patients with ISS and other chromosomal abnormalities. Using bioinformatics tools, we analyzed the gene content of these CNVs for enrichment in pathways of pathogenesis. Several important findings emerged. First, the gene content was enriched for the gene regulatory network involved in ventral forebrain development. Second, genes in pathways of synaptic function were overrepresented, significantly those involved in synaptic vesicle transport. Evidence also suggested roles for GABAergic synapses and the postsynaptic density. Third, we confirm the association of ISS with duplication of 14q12 and maternally inherited duplication of 15q11q13, and report the association with duplication of 21q21. We also present a patient with ISS and deletion 7q11.3 not involving MAGI2. Finally, we provide evidence that ISS in deletion 1p36 may be associated with deletion of KLHL17 and expand the epilepsy phenotype in that syndrome to include early infantile epileptic encephalopathy. Several of the identified pathways share functional links, and abnormalities of forebrain synaptic growth and function may form a common biologic mechanism underlying both ISS and autism. This study demonstrates a novel approach to the study of gene content in subjects with ISS and copy number variation, and contributes further evidence to support specific pathways of pathogenesis.
PMCID: PMC3230360  PMID: 21694734
infantile spasms; autism; bioinformatics; copy number variation; deletion 1p36 syndrome
8.  Pitx1 haploinsufficiency causes clubfoot in humans and a clubfoot-like phenotype in mice 
Human Molecular Genetics  2011;20(20):3943-3952.
Clubfoot affects 1 in 1000 live births, although little is known about its genetic or developmental basis. We recently identified a missense mutation in the PITX1 bicoid homeodomain transcription factor in a family with a spectrum of lower extremity abnormalities, including clubfoot. Because the E130K mutation reduced PITX1 activity, we hypothesized that PITX1 haploinsufficiency could also cause clubfoot. Using copy number analysis, we identified a 241 kb chromosome 5q31 microdeletion involving PITX1 in a patient with isolated familial clubfoot. The PITX1 deletion segregated with autosomal dominant clubfoot over three generations. To study the role of PITX1 haploinsufficiency in clubfoot pathogenesis, we began to breed Pitx1 knockout mice. Although Pitx1+/− mice were previously reported to be normal, clubfoot was observed in 20 of 225 Pitx1+/− mice, resulting in an 8.9% penetrance. Clubfoot was unilateral in 16 of the 20 affected Pitx1+/− mice, with the right and left limbs equally affected, in contrast to right-sided predominant hindlimb abnormalities previously noted with complete loss of Pitx1. Peroneal artery hypoplasia occurred in the clubfoot limb and corresponded spatially with small lateral muscle compartments. Tibial and fibular bone volumes were also reduced. Skeletal muscle gene expression was significantly reduced in Pitx1−/− E12.5 hindlimb buds compared with the wild-type, suggesting that muscle hypoplasia was due to abnormal early muscle development and not disuse atrophy. Our morphological data suggest that PITX1 haploinsufficiency may cause a developmental field defect preferentially affecting the lateral lower leg, a theory that accounts for similar findings in human clubfoot.
PMCID: PMC3177645  PMID: 21775501
9.  Variants in genes that encode muscle contractile proteins influence risk for isolated clubfoot 
Isolated clubfoot is a relatively common birth defect that affects approximately 4,000 newborns in the US each year. Calf muscles in the affected leg(s) are underdeveloped and remain small even after corrective treatment. This observation suggests that variants in genes that influence muscle development are priority candidate risk factors for clubfoot. This contention is further supported by the discovery that mutations in genes that encode components of the muscle contractile complex (MYH3, TPM2, TNNT3, TNNI2, and MYH8) cause congenital contractures, including clubfoot, in distal arthrogryposis (DA) syndromes. Interrogation of fifteen genes encoding proteins that control myofiber contractility in a cohort of both nonHispanic white (NHW) and Hispanic families, identified positive associations (p<0.05) with SNPs in twelve genes; only one was identified in a family-based validation dataset. Six SNPs in TNNC2 deviated from Hardy Weinberg Equilibrium (HWE) in mothers in our NHW discovery dataset. Relative risk and likelihood ratio tests showed evidence for a maternal genotypic effect with TNNC2/rs383112 and an inherited/child genotypic effect with two SNPs, TNNC2/rs4629 and rs383112. Associations with multiple SNPs in TPM1 were identified in the NHW discovery (rs4075583, p=0.01), family-based validation (rs1972041, p=0.000074) and case-control validation (rs12148828, p=0.04) datasets. Gene interactions were identified between multiple muscle contraction genes with many of the interactions involving at least one potential regulatory SNP. Collectively, our results suggest that variation in genes that encode contractile proteins of skeletal myofibers may play a role in the etiology of clubfoot.
PMCID: PMC3158831  PMID: 21834041
clubfoot; genetics; muscle; contraction; distal arthrogryposis; association study
10.  Vascular Abnormalities Correlate with Decreased Soft Tissue Volumes in Idiopathic Clubfoot 
Lower extremity vascular anomalies have been described for patients with clubfoot but few imaging studies have investigated effects on soft tissues such as fat and muscle. To make these assessments we need noninvasive, noncontrast agents to more safely image children.
We describe a novel noninvasive imaging protocol to identify vascular and soft tissue abnormalities in the lower limbs of patients with clubfoot and determine whether these abnormalities are present in patients who had recurrent clubfoot.
Patients and Methods
Three-dimensional noncontrast-enhanced MR angiography was used to identify vascular, bone, and soft tissue abnormalities in patients with clubfoot. We determined whether these abnormalities were more common in patients who had experienced recurrent clubfoot.
Four patients with isolated unilateral clubfoot had arterial anomalies in the clubfoot limb. All patients had less muscle volume in the affected limb, and nine of 11 patients (82%) had less subcutaneous fat, with a mean difference of 0.56 cm3 ± 0.36 cm3 (range, 0.08–1.12 cm3). Vascular anomalies and decreased fat and muscle volumes were present in all three patients with recurrent clubfoot.
We found a high frequency of vascular and soft tissue anomalies in the affected limbs of patients with unilateral clubfoot that may correlate with response to treatment.
Clinical Relevance
This approach has the potential to enhance our understanding of the anatomy of clubfoot and lead to a larger MRI study that may allow more accurate prediction of the risk of recurrent clubfoot.
PMCID: PMC3069258  PMID: 21042891
11.  Mutations in the novel protein PRRT2 cause paroxysmal kinesigenic dyskinesia with infantile convulsions 
Cell reports  2011;1(1):2-12.
Paroxysmal Kinesigenic Dyskinesia with Infantile Convulsions (PKD/IC) is an episodic movement disorder with autosomal dominant inheritance and high penetrance, but the causative gene is unknown. We have now identified four truncating mutations involving the PRRT2 gene in the vast majority (24/25) of well characterized families with PKD/IC. PRRT2 truncating mutations were also detected in 28 of 78 additional families. The PRRT2 gene encodes a proline-rich transmembrane protein of unknown function that has been reported to interact with the t-SNARE, SNAP25. PRRT2 localizes to axons but not to dendritic processes in primary neuronal culture and mutants associated with PKD/IC lead to dramatically reduced PRRT2 protein levels leading ultimately to neuronal hyperexcitability that manifests in vivo as PKD/IC.
PMCID: PMC3334308  PMID: 22832103
12.  Skeletal Muscle Abnormalities and Genetic Factors Related to Vertical Talus 
Congenital vertical talus is a fixed dorsal dislocation of the talonavicular joint and fixed equinus contracture of the hindfoot, causing a rigid deformity recognizable at birth. The etiology and epidemiology of this condition are largely unknown, but some evidence suggests it relates to aberrations of skeletal muscle. Identifying the tissue abnormalities and genetic causes responsible for vertical talus has the potential to lead to improved treatment and preventive strategies.
We therefore (1) determined whether skeletal muscle abnormalities are present in patients with vertical talus and (2) identified associated congenital anomalies and genetic abnormalities in these patients.
We identified associated congenital anomalies and genetic abnormalities present in 61 patients affected with vertical talus. We obtained abductor hallucis muscle biopsy specimens from the affected limbs of 11 of the 61 patients and compared the histopathologic characteristics with those of age-matched control subjects.
All muscle biopsy specimens (n = 11) had abnormalities compared with those from control subjects including combinations of abnormal variation in muscle fiber size (n = 7), type I muscle fiber smallness (n = 6), and abnormal fiber type predominance (n = 5). Isolated vertical talus occurred in 23 of the 61 patients (38%), whereas the remaining 38 patients had associated nervous system, musculoskeletal system, and/or genetic and genomic abnormalities. Ten of the 61 patients (16%) had vertical talus in one foot and clubfoot in the other. Chromosomal abnormalities, all complete or partial trisomies, were identified in three patients with vertical talus who had additional congenital abnormalities.
Vertical talus is a heterogeneous birth defect resulting from many diverse etiologies. Abnormal skeletal muscle biopsies are common in patients with vertical talus although it is unclear whether this is primary or secondary to the joint deformity. Associated anomalies are present in 62% of all cases.
PMCID: PMC3048242  PMID: 20645034
13.  Genome-wide association studies of adolescent idiopathic scoliosis suggest candidate susceptibility genes 
Human Molecular Genetics  2011;20(7):1456-1466.
Adolescent idiopathic scoliosis (AIS) is an unexplained and common spinal deformity seen in otherwise healthy children. Its pathophysiology is poorly understood despite intensive investigation. Although genetic underpinnings are clear, replicated susceptibility loci that could provide insight into etiology have not been forthcoming. To address these issues, we performed genome-wide association studies (GWAS) of ∼327 000 single nucleotide polymorphisms (SNPs) in 419 AIS families. We found strongest evidence of association with chromosome 3p26.3 SNPs in the proximity of the CHL1 gene (P < 8 × 10−8 for rs1400180). We genotyped additional chromosome 3p26.3 SNPs and tested replication in two follow-up case–control cohorts, obtaining strongest results when all three cohorts were combined (rs10510181 odds ratio = 1.49, 95% confidence interval = 1.29–1.73, P = 2.58 × 10−8), but these were not confirmed in a separate GWAS. CHL1 is of interest, as it encodes an axon guidance protein related to Robo3. Mutations in the Robo3 protein cause horizontal gaze palsy with progressive scoliosis (HGPPS), a rare disease marked by severe scoliosis. Other top associations in our GWAS were with SNPs in the DSCAM gene encoding an axon guidance protein in the same structural class with Chl1 and Robo3. We additionally found AIS associations with loci in CNTNAP2, supporting a previous study linking this gene with AIS. Cntnap2 is also of functional interest, as it interacts directly with L1 and Robo class proteins and participates in axon pathfinding. Our results suggest the relevance of axon guidance pathways in AIS susceptibility, although these findings require further study, particularly given the apparent genetic heterogeneity in this disease.
PMCID: PMC3049353  PMID: 21216876
14.  Axial distribution of myosin binding protein-C is unaffected by mutations in human cardiac and skeletal muscle 
Myosin binding protein-C (MyBP-C), a major thick filament associated sarcomeric protein, plays an important functional and structural role in regulating sarcomere assembly and crossbridge formation. Missing or aberrant MyBP-C proteins (both cardiac and skeletal) have been shown to cause both cardiac and skeletal myopathies, thereby emphasising its importance for the normal functioning of the sarcomere. Mutations in cardiac MyBP-C are a major cause of hypertrophic cardiomyopathy (HCM), while mutations in skeletal MyBP-C have been implicated in a disease of skeletal muscle—distal arthrogryposis type 1 (DA-1). Here we report the first detailed electron microscopy studies on human cardiac and skeletal tissues carrying MyBP-C gene mutations, using samples obtained from HCM and DA-1 patients. We have used established image averaging methods to identify and study the axial distribution of MyBP-C on the thick filament by averaging profile plots of the A-band of the sarcomere from electron micrographs of human cardiac and skeletal myopathy specimens. Due to the difficulty of obtaining normal human tissue, we compared the distribution to the A-band structure in normal frog skeletal, rat cardiac muscle and in cardiac muscle of MyBP-C-deficient mice. Very similar overall profile averages were obtained from the C-zones in cardiac HCM samples and skeletal DA-1 samples with MyBP-C gene mutations, suggesting that mutations in MyBP-C do not alter its mean axial distribution along the thick filament.
PMCID: PMC3351610  PMID: 22415774
Hypertrophic cardiomyopathy; Distal arthrogryposis type 1; C-protein; MyBP-C; Myosin binding protein-C mutations; Thick filament
15.  Evaluation of GPR50, hMel-1B, and ROR-Alpha Melatonin-Related Receptors and the Etiology of Adolescent Idiopathic Scoliosis 
Journal of pediatric orthopedics  2010;30(6):539-543.
Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity in children. Studies have shown low melatonin levels resulting from pinealectomy in chickens and mice result in the development scoliosis, while supplementation with melatonin after the pinealectomy prevented it. The mere characterization of low melatonin levels is not sufficient to explain the development of idiopathic scoliosis in primates and humans, but we hypothesize that a mutation in melatonin-related receptors may be involved with the development of scoliosis.
The coding, splice-site, and promoter regions of three melatonin-related receptors (hMel-1B, RORα, and GPR50) were evaluated by DNA sequencing for variants associated with the phenotype of adolescent idiopathic scoliosis. An initial screening of 50 scoliosis patients with adolescent idiopathic scoliosis was compared with 50 controls by DNA sequencing of the three receptors. Additional cases and controls were evaluated when genetic variants were observed (for a total of 885 individuals).
No significant differences were found in the hMel-1B and RORα receptors. We found two cSNPs in GPR50 (rs561077 and rs13440581) in the initial 50 patients. To evaluate the significance of these cSNPs, an additional 356 patients and 429 controls were analyzed. When the combined groups were analyzed, no significant associations were observed.
Despite the observed relationship between melatonin and scoliosis, there is no significant association between mutations found in any known melatonin-related receptors with adolescent idiopathic scoliosis. The strong evidence of a melatonin-related cause for the development of idiopathic scoliosis still encourages research into undiscovered melatonin-related receptors, melatonin-related hormones, and the catalytic enzymes for the serotonin-melatonin pathway.
Clinical Relevance
This investigation is a genetic testing of the remaining currently known melatonin-related receptors that have not previously been analyzed for association with AIS. Given the support in the literature of a relationship between melatonin and AIS, we have shown no mutations in any of the known melatonin-related receptor in patients with AIS.
PMCID: PMC2928583  PMID: 20733416
adolescent idiopathic scoliosis; melatonin; genetics
16.  Common polymorphisms in human lysyl oxidase genes are not associated with the adolescent idiopathic scoliosis phenotype 
BMC Medical Genetics  2011;12:92.
Although adolescent idiopathic scoliosis affects approximately 3% of adolescents, the genetic contributions have proven difficult to identify. Work in model organisms, including zebrafish, chickens, and mice, has implicated the lysyl oxidase family of enzymes in the development of scoliosis. We hypothesized that common polymorphisms in the five human lysyl oxidase genes (LOX, LOXL1, LOXL2, LOXL3, and LOXL4) may be associated with the phenotype of adolescent idiopathic scoliosis.
This was a case-control genetic association study. A total of 112 coding and tag SNPs in LOX, LOXL1, LOXL2, LOXL3, and LOXL4 were genotyped in a discovery cohort of 138 cases and 411 controls. Genotypes were tested for association with adolescent idiopathic scoliosis by logistic regression with a two degree of freedom genotypic model and gender as a covariate. Fourteen SNPs with p < 0.1 in the discovery phase were genotyped in an independent replication cohort of 400 cases and 506 controls.
No evidence for significant association was found between coding or tag SNPs in LOX, LOXL1, LOXL2, LOXL3, and LOXL4 and the phenotype of adolescent idiopathic scoliosis.
Despite suggestive evidence in model organisms, common variants and known coding SNPs in the five human lysyl oxidase genes do not confer increased genotypic risk for adolescent idiopathic scoliosis. The above methodology does not address rare variants or individually private mutations in these genes, and future research may focus on this area.
PMCID: PMC3154146  PMID: 21740577
18.  Myosin binding protein C1: a novel gene for autosomal dominant distal arthrogryposis type 1 
Human Molecular Genetics  2010;19(7):1165-1173.
Distal arthrogryposis type I (DA1) is a disorder characterized by congenital contractures of the hands and feet for which few genes have been identified. Here we describe a five-generation family with DA1 segregating as an autosomal dominant disorder with complete penetrance. Genome-wide linkage analysis using Affymetrix GeneChip Mapping 10K data from 12 affected members of this family revealed a multipoint LODmax of 3.27 on chromosome 12q. Sequencing of the slow-twitch skeletal muscle myosin binding protein C1 (MYBPC1), located within the linkage interval, revealed a missense mutation (c.706T>C) that segregated with disease in this family and causes a W236R amino acid substitution. A second MYBPC1 missense mutation was identified (c.2566T>C)(Y856H) in another family with DA1, accounting for an MYBPC1 mutation frequency of 13% (two of 15). Skeletal muscle biopsies from affected patients showed type I (slow-twitch) fibers were smaller than type II fibers. Expression of a green fluorescent protein (GFP)-tagged MYBPC1 construct containing WT and DA1 mutations in mouse skeletal muscle revealed robust sarcomeric localization. In contrast, a more diffuse localization was seen when non-fused GFP and MYBPC1 proteins containing corresponding MYBPC3 amino acid substitutions (R326Q, E334K) that cause hypertrophic cardiomyopathy were expressed. These findings reveal that the MYBPC1 is a novel gene responsible for DA1, though the mechanism of disease may differ from how some cardiac MYBPC3 mutations cause hypertrophic cardiomyopathy.
PMCID: PMC2838534  PMID: 20045868
19.  Altered Transmission of HOX and Apoptotic SNPs Identify a Potential Common Pathway for Clubfoot 
Clubfoot is a common birth defect that affects 135,000 newborns each year worldwide. It is characterized by equinus deformity of one or both feet and hypoplastic calf muscles. Despite numerous study approaches, the cause(s) remains poorly understood although a multifactorial etiology is generally accepted. We considered the HOXA and HOXD gene clusters and insulin-like growth factor binding protein 3 (IGFBP3) as candidate genes because of their important roles in limb and muscle morphogenesis. Twenty SNPs from the HOXA and HOXD gene clusters and 12 SNPs in IGFBP3 were genotyped in a sample composed of nonHispanic white and Hispanic multiplex and simplex families (discovery samples) and a second sample of nonHispanic white simplex trios (validation sample). Four SNPs (rs6668, rs2428431, rs3801776 and rs3779456) in the HOXA cluster demonstrated altered transmission in the discovery sample, but only rs3801776, located in the HOXA basal promoter region, showed altered transmission in both the discovery and validation samples (p=0.004 and p=0.028). Interestingly, HOXA9 is expressed in muscle during development. A SNP in IGFBP3, rs13223993, also showed altered transmission (p=0.003) in the discovery sample. Gene-gene interactions were identified between variants in HOXA, HOXD and IGFBP3 and with previously associated SNPs in mitochondrial-mediated apoptotic genes. The most significant interactions were found between CASP3 SNPS and variants in HOXA, HOXD and IGFBP3. These results suggest a biologic model for clubfoot in which perturbation of HOX and apoptotic genes together affect muscle and limb development, which may cause the downstream failure of limb rotation into a plantar grade position.
PMCID: PMC2795347  PMID: 19938081
association study; genotyping; complex disease; clubfoot; HOXA; IGFBP3
20.  Genome-Wide Copy Number Variation in Epilepsy: Novel Susceptibility Loci in Idiopathic Generalized and Focal Epilepsies 
PLoS Genetics  2010;6(5):e1000962.
Epilepsy is one of the most common neurological disorders in humans with a prevalence of 1% and a lifetime incidence of 3%. Several genes have been identified in rare autosomal dominant and severe sporadic forms of epilepsy, but the genetic cause is unknown in the vast majority of cases. Copy number variants (CNVs) are known to play an important role in the genetic etiology of many neurodevelopmental disorders, including intellectual disability (ID), autism, and schizophrenia. Genome-wide studies of copy number variation in epilepsy have not been performed. We have applied whole-genome oligonucleotide array comparative genomic hybridization to a cohort of 517 individuals with various idiopathic, non-lesional epilepsies. We detected one or more rare genic CNVs in 8.9% of affected individuals that are not present in 2,493 controls; five individuals had two rare CNVs. We identified CNVs in genes previously implicated in other neurodevelopmental disorders, including two deletions in AUTS2 and one deletion in CNTNAP2. Therefore, our findings indicate that rare CNVs are likely to contribute to a broad range of generalized and focal epilepsies. In addition, we find that 2.9% of patients carry deletions at 15q11.2, 15q13.3, or 16p13.11, genomic hotspots previously associated with ID, autism, or schizophrenia. In summary, our findings suggest common etiological factors for seemingly diverse diseases such as ID, autism, schizophrenia, and epilepsy.
Author Summary
Epilepsy, a common neurological disorder characterized by recurrent seizures, affects up to 3% of the population. In some cases, the epilepsy has a clear cause such as an abnormality in the brain or a head injury. However, in many cases there is no obvious cause. Numerous studies have shown that genetic factors are important in these types of epilepsy, but although several epilepsy genes are known, we can still only identify the genetic cause in a very small fraction of cases. In order to identify new genes that contribute to the genetic causes of epilepsy, we searched the human genome for deletions (missing copies) and duplications (extra copies) of genes in ∼500 patients with epilepsy that are not found in control individuals. Using this approach, we identified several large deletions that are important in at least 3% of epilepsy cases. Furthermore, we found new candidate genes, some of which are also thought to play a role in other related disorders such as autism and intellectual disability. These genes are candidates for further studies in patients with epilepsy.
PMCID: PMC2873910  PMID: 20502679
21.  Magnetic Resonance Angiography in Clubfoot and Vertical Talus: A Feasibility Study 
Congenital vascular alterations of the normal adult arterial pattern have been associated with multiple congenital limb deformities including clubfoot and vertical talus. Investigators have observed absence of the anterior tibial artery and dorsalis pedis artery in most patients with clubfoot, and absence of the posterior tibial artery in all patients with vertical talus. We used magnetic resonance angiography to define the lower extremity vascular anatomy of two patients with left-sided vertical talus and right-sided clubfoot and one patient with bilateral vertical talus and cartilage-derived morphogenetic protein-1 (CDMP-1) gene mutation. Of the three patients, one had bilateral posterior tibial artery deficiencies while the other had bilateral anterior tibial artery deficiencies. The third patient with bilateral vertical talus and CDMP-1 mutation had normal arterial structure bilaterally. Though clubfoot and vertical talus have distinctly different clinical phenotypes, the association of each with arterial abnormalities suggests a common etiology during development. The presence of normal arterial structure in our patient with vertical talus and CDMP-1 mutation suggests that other nonvascular etiologies may be responsible for some cases of foot deformities.
Level of Evidence: Level IV, prognostic case series. See the Guidelines for Authors for a complete description of levels of evidence.
PMCID: PMC2664419  PMID: 19127394
22.  Skeletal Muscle Contractile Gene (TNNT3, MYH3, TPM2) Mutations Not Found in Vertical Talus or Clubfoot 
Arthrogryposis presents with lower limb contractures that resemble clubfoot and/or vertical talus. Recently, mutations in skeletal muscle contractile genes MYH3 (myosin heavy chain 3), TNNT3 (troponin T3), and TPM2 (tropomyosin 2) were identified in patients with distal arthrogryposis DA2A (Freeman-Sheldon syndrome) or DA2B (Sheldon-Hall syndrome). We asked whether the contractile genes responsible for distal arthrogryposis are also responsible for cases of familial clubfoot or vertical talus. We determined the frequency of MYH3, TNNT3, and TPM2 mutations in patients with idiopathic clubfoot, vertical talus, and distal arthrogryposis type 1 (DA1). We resequenced the coding exons of the MYH3, TNNT3, and TPM2 genes in 31 patients (five with familial vertical talus, 20 with familial clubfoot, and six with DA1). Variants were evaluated for segregation with disease in additional family members, and the frequency of identified variants was determined in a control population. In one individual with DA1, we identified a de novo TNNT3 mutation (R63H) previously identified in an individual with DA2B. No other causative mutations were identified, though we found several previously undescribed single-nucleotide polymorphisms of unknown importance. Although mutations in MYH3, TNNT3, and TPM2 are frequently associated with distal arthrogryposis syndromes, they were not present in patients with familial vertical talus or clubfoot. The TNNT3 R63H recurrent mutation identified in two unrelated individuals may be associated with either DA1 or DA2B.
Level of Evidence: Level II, prospective study. See the Guidelines for Authors for a complete description of levels of evidence.
PMCID: PMC2664426  PMID: 19142688
23.  Update on Clubfoot: Etiology and Treatment 
Although clubfoot is one of the most common congenital abnormalities affecting the lower limb, it remains a challenge not only to understand its genetic origins but also to provide effective long-term treatment. This review provides an update on the etiology of clubfoot as well as current treatment strategies. Understanding the exact genetic etiology of clubfoot may eventually be helpful in determining both prognosis and the selection of appropriate treatment methods in individual patients. The primary treatment goal is to provide long-term correction with a foot that is fully functional and pain-free. To achieve this, a combination of approaches that applies the strengths of several methods (Ponseti method and French method) may be needed. Avoidance of extensive soft-tissue release operations in the primary treatment should be a priority, and the use of surgery for clubfoot correction should be limited to an “a la carte” mode and only after failed conservative methods.
Level of Evidence: Level V, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
PMCID: PMC2664438  PMID: 19224303
24.  Genetic Linkage Localizes an Adolescent Idiopathic Scoliosis and Pectus Excavatum Gene to the 18q Chromosome 
Spine  2009;34(2):E94-100.
Study Design
A single large family, in which adolescent idiopathic scoliosis (AIS) and pectus excavatum (PE) segregate as an autosomal dominant condition, was evaluated. Genome-wide linkage analysis and candidate gene sequencing were performed.
To map the disease-causing locus in a large Caucasian family in which AIS and PE co-segregate.
Summary of Background Data
AIS and PE are common musculoskeletal conditions known to have a genetic component, though few genes have been identified for either. Genetic studies have been confounded by a lack of large families in which the disorders segregate.
Clinical examinations were performed on the proband, who underwent posterior spinal fusion, and twelve additional affected family members. To map a gene causing AIS and PE, a genome-wide linkage analysis was performed with the Affymetrix Mapping 10K XbaI array on thirteen affected and ten unaffected family members. Candidate genes were sequenced.
AIS was present in thirteen female family members and PE was present in three males and one female. Genome-wide linkage analysis resulted in a linkage peak on chromosome 18q with a maximum parametric multipoint logarithm of the odds (LOD) score of 3.86. Recombinants delineated the critical genetic region to an interval of 6.4 cM between SNP_A-1519369 and SNP_A-1507702, corresponding to a 7.06 Mb region (hg18: chr18:26342508-34395660). The chromosome 18q linkage region contains more than 30 genes. Re-sequencing of the coding regions of 21 candidate genes in the region did not reveal any causative mutation.
Linkage analysis in this large family demonstrated a novel locus for AIS and PE on chromosome 18q. Because of the increased frequency of PE in family members of AIS patients, consideration of family members with PE as affected may increase the power of AIS genetic linkage studies.
PMCID: PMC2638207  PMID: 19139660
idiopathic scoliosis; pectus excavatum; genetics; linkage analysis
25.  A Role of SCN9A in Human Epilepsies, As a Cause of Febrile Seizures and As a Potential Modifier of Dravet Syndrome 
PLoS Genetics  2009;5(9):e1000649.
A follow-up study of a large Utah family with significant linkage to chromosome 2q24 led us to identify a new febrile seizure (FS) gene, SCN9A encoding Nav1.7. In 21 affected members, we uncovered a potential mutation in a highly conserved amino acid, p.N641Y, in the large cytoplasmic loop between transmembrane domains I and II that was absent from 586 ethnically matched population control chromosomes. To establish a functional role for this mutation in seizure susceptibility, we introduced the orthologous mutation into the murine Scn9a ortholog using targeted homologous recombination. Compared to wild-type mice, homozygous Scn9aN641Y/N641Y knockin mice exhibit significantly reduced thresholds to electrically induced clonic and tonic-clonic seizures, and increased corneal kindling acquisition rates. Together, these data strongly support the SCN9A p.N641Y mutation as disease-causing in this family. To confirm the role of SCN9A in FS, we analyzed a collection of 92 unrelated FS patients and identified additional highly conserved Nav1.7 missense variants in 5% of the patients. After one of these children with FS later developed Dravet syndrome (severe myoclonic epilepsy of infancy), we sequenced the SCN1A gene, a gene known to be associated with Dravet syndrome, and identified a heterozygous frameshift mutation. Subsequent analysis of 109 Dravet syndrome patients yielded nine Nav1.7 missense variants (8% of the patients), all in highly conserved amino acids. Six of these Dravet syndrome patients with SCN9A missense variants also harbored either missense or splice site SCN1A mutations and three had no SCN1A mutations. This study provides evidence for a role of SCN9A in human epilepsies, both as a cause of FS and as a partner with SCN1A mutations.
Author Summary
Febrile seizures are the most common seizure disorder of early childhood, and exhibit a prevalence of 2%–5% in European and North American children. While the genetic basis of febrile seizures is well-documented, efforts to uncover these genes have yielded only a few genes in a small proportion of cases. In a genomic region on human chromosome 2 known to harbor the febrile seizure SCN1A sodium channel gene, we now report a disease-causing mutation in the adjacent gene, SCN9A (Nav1.7), in a large family with febrile seizures. We introduced the family mutation (N641Y) into the orthologous mouse gene to create a knockin mouse model, and tested seizure susceptibility in these mice. Compared to wild-type mice, our Scn9a knockin mice have a significantly lower threshold to electrically induced seizures and experience seizures at a significantly faster rate with repeated subthreshold stimulation. We also report novel missense SCN9A mutations in unrelated febrile seizure patients. Furthermore, we show that a subset of patients with the catastrophic early-onset Dravet syndrome who commonly have mutations in SCN1A also harbor mutations in SCN9A. This finding is important as it demonstrates for the first time mutational evidence for a modifying digenic mechanism of human epilepsy. For infants with Dravet syndrome, a genetic diagnosis will be of immediate benefit to guide therapeutics away from the sodium channel blocking class of anticonvulsant drugs that exacerbate seizures but are often the first administered.
PMCID: PMC2730533  PMID: 19763161

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