Kevin McCall

Myosin binding protein C1: a novel gene for autosomal dominant distal arthrogryposis type 1

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 LOD(max) 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.

Familial Isolated Clubfoot Is Associated with Recurrent Chromosome 17q23.1q23.2 Microduplications Containing TBX4

Clubfoot is a common musculoskeletal birth defect for which few causative genes have been identified. To identify the genes responsible for isolated clubfoot, we screened for genomic copy-number variants with the Affymetrix Genome-wide Human SNP Array 6.0. A recurrent chromosome 17q23.1q23.2 microduplication was identified in 3 of 66 probands with familial isolated clubfoot. The chromosome 17q23.1q23.2 microduplication segregated with autosomal-dominant clubfoot in all three families but with reduced penetrance. Mild short stature was common and one female had developmental hip dysplasia. Subtle skeletal abnormalities consisted of broad and shortened metatarsals and calcanei, small distal tibial epiphyses, and thickened ischia. Several skeletal features were opposite to those described in the reciprocal chromosome 17q23.1q23.2 microdeletion syndrome associated with developmental delay and cardiac and limb abnormalities. Of note, during our study, we also identified a microdeletion at the locus in a sibling pair with isolated clubfoot. The chromosome 17q23.1q23.2 region contains the T-box transcription factor TBX4, a likely target of the bicoid-related transcription factor PITX1 previously implicated in clubfoot etiology. Our result suggests that this chromosome 17q23.1q23.2 microduplication is a relatively common cause of familial isolated clubfoot and provides strong evidence linking clubfoot etiology to abnormal early limb development.

Pitx1 haploinsufficiency causes clubfoot in humans and a clubfoot-like phenotype in mice

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.

MYBPC1 mutations impair skeletal muscle function in zebrafish models of arthrogryposis

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.

Candidate genes for alcohol preference identified by expression profiling in alcohol-preferring and -nonpreferring reciprocal congenic rats

BackgroundSelectively bred alcohol-preferring (P) and alcohol-nonpreferring (NP) rats differ greatly in alcohol preference, in part due to a highly significant quantitative trait locus (QTL) on chromosome 4. Alcohol consumption scores of reciprocal chromosome 4 congenic strains NP.P and P.NP correlated with the introgressed interval. The goal of this study was to identify candidate genes that may influence alcohol consumption by comparing gene expression in five brain regions of alcohol-naïve inbred alcohol-preferring and P.NP congenic rats: amygdala, nucleus accumbens, hippocampus, caudate putamen, and frontal cortex.ResultsWithin the QTL region, 104 cis-regulated probe sets were differentially expressed in more than one region, and an additional 53 were differentially expressed in a single region. Fewer trans-regulated probe sets were detected, and most differed in only one region. Analysis of the average expression values across the 5 brain regions yielded 141 differentially expressed cis-regulated probe sets and 206 trans-regulated probe sets. Comparing the present results from inbred alcohol-preferring vs. congenic P.NP rats to earlier results from the reciprocal congenic NP.P vs. inbred alcohol-nonpreferring rats demonstrated that 74 cis-regulated probe sets were differentially expressed in the same direction and with a consistent magnitude of difference in at least one brain region.ConclusionsCis-regulated candidate genes for alcohol consumption that lie within the chromosome 4 QTL were identified and confirmed by consistent results in two independent experiments with reciprocal congenic rats. These genes are strong candidates for affecting alcohol preference in the inbred alcohol-preferring and inbred alcohol-nonpreferring rats.

A polygenic burden of rare variants across extracellular matrix genes among individuals with adolescent idiopathic scoliosis

Adolescent idiopathic scoliosis (AIS) is a complex inherited spinal deformity whose etiology has been elusive. While common genetic variants are associated with AIS, they explain only a small portion of disease risk. To explore the role of rare variants in AIS susceptibility, exome sequence data of 391 severe AIS cases and 843 controls of European ancestry were analyzed using a pathway burden analysis in which variants are first collapsed at the gene level then by Gene Ontology terms. Novel non-synonymous/splice-site variants in extracellular matrix genes were significantly enriched in AIS cases compared with controls (P = 6 × 10(-9), OR = 1.7, CI = 1.4-2.0). Specifically, novel variants in musculoskeletal collagen genes were present in 32% (126/391) of AIS cases compared with 17% (146/843) of in-house controls and 18% (780/4300) of EVS controls (P = 1 × 10(-9), OR = 1.9, CI = 1.6-2.4). Targeted resequencing of six collagen genes replicated this association in combined 919 AIS cases (P = 3 × 10(-12), OR = 2.2, CI = 1.8-2.7) and revealed a highly significant single-gene association with COL11A2 (P = 6 × 10(-9), OR = 3.8, CI = 2.6-7.2). Importantly, AIS cases harbor mainly non-glycine missense mutations and lack the clinical features of monogenic musculoskeletal collagenopathies. Overall, our study reveals a complex genetic architecture of AIS in which a polygenic burden of rare variants across extracellular matrix genes contributes strongly to risk.

Deletions of 5′ HOXC genes are associated with lower extremity malformations, including clubfoot and vertical talus

Background Deletions of the HOXC gene cluster result in variable phenotypes in mice, but have been rarely described in humans. Objective To report chromosome 12q13.13 microdeletions ranging from 13 to 175 kb and involving the 5′ HOXC genes in four families, segregating congenital lower limb malformations, including clubfoot, vertical talus and hip dysplasia. Methods Probands (N=253) with clubfoot or vertical talus were screened for point mutations and copy number variants using multiplexed direct genomic selection, a pooled BAC targeted capture approach. SNP genotyping included 1178 probands with clubfoot or vertical talus and 1775 controls. Results The microdeletions share a minimal non-coding region overlap upstream of HOXC13, with variable phenotypes depending upon HOXC13, HOXC12 or the HOTAIR lncRNA inclusion. SNP analysis revealed HOXC11 p.Ser191Phe segregating with clubfoot in a small family and enrichment of HOXC12 p.Asn176Lys in patients with clubfoot or vertical talus (rs189468720, p=0.0057, OR=3.8). Defects in limb morphogenesis include shortened and overlapping toes, as well as peroneus muscle hypoplasia. Finally, HOXC and HOXD gene expression is reduced in fibroblasts from a patient with a 5′ HOXC deletion, consistent with previous studies demonstrating that dosage of lncRNAs alters expression of HOXD genes in trans. Conclusions Because HOXD10 has been implicated in the aetiology of congenital vertical talus, variation in its expression may contribute to the lower limb phenotypes occurring with 5′ HOXC microdeletions. Identification of 5′ HOXC microdeletions highlights the importance of transcriptional regulators in the aetiology of severe lower limb malformations and will improve their diagnosis and management.

Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine

Large-scale mutagenesis of target DNA sequences allows researchers to comprehensively assess the effects of single-nucleotide changes. Here we demonstrate the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP). We created a mutational library containing every possible single-nucleotide mutation surrounding the active site of the TEM-1 β-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation.

Identification of a novel cytosolic aldehyde dehydrogenase allele, ALDHIAI*4

This paper reports the identification of a novel cytosolic aldehyde dehydrogenase 1 (ALDHIAI) allele. One hundred and sixty-two Indo-Trinidadian and 85 Afro-Trinidadian individuals were genotyped. A novel ALDHIAI allele, ALDHIAI*4, was identified in an Indo-Trinidadian alcoholic with an A inserted at position -554 relative to the translational start site, +1. It was concluded that a wider cross-section of individuals needs to be evaluated in order to determine the representative frequency of the allele, and to see if it is associated with risk of alcoholism.

A missense variant in SLC39A8 is associated with severe idiopathic scoliosis

Genetic factors predictive of severe adolescent idiopathic scoliosis (AIS) are largely unknown. To identify genetic variation associated with severe AIS, we performed an exome-wide association study of 457 severe AIS cases and 987 controls. We find a missense SNP in SLC39A8 (p.Ala391Thr, rs13107325) associated with severe AIS (P = 1.60 × 10−7, OR = 2.01, CI = 1.54–2.62). This pleiotropic SNP was previously associated with BMI, blood pressure, cholesterol, and blood manganese level. We replicate the association in a second cohort (841 cases and 1095 controls) resulting in a combined P = 7.02 × 10−14, OR = 1.94, CI = 1.63–2.34. Clinically, the minor allele of rs13107325 is associated with greater spinal curvature, decreased height, increased BMI and lower plasma manganese in our AIS cohort. Functional studies demonstrate reduced manganese influx mediated by the SLC39A8 p.Ala391Thr variant and vertebral abnormalities, impaired growth, and decreased motor activity in slc39a8 mutant zebrafish. Our results suggest the possibility that scoliosis may be amenable to dietary intervention.The majority of scoliosis is considered idiopathic with onset in adolescence (AIS) and has a genetic contribution. Here, the authors perform an exome wide association study of data from 457 severe AIS cases and 987 controls, and find a missense variant in SLC39A8 is associated with AIS.