Analysis of Variation in Clubfoot Candidate Genes

Isolated clubfoot, a common birth defect occurring in more than 135,000 livebirths worldwide each year, is associated with significant health care and financial burdens. Clubfoot is defined by forefoot adduction, hindfoot varus, midfoot cavus and hindfoot equinus. Isolated clubfoot, which is the focus of these studies, is distinct from syndromic clubfoot because there are no other associated malformations. Population, family, twin and segregation analysis studies provide evidence that genetic and environmental factors play an etiologic role in isolated clubfoot. The studies described in this thesis were performed to define the role of genetic variation in isolated clubfoot. Interrogation of a deletion region associated with syndromic clubfoot, suggested that CASP8 and CASP10, two apoptotic genes, play a role in isolated clubfoot. To explore the role of apoptotic genes in clubfoot, SNPs spanning genes involved in the apoptotic pathway in the six chromosomal deletion regions, and limb patterning genes, HOXD and HOXA, were interrogated. SNPs in mitochondrial mediated apoptotic genes and several SNPs in HOXA and HOXD genes were modestly associated with clubfoot with the most significant SNP, rs3801776, located in the basal promoter of HOXA9. Several significant associations were found with SNPs in NFAT2 and TNIP2. Significant gene interactions were detected between SNPs in HOX and apoptotic genes. These findings suggest a model for clubfoot in which variation in one gene is not sufficient to cause the malformation but requires variation several genes to perturb protein expression sufficiently to alter muscle and foot development. These results significantly impact our knowledge base by delineating underlying mechanisms causing clubfoot.

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 non-Hispanic white and Hispanic multiplex and simplex families (discovery samples) and a second sample of non-Hispanic 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 0.028). Interestingly, HOXA9 is expressed in muscle during development. An 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.

IDENTIFYING GENETIC VARIANTS AND CHARACTERIZING THEIR ROLE IN CLUBFOOT

Clubfoot is a common, complex birth defect affecting 4,000 newborns in the United States and 135,000 world-wide each year. The clubfoot deformity is characterized by inward and rigid downward displacement of one or both feet, along with persistent calf muscle hypoplasia. Despite strong evidence for a genetic liability, there is a limited understanding of the genetic and environmental factors contributing to the etiology of clubfoot. The studies described in this dissertation were performed to identify variants and/or genes associated with clubfoot. Genome-wide linkage scan performed on ten multiplex clubfoot families identified seven new chromosomal regions that provide new areas to search for clubfoot genes. Troponin C (TNNC2) the strongest candidate gene, located in 20q12-q13.11, is involved in muscle contraction. Exon sequencing of TNNC2 did not identify any novel coding variants. Interrogation of fifteen muscle contraction genes found strong associations with SNPs located in potential regulatory regions of TPM1 (rs4075583 and rs3805965), TPM2 (rs2025126 and rs2145925) and TNNC2 (rs383112 and rs437122). In previous studies, a strong association was found with rs3801776 located in the basal promoter of HOXA9, a gene also involved in muscle development and patterning. Altogether, this data suggests that SNPs located in potential regulatory regions of genes involved in muscle development and function could alter transcription factor binding leading to changes in gene expression. Functional analysis of 3801776/HOXA9, rs2025126/TPM2 and rs2145925/TPM2 showed altered protein binding, which significantly influenced promoter activity. Although the ancestral allele (G) of rs4075583/TPM1 creates a DNA-protein complex, it did not affect TPM1 promoter activity. However and importantly, in the context of a haplotype, rs4075583/G significantly decreased TPM1 promoter activity. These results suggest dysregulation of multiple skeletal muscle genes, TPM1, TPM2, TNNC2 and HOXA9, working in concert may contribute to clubfoot. However, specific allelic combinations involving these four regulatory SNPs did not confer a significantly higher risk for clubfoot. Other combinations of these variants are being evaluated. Moreover, these variants may interact with yet to be discovered variants in other genes to confer a higher clubfoot risk. Collectively, we show novel evidence for the role of skeletal muscle genes in clubfoot indicating that there are multiple genetic factors contributing to this complex birth defect.