Studies of TBX4 and chromosome 17q23.1q23.2: an uncommon cause of nonsyndromic clubfoot

Abstract

Clubfoot is a common birth defect characterized by inward posturing and rigid downward displacement of one or both feet. The etiology of syndromic forms of clubfoot is varied and the causes of isolated clubfoot are not well understood. A microduplication of 2.2 Mb on chromosome 17q23.1q23.2 which includes T-box 4 (TBX4), a hindlimb-specific gene, and 16 other genes was recently identified in 3 of 66 families reported as nonsyndromic clubfoot, but additional non-foot malformations place them in the syndromic clubfoot category. Our study assesses whether variation in or around TBX4 contributes to nonsyndromic clubfoot. To determine whether this microduplication was a common cause of nonsyndromic clubfoot, 605 probands (from 148 multiplex and 457 simplex families) with nonsyndromic clubfoot were evaluated by copy number and oligonucleotide array CGH testing modalities. Only one multiplex family (0.68%) that had 16 with clubfoot and 9 with other foot anomalies, had a 350 kb microduplication, which included the complete duplication of TBX4 and NACA2 and partial duplication of BRIP1. The microduplication was transmitted in an autosomal dominant pattern and all with the microduplication had a range of phenotypes from short wide feet and toes to bilateral clubfoot. Minimal evidence was found for an association between TBX4 and clubfoot and no pathogenic sequence variants were identified in the two known TBX4 hindlimb enhancer elements. Altogether, these results demonstrate that variation in and around the TBX4 gene and the 17q23.1q23.2 microduplication are not a frequent cause of this common orthopedic birth defect and narrows the 17q23.1q23.2 nonsyndromic clubfoot-associated region.

Figure. 1. Pedigree of clubfoot family

Filled circles or squares indicate clubfoot and half filled circles or squares designate short, wide feet. WT indicates wild type or two copies and CNG indicates copy number gain or three copies of the chromosome 17q22.3 region.

Figure. 2. Foot anomalies in individuals with microduplication

Variability of foot anomalies in microduplication individuals is shown for individuals transmitting the microduplication. Severe clubfoot deformity with muscle hypoplasia is seen in V-5, while his siblings (V-6 and V-7), father (VI-9) and grandfather (III-7) have short, wide feet. The great toes are short in those with foot anomalies and hallux varus is present in V-6. The hands are normal. WT indicates wild type or two copies and CNG indicates copy number gain or three copies of the chromosome 17q22.3 region.

Figure 3. Schematic of chromosome 17q23.2 copy number gain region

This region, spanning from 59,487,443 to 59,835,772 bps (Genomic coordinates hg19), contains three genes. TBX4 and NACA2 are duplicated; and exons 15 to 20 of BRIP1 are duplicated.

Figure 4. Segregation of hindlimb enhancer variants in clubfoot family members

A. Chromatogram shows the heterozygote C>T variant in HLEA in Family 1 (F1). This variant does not segregate with the nonsyndromic clubfoot phenotype. B. Chromatograms showing the C deletion in HLEB with both forward and reverse sequences. This polymorphism was present in three families (F2-4) and does not segregate with the nonsyndromic clubfoot phenotype. Het = heterozygote. WT = wild type, F = forward sequence, R = reverse sequence.

Figure 4. Segregation of hindlimb enhancer variants in clubfoot family members

A. Chromatogram shows the heterozygote C>T variant in HLEA in Family 1 (F1). This variant does not segregate with the nonsyndromic clubfoot phenotype. B. Chromatograms showing the C deletion in HLEB with both forward and reverse sequences. This polymorphism was present in three families (F2-4) and does not segregate with the nonsyndromic clubfoot phenotype. Het = heterozygote. WT = wild type, F = forward sequence, R = reverse sequence.