Rare and <i>de novo</i> duplications containing <i>SHOX</i> in clubfoot.

Abstract

Congenital clubfoot is a common birth defect that affects at least 0.1% of all births. Nearly 25% cases are familial and the remaining are sporadic in inheritance. Copy number variants (CNVs) involving transcriptional regulators of limb development, including <i>PITX1</i> and <i>TBX4</i>, have previously been shown to cause familial clubfoot, but much of the heritability remains unexplained. Exome sequence data from 816 unrelated clubfoot cases and 2645 in-house controls were analysed using coverage data to identify rare CNVs. The precise size and location of duplications were then determined using high-density Affymetrix Cytoscan chromosomal microarray (CMA). Segregation in families and <i>de novo</i> status were determined using qantitative PCR. Chromosome Xp22.33 duplications involving <i>SHOX</i> were identified in 1.1% of cases (9/816) compared with 0.07% of in-house controls (2/2645) (p=7.98×10^-5, OR=14.57) and 0.27% (38/13592) of Atherosclerosis Risk in Communities/the Wellcome Trust Case Control Consortium 2 controls (p=0.001, OR=3.97). CMA validation confirmed an overlapping 180.28 kb duplicated region that included <i>SHOX</i> exons as well as downstream non-coding regions. In four of six sporadic cases where DNA was available for unaffected parents, the duplication was <i>de novo</i>. The probability of four <i>de novo</i> mutations in <i>SHOX</i> by chance in a cohort of 450 sporadic clubfoot cases is 5.4×10^-10. Microduplications of the pseudoautosomal chromosome Xp22.33 region (PAR1) containing <i>SHOX</i> and downstream enhancer elements occur in ~1% of patients with clubfoot. <i>SHOX</i> and regulatory regions have previously been implicated in skeletal dysplasia as well as idiopathic short stature, but have not yet been reported in clubfoot. <i>SHOX</i> duplications likely contribute to clubfoot pathogenesis by altering early limb development.