Polyalamine repeat expansion mutations in the HOXD13 gene in Pakistani families with synpolydactyly

To the Editor : Synpolydactyly (SPD; MIM 186000) is a rare autosomal dominant limb deformity, with a distinctive combination of syndactyly and polydactyly. The main features of SPD are the webbing of the Third or Fourth fingers and Fourth or Fifth toes, with partial or complete digital duplication within the syndactylous web (1, 2). Currently, SPD is classified into three types, SPD1–SPD3. Of these, SPD1 is caused by polyalanine expansion repeats in the HOXD13 gene on chromosome 2q31 (1). HOXD13 is a member of the HOX family that encodes for a transcription factor with a crucial role in limb development (3). N-terminal polyalanine repeats of +7 to 14 polyalanine repeats are the mutations described in HOXD13 (4) with the normal number of polyalanine repeats being 15 (5). Genotype–phenotype correlation suggests that short polyalanine repeats (i.e., +7 repeats) are associated with lower penetrance and expressivity and thus resulting in variable phenotypesm, whereas long repeats (+10 and more) are associated with higher penetrance and more severe disease (6). The role of HOXD13 during embryogenesis is crucial in limb development (7, 8). The pathophysiology of the perturbed function of HOXD13 is such that the mutant protein with additional polyalanine repeats is predicted to destabilize the normal protein configuration, leading to its aggregation. This impedes protein translocation from the cytoplasm to the nucleus where it acts as a transcription factor, although the pathway and the downstream genes it regulates are still unknown (9, 10). In addition, the mutant protein may aggregate and possibly inhibit the wild-type protein from translocating into the nucleus, thus the polyalanine repeat expansions in the HOXD13 gene can show dominant-negative activity (11). To date, only a limited number of families with HOXD13 mutations have been reported (1). Here, we studied four Pakistani families with SPD. After obtaining informed consent, we extracted genomic DNA from peripheral blood samples from members of the four families (under institutional approval and in adherence to the Declaration of Helsinki Principles). All four families showed an autosomal dominant inheritance pattern (Fig. 1a–d). All patients were heterozygotes except for one patient in Family 1 who was homozygous. The clinical manifestations were variable, such as syndactyly and webbing involving the third and fourth fingers (Fig. 1e,k,n), syndactyly involving the fourth and fifth toes with digital duplication (Fig. 1f,m). In addition, some affected individuals showed finger deviations and contractures (Fig. 1 h,i,l), and others displayed incomplete formation of the limb, digits, and brachydactyly (Fig. 1q–t). As expected, the homozygous patient in Family 1 showed a severe phenotype (Fig. 1q,r), but interestingly one of the heterozygotes in Family 1 (Fig. 1s,t) showed a phenotype as severe as the homozygous patient, be consistent with variable expressivity of the disease. External genitalia appeared normal. Using the genomic DNA of the four families, we first performed genotyping using microsatellite markers flanking to HOXD13 gene on chromosome 2q31. Our results suggested linkage of all four families to the HOXD13 gene. Subsequently, we performed direct sequencing of the amplified PCR products. We identified a duplication mutation c.184_210dup in exon 1 of the HOXD13 gene of affected individuals in Families 1 and 3 (Table 1). The mutation results in the expansion of +9 alanine residues in the Nterminal domain of the HOXD13 protein thus leading to a total of 24 polyalanine repeats. We also found another duplication mutation c.187_207dup in exon 1 of the HOXD13 gene of affected individuals in Families 2 and 4, which generate to an expansion of +7 alanine residues (Table 1) leading