Syndactyly is one of the most common congenital limb malformations and is associated with webbing of adjacent fingers/toes (1). It is a heterogeneous hereditary disease, with a prevalence of between 3 and 10 in every 10,000 births; however, a higher prevalence of between 10 and 40 in every 10,000 births has been reported (1,2). In addition, it is twice as common in males, indicating that this disease may demonstrate incomplete penetrance (3). Syndactyly exists either as an isolated non-syndromic anomaly or as part of >300 syndromic abnormalities. Non-syndromic syndactyly may be cutaneous or bony and unilateral or bilateral (4). To date, at least nine types (I–IX) of phenotypically diverse non-syndromic syndactyly have been identified. Five primary types (type I–V) are inherited as an autosomal dominant trait with variable disease phenotypes and incomplete penetrance (4); while the remaining types and subtypes may demonstrate autosomal recessive or X-linked hereditary patterns of inheritance (3). The phenotype of a specific patient appears to be dependent on a specific gene, as well as their genetic background and the subsequent signaling pathways involved in limb formation (3). Non-syndromic syndactyly is genetic in origin (5). At present, at least 11 disease loci and eight disease-associated genes have been identified, including the homeobox D13 gene (HOXD13), the fibulin 1 gene, the gap junction protein alpha 1 gene, the limb development membrane protein 1 gene, the LDL receptor related protein 4 gene, the gremlin 1 gene, the formin 1 gene and the fibroblast growth factor 16 gene (1,6). Mutations in the HOXD13 gene are responsible for the development of syndactyly type II-a and syndactyly type V (7,8). The typical clinical features of syndactyly type I-c include bilateral cutaneous or bony fusion of the third and fourth fingers and occasionally of the third to fifth fingers, with normal feet (2).

The aim of the present study was to identify the genetic basis of syndactyly type I-c in four generations of a single Chinese family.

Exome sequencing of the proband (Fig. 1A: Generation IV, member 1) in the syndactyly type I-c-affected family was first performed. A total of 5.92 billion bases of 90-bp paired-end read sequences for the patient were generated, and 106,958 genetic variants, including 14,830 non-synonymous alterations, were identified in the coding regions or the splicing sites. A prioritization scheme was applied to identify the pathogenic mutation, using similar methods described in a previous study (18). Given that the frequency of syndactyly type I is 0.03% (2), known variants identified in the dbSNP (build, 137), the 1000 Genomes Project, the International HapMap project and the YanHuang project with a minor allele frequency value of >0.50% in each database, were excluded. Using the aforementioned criteria, the number of candidate genes were reduced by >92.99%. Variants not annotated in public databases were prioritized for further analysis.

Following validation by Sanger sequencing, a heterozygous mutation in the HOXD13 gene (c.917G>A, p.R306Q) was observed in the proband (Fig. 2), however, no other variants in the known disease-causing genes for syndactyly were identified by exome sequencing. The mutation co-segregated among the affected family members, and was absent in the unaffected family members, as well as in the 100 normal controls. This suggests that the c.917G>A mutation in HOXD13 may have been the pathogenic mutation. In further support of this notion, this mutation was also absent in an in-house database from the BGI-Shenzhen (Shenzhen, China) with 2,375 ethnically-matched controls. The results indicate that the c.917G>A (p.R306Q) variant of the HOXD13 gene may be the disease-associated mutation in this family. The c.917G>A mutation is located in exon 2, which encodes the homeodomain in the C-terminal region (a highly-conserved DNA binding motif) that is responsible for binding to consensus DNA sequences (19). The mutation alters the amino acid sequence at position 306 from Arg to Gln at residue 31 of this homeodomain. This position was observed to be highly conserved among different species (Fig. 3), indicating its structural and functional importance. This mutation was predicted to be disease-associated and affect protein function using the MutationTaster and SIFT prediction algorithms, and potentially damaging using the PolyPhen-2 algorithm (score, 1.00).

Syndactyly displays marked genetic heterogeneity and high clinical variability, and is characterized by limb malformation (1). Syndactyly type I-c is the rarest type among the four subtypes of syndactyly type I (I-a, I-b, I-c and I-d), which is the most common form of non-syndromic syndactyly (2). Two mutations (p.R306Q and p.R306 G) in the HOXD13 homeodomain have been identified in two independent Chinese families diagnosed with syndactyly type I-c in a previous study (20). However, inconsistencies between the clinical phenotypes and diagnostic criteria in selected patients from the two families were observed, including a mild synpolydactyly phenotype, involvement of the feet, clinodactyly and fusion of the second and third fingers, which questions the accuracy of the clinical diagnosis (20). The present study included a Chinese Han family consisting of members diagnosed with typical syndactyly type I-c that presented with bilateral cutaneous webbing of the third and fourth fingers, normal toes, and an absence of additional abnormalities. The results, together with those obtained from the different prediction programs, identified the HOXD13 c.917G>A (p.R306Q) variant as the potential pathogenic mutation for this family. Given that G>A or C>T transitions within CpG dinucleotides are reportedly responsible for one-third of single base-pair mutations in gene coding regions in human genetic diseases (21), the c.917G>A mutation may be caused by cytosine methylation followed by deamination to thymine (C>T) on the complementary strand.

To the best of our knowledge, this is the first study to present a family diagnosed with syndactyly type I-c, with affected members harboring the HOXD13 p.R306Q mutation with complete penetrance. The two independent Chinese families with syndactyly reported previously (20), may have been diagnosed with unknown complex types of non-syndromic syndactyly or syndactyly type I-c variant types with incomplete penetrance and atypical phenotypes. As two different amino acid substitutions from different families have been identified at position 306, this position may be a mutation hotspot. In addition, in a previous study, common mutation screening was unable to exclude the genes involved in finger/toe development, which was excluded by exome sequencing used in the present study (20).

The HOXD13 gene is a member of the HOX gene family, and encodes a transcription factor that serves a crucial role in limb development (22). HOXD13 is composed of 2 exons and encodes a 343-amino acid polypeptide sequence (23). At least 36 different HOXD13 mutations, including 17 missense/nonsense mutations, 8 repeats, 6 small deletions, 2 splicing mutations, a small insertion, a gross deletion, as well as a gross insertion/duplication mutation, have been described in the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php). The majority of these mutations are observed in patients with limb malformations, including syndactyly, synpolydactyly and brachydactyly (24). Mouse models with different genetic deficiencies in the HOXD13 gene have been described previously in the literature. The majority of these mouse models mimic some aspects of human synpolydactyly phenotypes, further supporting the importance of the HOXD13 gene in limb development (25–27).

In conclusion, the results of the present study indicate that the c.917G>A (p.R306Q) mutation in the HOXD13 gene may be the genetic basis of syndactyly type I-c in a single Chinese family, which increases the phenotypic spectrum of the HOXD13 gene. Whole exome sequencing provides a cost-effective and expedited approach for identifying disease-associated genes, and excludes the effects of background genes in disorders displaying high clinical heterogeneity. The results of the present study may provide novel insights into the genetic diagnosis of syndactyly, and may have implications for genetic counseling and the clinical management of this condition.