Synpolydactyly (SPD) is mainly caused by mutations of polyalanine expansion (PAE) in the transcription factor gene HOXD13 and the involved cell types and signal pathway are still not clear possible pathways and single-cell expression characteristics of limb bud in HOXD13 PAE mice was analyzed in this study. We investigated a previous study of a mouse model with SPD and conducted weighted gene co-expression network analysis (WGCNA) using a single-cell RNA sequencing dataset from limb bud cells of SPD mouse model of HOXD13 + 7A heterozygote. Analysis of WGCNA revealed that synpolydactyly-associated Hoxd13 PAEs alter the immune response and osteoclast differentiation, and enhance DNA replication. Bmp4, Hand2, Hoxd12, Lnp, Prrx1, Gmnn, and Cdc6 were found to play potentially key roles in synpolydactyly. These findings evaluated the main genes related to SPD with PAE mutations in HOXD13 and advance our understanding of human limb development.

HOXD13 is an important regulator of limb development. Pathogenic variants in HOXD13 cause synpolydactyly type 1 (SPD1). How different types and positions of HOXD13 variants contribute to genotype-phenotype correlations, penetrance, and expressivity of SPD1 remains elusive. Here, we present a novel cohort and a literature review to elucidate HOXD13 phenotype-genotype correlations. Patients with limb anomalies suggestive of SPD1 were selected for analysis of HOXD13 by Sanger sequencing, repeat length analysis, and next-generation sequencing. Literature was reviewed for HOXD13 heterozygotes. Variants were annotated for phenotypic data. Severity was calculated, and cluster and decision-tree analyses were performed. We identified 98 affected members of 38 families featuring 11 different (likely) causative variants and 4 variants of uncertain significance. The most frequent (25/38) were alanine repeat expansions. Phenotypes ranged from unaffected heterozygotes to severe osseous synpolydactyly, with intra- and inter-familial heterogeneity and asymmetry. A literature review provided 160 evaluable affected members of 49 families with SPD1. Computer-aided analysis only corroborated a positive correlation between alanine repeat length and phenotype severity. Our findings support that HOXD13-protein condensation in addition to haploinsufficiency is the molecular pathomechanism of SPD1. Our data may, also, facilitate the interpretation of synpolydactyly radiographs by future automated tools.

The mutations of HOXD13 gene have been involved in synpolydactyly (SPD), and the polyalanine extension mutation of Hoxd13 gene could lead to SPD in mice. In this study, a novel missense mutation of Hoxd13 (NM_000523: exon2: c.G917T: p.R306L) was identified in a Chinese family with SPD. The mice carrying the corresponding Hoxd13mutation were generated. The results showed that the homozygous mutation of Hoxd13 also caused SPD, but heterozygous mutation did not affect limbs development, which was different from that of SPD patients. With the increasing generation, the mice with homozygous Hoxd13 mutation presented more severe syndactyly. Western blotting showed that this mutation did not affect the protein expression of Hoxd13, suggesting that this mutation did not result in haploinsufficiency. Further analysis demonstrated that this homozygous Hoxd13mutation promoted osteoclast differentiation and bone loss, and enhanced the mRNA and protein expression of osteoclast-related genes Rank, c-Fos, and p65. Meanwhile, this homozygous Hoxd13 mutation elevated the level of phosphorylated Smad5 (pSmad5). Co-immunoprecipitation verified that this mutation attenuated the interaction between pSmad5 and HOXD13, suggesting that this mutation released more pSmad5. Inhibition of pSmad5 reduced the expression of Rank, c-Fos, and p65 despite in the mutation group. In addition, inhibition of pSmad5 repressed the osteoclast differentiation. ChIP assay confirmed that p65 and c-Fos could bind to the promoter of Rank. These results suggested that this novel Hoxd13 mutation promoted osteoclast differentiation by regulating Smad5/p65/c-Fos/Rank axis, which might provide a new insight into SPD development.

Synpolydactyly (SPD) is caused by mutations in the transcription factor gene HOXD13. Such mutations include polyalanine expansion (PAE), but further study is required for the phenotypic spectrum characteristics of HOXD13 PAE. We investigated four unrelated Chinese families with significant limb malformations. Three PAEs were found in the HOXD13 polyalanine coding region: c.172_192dup (p.Ala58_Ala64dup) in Family 1, c.169_192dup (p.Ala57_Ala64dup) in Family 2, and c.183_210dup (p.Ala62_Ala70dup) in Family 3 and Family 4. Interestingly, we identified a new manifestation of preaxial polydactyly in both hands in a pediatric patient with an expansion of seven alanines, a phenotype not previously noted in SPD patients. Comparing with the wild-type cells and mutant cells with polyalanine contractions (PACs), the HOXD13 protein with a PAE of nine-alanine or more was difficult to enter the nucleus, and easy to form inclusion bodies in the cytoplasm, and with the increase of PAE, the more inclusion bodies were formed. This study not only expanded the phenotypic spectrum of SPD, but also enriched our understanding of its pathogenic mechanisms.

Synpolydactyly is a rare congenital anomaly characterized by syndactyly and polydactyly in the central hand. Limited treatment guidelines exist for this complex condition. A retrospective review of synpolydactyly patients was conducted at a large, tertiary pediatric referral center to describe our surgical experience and evolution of management. The Wall classification system was used to categorize cases. Eleven patients (21 hands) with synpolydactyly were identified. Most of the patients were White and had at least one first-degree relative who also had synpolydactyly. The Wall classification yielded the following results: 7 type 1A hands, 4 type 2B hands, 6 type 3 hands, and 4 uncategorizable hands. Each patient had an average of 2.6 surgeries and an average follow-up time of 5.2 years. The rates of postoperative angulation and flexion deformities were 24% and 38%, respectively, with many of these cases also demonstrating preoperative alignment abnormalities. These cases often required additional surgeries including osteotomies, capsulectomies, and/or soft tissue releases. The rate of web creep was 14% with 2 of these patients requiring revision surgery. Despite these findings, at the time of final follow-up, most patients had favorable functional outcomes, were able to engage in bimanual tasks, and were able to perform activities of daily living independently. Synpolydactyly is a rare congenital hand anomaly with a significant degree of variability in clinical presentation. The rates of angulation and flexion deformities as well as web creep are not insignificant. We have learned to prioritize correcting contractures, angulation deformities, and skin fusion, over simply trying to delete the "extra" number of bones as this may destabilize the digit(s).