A Novel HOXD13 Mutation Causes Synpolydactyly And Promotes Osteoclast Differentiation By PSMAD5/RANK/p65/c-Fos Axis

Congenital syndactyly contains simple syndactyly and complex syndactyly.Simple syndactyly,also known as cutaneous syndactyly,refers to only skin and soft tissue.Complex syndactyly,also named osseous syndactyly,refers to bone fusion or neurovascular connection between two or more fingers.Syndactyly is an autosomal dominant disorder with the significant clinical heterogeneity.Mutations in HOXD13 gene are widely involved in Synpolydactyly（SPD）.HOXD13 is a member of the homeobox transcription factor family and plays an important role in embryonic development.Polyalanine extension in the HOXD13 gene has been shown to induce an SPD phenotype in mice by reducing retinoic acid synthesis.Missense mutations in the HOXD13（G220V）gene also cause a SPD phenotype and impair the transcriptional activity of HOXD13.HOXD13 and HOXA13 are located at the 5 ’end of their respective gene clusters and together coordinate the development of distal limb organs.HOXD13 mutation has been widely confirmed to be the most important cause of familial syndactyly.Bone development,remodeling and repair during embryonic development and after birth are finely regulated by osteoblasts and osteoclasts.Osteoblasts secrete bone matrix proteins（BMPs）,and osteoclasts are responsible for bone resorption and clearance.In this study,a SPD family with 48 members in 4 generations was collected,of which 28 members were included in the analysis,including 15 patients and 13 normal controls in the family.HOXD13（NM₀00523:exon2:c.G917T:P.R3061）.Although the role of HOXD13 gene mutation in syndactyly is well known,the mechanism of HOXD13 gene mutation in syndactyly needs to be further studied.To further investigate the mechanism of this novel mutation in syndactyly,we found that the mutation site（G917T）is located in the homeobox domain of exon 2.Based on sequence alignment,we generated HOXD13 mutant（G905T）mice,homozygous mutation of which leads to obvious syndactyly phenotype.With the increase of generations of mice,syndactyly gradually developed from skin syndactyly to bone syndactyly,which was gradually aggravated,mimicking the development trend of clinical syndactyly families.Bone marrow-derived monocytes（BMMs）isolated from HOXD13 wild-type mice and HOXD13 homozygous mutant mice were induced by stimulating factors M-CSF and RANKL,respectively.Real-time PCR and Western blot analysis showed that,Homozygous HOXD13 mutation enhanced the expression of RANK,p65 and c-Fos at both mRNA and protein levels.TRAP staining and Micro CT analysis of femora from HOXD13 wild-type and homozygous mutant mice revealed increased osteoclast differentiation and osteoporosis in HOXD13 mutant mice.To explore the regulatory mechanism of HOXD13 in the expression of RANK,c-Fos and p65,Western blot,TRAP staining,protein immunoprecipitation and chromatin immunoprecipitation analysis showed that HOXD13 mutation impaired the interaction between HOXD13 and pSmad5,and increased free pSmad5.It induces the expression of c-Fos and p65,which can bind to the RANK promoter to activate RANK expression and promote osteoclast differentiation.In conclusion,HOXD13 mutation promotes osteoclast differentiation by regulating the Smad5/p65/c-Fos/RANK axis,providing new insights into the occurrence and development of SPD.