一、Establishment Of Syndactyly Type Ⅴ Mice Model And Its Morphological Analysis 二、Mutation Spectrum Of Pathogenic Genes In Chinese Patients With Osteogenesis Imperfecta

Syndactyly is a digital malformation in which adjacent fingers and/or toes are webbed because they fail to separate during limb development. It is one of the most common hereditary limb malformations depicting a prevalence of 3-10 in 10000 births. The hallmark of syndactyly type Ⅴ is the fusion of fourth and fifth metacarpals. Additional symptoms may involve shortening of fused fourth and fifth metacarpals, ulnar deviation of fingers from second to fifth, interdigital cleft between third and fourth fingers, camptodactyly of fifth finger, short distal phalanges, and absent distal interphalangeal creases of the affected fingers.In the feet, there is hyperplasia of first ray/metatarsal, and shortening of metatarsals from second to fifth, resulting in varus deviation of metatarsals and valgus deviation of toes/phalanges. Type Ⅴ syndactyly is inherited as an autosomal dominant entity, and it has been attributed to a missense mutation in HOXD13 in a Chinese family by our previous work. In the family with syndactyly type Ⅴ, we identified a missense mutation in the HOXD13 homeodomain, c.950A>G (p.Q317R), which leads to substituting an arginine (R) for the highly conserved Q50. Experimental results shows that mutation can decrease the DNA-binding specificity and affinity in vitro. HOXD13 belongs to a large family ofhomeobox transcription factors that play important roles during embryonic pattern formation. In this study, in order to understand the relationship between Hoxd13-Q50R mutations and limb malformation and to analyze the mechanism of syndactyly type V, TALEN (Transcription activator-like effector nuclease) were first time used to develop the Hoxd13-Q50R mutant mice. We propose to use phenotypic and molecular biology analysis of model mice to slove the problem above. So far, limb malformation has been confirmed by appearance observation and computed tomography (CT) in heterozygous and homozygous mutant mice, respectively. Our results preliminarily prove that the Hoxd13-Q50R mutation can cause syndactyly type V.Osteogenesis Imperfecta (OI) was commonly known as’brittle bone disease’.Patients with OI have fragile bones with increased risk of fracture and are described with extensive variability in the severity of their phenotype. Bone deformities, dentinogenesis imperfecta (DI), deafness, and grayish-blue sclera are often associated with OI. OI totally has 13 candidate genes and more than 90% OI patients are detected with heterozygous mutations in COL1A1 or COL1A2 which encode type I procollagen. The remaining OI cases carry homozygous pathogenic changes in genes can cause overmineralization or undermineralization defects or genes directly involved in collagen type-I postranslational modifications, folding, secretion, or processing or in genes related to osteoblast development. In this study, a combination of PCR-sanger sequencing、Next generation sequencing (NGS) and MLPA technique were used to detect pathogenic mutations in the two major candidate genes mentioned above and PCR-DNA sequencing was used to detect mutations in other autosomal recessive genes. Genomic DNA of all available family members was extracted from blood through the standard phenol/chloroform method. Fragments covering the exons and splicing sites of COL1A1/A2 were amplified and sequenced directly. MLPA analysis was performed to detect copy number variations (CNVs) in patients without mutations detected through sequencing. Finally PCR-DNA sequencing was performed to detect mutations in autosomal recessive genes in the OI affecteds without mutations in COL1A1/A2. In this study, we found 132 point mutations and indels of COL1A1/A2 in 200 OI cases (78 in COL1A1 and 54 in COL1A2). In the 132 mutations above,46 mutations were detected by NGS and 84 mutations were detected through PCR-sanger sequencing. Most of them are missense mutations at glysine sites, nonsense mutations and splicing site mutations. In addition, two gross deletions were found through MLPA method in COL1A1/A2 and a compound heterozygous mutation of c.397G>A(Alal33Thr)/c.677C>T (Ser226Leu) was found in gene WNT1. By application of multiple techniques to establish osteogenesis imperfecta screening platform, we can increase the detection rate of OI causative mutations, meanwhile our experimental results has enriched the Chinese mutation spectrum of OI as well.