Mutation Identification Of Pathogenic Gene For Two Families With Limb Malformation

Congenital limb deformity is one of the most common birth defects in newborns and the incidence is about1‰to2‰. Variation of genetic materials and environmental factors during embryo development (such as virus infection, chemical drugs, ionizing radiation, abnormal intrauterine environment, etc.) are two main reasons for limb abnormalities. A variety of gene mutations and chromosome aberrations can directly lead to acra teratogenesis, which are refers to the number of fingers or toes, length and anatomical morphological abnormalities, such as polydactyly(PD), ectrodactyly (ED), brachydactyly (BD) and syndactyly (SD). Different types of acra abnormalities can occur alone, also can cross exist, such as synpolydactyly (SPD) and brachydactyly with syndactyly, etc. To research disease-causing gene mutation and pathogenesis mechanism of congenital limb malformation is helpful for understanding regulatory mechanism of the acra development and has very important significance and application value for guiding genetic improvement and providing prenatal counseling. We took one family with synpolydactyly and another affected with split-hand/split-foot malformation as research objects, did the study of pathogenic gene screening and mutation detection respectively. In addition, the preliminary function research for the pathogenic mutation of one family with rare ectrodactyly phenotype had been carried on.Part One Pathogenic Mutation Analysis of HOXD13Gene for a Chinese Family with SynpolydactylySynpolydactyly is a rare autosomal dominant acra malformation, belonging to the syndactyly Ⅱ. It is characterized by syndactyly of3or4fingers and4or5toes. The lingers or toes are webbed and not separated with part or full copy of additional fingers or toes between the web with or without osseous fusion. SPD is a highly clinical heterogeneity and genetic heterogeneity limb deformity. The expressivity and penetrance of the patients have significant difference in different families or the different patients or the same patient with hand or foot deformity in the same family. Patients can only show one hand (foot) or two hands (feet) abnormalities. The more severe symptoms can involve metacarpal or metatarsal deformities, at the same time with bone morphological deformities such as BD and curving fingers or toes, etc. According to the genetic heterogeneity of SPD, it can be divided into three subtypes: SPD1(MIM186000), SPD2(MIM608180) and SPD3(MIM610234) respectively, located on chromosome2q31,22q13.31and14q11.2-q12.HOX gene cluster encode a highly conservative transcription factors family, which send different location information to different parts of the body and has an important regulatory role in differentiation and other physiological processes during acra development and morphogenesis process of vertebrate embryo. HOXD13is mapped on2q31and is the first HOX gene that is found associated with human limb development. Four types of HOXD13mutation can lead to SPD:poly alanine chain elongation (PAE) mutation, deletion, missense mutation and nonsense mutation. Among them, the PAE mutation in exonl of HOXD13is responsible for most of the typical SPD cases. This study aimed to explore the phenotype and genetic characteristics of this family with typical SPD in Shandong province, and perform disease-causing gene screening and mutation analysis.First of all, diagnosis of SPD for the family was identified by research on detailed clinical and radiographic examination for patients with SPD in this family; Then, candidate genes was selected for mutation detection according to reports. To amplify HOXD13coding exons and flanking sequence by polymerase chain reaction and to detect pathogenic mutation by direct sequencing the amplification products and TA cloning products. The results of the sequencing were compared with reference sequences provided by GenBank to identify the mutation type. Finally, the gene mutation was verified co-segregated with the phenotype of patients with SPD within the family and the60local healthy people, so as to identify the pathogenic mutation for the family.There were patients with SPD each generation in the five-generation family with no atavism phenomenon, and the men and women were both affected by SPD. It accorded with autosomal dominant characteristics. Family members displayed complex and varied clinical phenotype, and the diagnosis of SPD was definite. Gene cloning and sequencing results showed that all patients carry a heterozygous insertion mutation:24bp not fully trinucleotide repeat sequence in the exonl of HOXD13, resulting poly alanines chain extending eight extra alanine residues. But it was not observed in the normal members of the family with SPD and60local healthy controls.In this study, phenotypic differences of the patients in the family with SPD are large, conforming to autosomal dominant inheritance.8alanine residues elongation mutation in HOXD13gene is co-segregated with the phenotype of patients in the family and is the pathogenic mutation for the family. Part Two Identifying the Disease—℃ausing Gene Responsible for the Family with Split-Hand/Split-Foot Malformation Using Exome SequencingSplit-hand/split-foot malformation(SHFM), also known as ectrodactyly, is a congenital autopod malformation characterized by cleft of the hand and/or foot due to the absence of the central rays, ectrodactyly, syndactyly and dysplasia of metacarpale and metatarsal bones. It may occur as an isolated entity or as part of a syndrome. SHFM is highly genetic heterogeneity. So far, six loci for SHFM have been identified, including SHFM1(MIM183600), SHFM2(MIM313350), SHFM3(MIM600095), SHFM4(MIM605289), SHFM5(MIM606708) and SHFM6(MIM225300), mapped on human chromosome regions7q21、Xq26、10q2、3q28、2q31and12q13respectively. Among them, the pathogenic genes for SHFMK SHFM4and SHFM6have been identified:DLX5, TP63and WNT1Ob respectively.DLX5gene is located on chromosome7q21, belonging to the DLX homeobox gene family, encoding transcription factors mainly expressed in the early development of embryo, which regulate the development and morphogenesis of vertebrate limb. According to the report, DLX5acts as oncogene and can combine with two different sites in the promoter of MYC gene and activate transcription of MYC. We found a Chinese family with SHFM in Linyi, Shandong province. First of all, the diagnosis of non-syndromic SHFM was made by detailed clinical and X-ray examination for patients with SHFM in the family. Then mutation detection for candidate gene TP63was performed by using polymerase chain reaction combined with direct sequencing. But there was no mutation. So we took the exome sequencing for the proband in the family and the sequencing results were compared with referred gene sequence and screened using databases, such as dbSNP database, Hapmap database, one thousand-genome project (1000-genome project), combined with variations screening of candidate genes in the identified loci. Next, we confirmed the mutation was co-segregated with the phenotype of patients with SHFM in the family by direct sequencing. In addition, the same work was carried out in the200local health crowd to rule out the possibility the mutation was a rare polymorphism. Finally,we performed cell transfection and luciferase activity detection using human DLX5wild type vector, mutant expression vector involving the mutation we found and MYC promoter luciferase expression vectors. The data revealed the function of mutant gene for transcription activation of MYC had significantly reduced, and further confirmed the mutation was pathogenic.There were2patients (1female,1male) in the SHFM family of three generations of eight members, conforming to autosomal dominant inheritance with complete penetrance. The difference of phenotype expressivity in the family was not very large. The feet of patients displayed consistent typical split malformation and with triphalangeal thumbs of the hands, but with approximate normal fingers. The heterozygous mutation in DLX5(c.558G>T) was the disease-causing mutation for the family with SHFM. The index person was the first one taking this mutation in the family. The results of population confirmation and lucilerase activity detection showed the mutation was a pathogenic mutation for SHFM, rather than a benign polymorphism. In this study, we first found the intragenic DLX5mutation was responsible for autosomal dominant isolated SHFM and laid the foundation for further research on pathogenesis of SHFM.