The Phenotype And Gene Mutation Analysis Of HOXD13 In A Congenital Synpolydactyly Pedigree

Background and ObjectiveSynpolydactyly (SPD;MIM# 186000) is an rare, autosomal dominant inherited limb malformation, it belongs to syndactly type II. Typically, affected individuals have 3/4 finger and 4/5 toe syndactyly, with a duplicated digit in the syndactylous web. As a lot of other of dominantly disorders, SPD display incomplete penetrance and variable expressivity both between and within affect families. One to four limbs can be involved, and the severity of involvement varies from partial skin syndactyly to almost complete digit duplication, extending proximally as far as the metacarpals/metatarsals.HOXD13, the homeobox-containing gene located at the most 5' end of the HOXD cluster, plays a critical role in limb development, HOXD13 located on chromosome 2q31.In 1996, Muragahed ect identified the PAE mutation of HOXD13 in three SPD families and confirmed that it is the mutation in HOXD13 that caused SPD. This research first suggested that PAE may play an important role in the development of inherited disease. In 1998, Goodman ect reported a Belgian family with a complex type of SPD cosegregating with a balanced t(12;22), and later researches show that the disrupted FBLN1 caused by t(12;22) may be the molecular pathogenesis of this type of SPD.During last ten years, a lot of researches about SPD had been done, however, until now, the data is still scarce of the mutation in Chinese SPD pedigree. As SPD display incomplete penetrance and variable expressivity both between and within affect families, gene diagnosis can help us to confirm the clinical diagnosis. Through the study of a large Chinese SPD, our study aimed to analysis and summarized all the clinical features in detail; establish method of gene diagnosis for prenatal carriers and presymptomatic patients ;to identify the mutation of HOXD13 gene in the pedigree; to explore the role of PAE in HOXD13 in the development of SPD.Subjects and methodsThe pedigree was a five-generation Chinese family, including 13 affected individuals. All the affected individuals have congenital synpolydactyly. We examined the effected individuals that still alive clinically, and radiographs of both hands and feet were obtained from five of these individuals.13 blood samples of the affected invidividuals and 11 of the patients' relatives were obtained with informed consent. Chromosome caryotype analysis was carried on for the proband and his father. Standard salting-out method was adopted in extraction genomic DNA from 5ml peripheral blood which was anticoagulated by ACD. To screen the expansions of HOXD13 polyalanine tract, we perform design to amplify a 161-bp fragment of the first exon of HOXD13, which contains the imperfect trinucleotide repeat sequence coding the tract. The mutation was first screened by agarose gel electrophoresis to detected mutation. Then the mutate fragment was separated from the mixed products by 8% polyacrylamide gel electrophoresis. After purifying the PCR production with negative result, PCR was carried out to amplify the mutate alley. The PCR product of normal and mutant alleles were directly cycle sequenced by shanghai bioasia biological technology company using applied biosystem 377.Result1. SPD was autosomal dominant inherited and the numbers of the women patients and men patients are equal.2. The severity of involvement varies between the affected individuals, SPD in this pedigree display variable expressivity within affect inviduals while the penetrance is 100%.3. The chromosomes of the proband and his father are normal.4. All of the patients show mutations in HOXD13, they are all hetozygosities.5. Sequence result showed that there was an extra 9 alanine in the HOXD13, this made the polyalanine in the HOXD13 changed from 15 to 24.Conclusion1. Synpolydactyly in this pedigree was an autosomal dominant inherited limb malformation disease.The affected individuals show complex phenotype, SPD in this pedigree display variable expressivity.2. It is the mutation in HOXD13 that caused the SPD in this pedigree.There is an extra inserted alanine in the HOXD13 which changed the polyalanine tract from 15 to 24 in this pedigree.