Establishment And Application Of Preimplantation Genetic Diagnostic Technique For Hereditary Hearing Loss And Study Of Molecular Mechanism Of Inherited Deafness Pedigrees

Hearing impairment is the most common birth defect and sensorineural disorder. Based on the national survey of disabled population in 2006, the total number of hearing impairment in China has reached 27.8 millions. The number of newborn with congenital hearing impairment is 20-30 thousands every year. Therefore, diagnostic techniques and interventions should be established to reduce the risk of future recurrence of deafness. It has very important long-term significance of improving the quality of the population in China.In this study, we have established preimplantation genetic diagnostic (PGD) technique for hereditary hearing loss by multiple displacement amplification, nested PCR, real-time quantitative PCR and microsatellite marker. Using microsatellite marker, we have successfully identified a novel hearing loss locus and the gene in a family with autosomal dominant non-syndromic deafness. We Successful identify TCOF1 gene mutation in a He-Nan family with Treacher Collins syndrome.PART1:Establishment and application of PGD technique for hereditary hearing lossThe vast majority of genetic deafness was a single-gene disease. Therefore, it is in line with the medical indications for PGD, and can be effectively prevented abnormal embryos for therapeutic abortion, which would avoid the physical and psychological harm for patients. In the previous study of our research group, we have demonstrated that mutation in DIABLO underlying non-syndromic hearing impairment in a large Chinese family. The couple of the proband had very strong demand for PGD, and signed informed consent. We applied the multiple displacement amplification of whole genome, nested PCR, real-time fluorescent quantitative PCR and microsatellite marker to establish the PGD techniques from single lymphocytes of the couple of proband and superseded single blastomere. We have successfully performed an intact PGD cycle in the couple of proband and identified a healthy fetus for implantation. Unfortunately, this fetus did not successfully get nidation due to poor endometrium environment. PART 2:Identification of gene underlying autosomal-dominant nonsyndromic hearing loss in a Chinese familyIn this study, we identified the causative gene in an autosomal-dominant nonsyndromic hearing loss Chinese pedigree named SD-L030 and analyzed DFNA5 mutation. This Chinese family spanned four generations and comprised 30 members. Application of microsatellite markers on DFNA 23 loci preliminary screening of 25 genes, data were analyzed by linkage analysis. We mapped the locus to the region between D7S629 and D7S516 (two-point lod-score of 5.39) with application of 8 microsatellite markers. By direct sequencing of best candidate genes in mapping region, we identified a novel missense mutation ivs7-2 A>G in DFNA5 gene, which was faithfully cosegregated with hearing loss in the family. This mutation causes skipping of exon 8, resulting in premature termination of the open reading frame. In order to understand DFNA5 mechanisms and to gain more insights into the function changes of protein resulting from the missense mutation, we will establish effective experimental model in near future.PART 3:Mutational screening of TCOFl gene in patients with Treacher Collins syndromeTreacher Collins syndrome (TCS) is characterized by hypoplasia of the zygomatic bones and mandible, and so on. Almost 40%-50% of individuals have conductive hearing loss attributed most commonly to malformation (including ankylosis, hypoplasia, or absence) of the ossicles and hypoplasia of the middle ear cavities. We have collected two sporadic patients with conductive hearing loss and notching of the lower eyelid recruited from clinic of Department of otolaryngology of PLA General Hospital. By direct sequencing TCOFl, we identified a novel missense mutation c.146 T>C (p.I49T) in the second exon of TCOFl gene in one patient, but was not found in the gene in his parents. Furthermore, the amine acid of isoleucine in this site is highly conservative across eight species. This result can be used to conduct prenatal diagnosis in this family.