Study And Application Of New Diagnostic Techniques On Deafness Genes And Molecular Epidemiology Of Hot-spot Mutations Among Non-syndromic Hearing Loss Patients In China

Along with exciting development in genome medicine,there has made remarkable progress on hearing loss.Numerous auditory genes have been identified and genetic testing technologies for different forms of deafness have led to identify more and more inherited deafness patients.Diagnosis prevention and intervention of hearing loss have close relationship with national economy development and health promotion.We have discovered several hot-spot mutation in Chinese Han population.Furthermore,we developed a few genetic testing technologies with independent intellectual property rights by using instrumenttation in Genetic Testing Center for Deafness of PLA General Hospital,and visualize satisfactory clinical application.In addition,we also achieved new findings both in molecular epidemiology of human deafness and in hearing phenotype correlation. This thesis is divided into five main parts to present the results for the study.Part one:Study and application of new diagnostic techniques series on deafness genesEstablishment of molecular diagnostic techniques for hearing loss makes possible various diagnosis strategies.First,time spending is less and results more reliable.Second,once a diagnosis is made,the prevention and treatment of heating loss may be possible.ChapterⅠ: we establish a new detecting method for SLC26A4 IVS7-2 A＞G mutation using testing kit with PAGE silver staining and to evaluate the feasibility of the method as a fast diagnostic technique for enlarged vestibular aqueduct syndrome.ChapterⅡ:we use testing kit with PAGE silver staining to detect mt DNA A1555G mutation and conclude PAGE silver staining methods was more sensitive than ethidium bromide staining.ChapterⅢ:we perform the detection of GJB2 235delC heterozygote and mt DNA A1555G by PCR-Genescan technique.These techniques,based on DNA amplification by PCR, are relatively simple,and the testing costs are acceptable,leading to their adoption by many laboratories.Moreover,these systems may detect single or several mutations by simultaneous and preferential use and are highly sensitive and specific.Part two:the prevalence of genes mutations for hearing loss patients of different ethnic origins in XinjiangXinjiang Uigur Municipality is located in northwest China and is the largest province in China.The Han and Uigur are the two dominating ethnic groups in the province.Genetic testing include GJB2,SLC26A4 IVS7-2A＞G and mt DNA A1555G referred to the Molecular Otolaryngology Research Laboratories to establish a diagnosis of deafness.ChapterⅠ:Exon 2 of GJB2 was screened for coding sequence allele variants by direct sequencing on 127 NSHL patients(61Uigurs and 66Hans).We found Uigur population GJB2 mutations had a similar carrier frequency with Han population.GJB2 35delG was found only in Uigur deaf groups but not Han,whereas 235delC was identified in both Uigur and Han deaf groups.Allelic Frequency of the 35delG,235delC mutation in Uigur,Han deaf students were 7.4%(9/122),0(0/128)and 5.7%,8.6%,respectively. ChapterⅡ:RFLP showed the carder frequency of mt DNA A1555G mutation were 10.24%(21/205)in Xinjiang and 6.67%(5/75)in Binzhou,Shandong.Mt DNA A1555G has a higher carrier frequency in underdeveloped regions.ChapterⅢ:25 of 205 were found to carry SLC26A4 IVS7-2 A＞G mutation in Han group and none in Uigur.Molecular epidemiology of different nations may help us establish diverse diagnostic strategies in different region of China.As a consequence,it will improve the current diagnosis and therapeutical option.Part three:Molecular epidemiology of SLV26A4 IVS7-2A＞G mutation and its significance in diagnosis related to temporal bone malformation and non-syndromic hearing lossThe SLC26A4 IVS7-2 A＞G mutation was most frequently found in Chinese deaf groups.In present study,we carry out a molecular epidemiology study of SLC26A4 IVS7-2 A＞G mutation in a large Chinese deaf population and to provide evidence for fast screening and gene diagnosis of enlarged vestibular aqueduct syndrome(EVAS). A total of 3271 patients with non-syndromic hearing loss(NSHL) underwent RFLP and direct sequencing for IVS7-2A＞G mutation detection of SLC26A4 gene.245 patients(12.38%)with homozygotous and heterozygotous IVS7-2 A＞G mutation were found among the 3271 NSHL It showed statistically significant difference in regions and nations.We found a high SLC26A4 IVS7-2 A＞G mutation frequency for deafness in Chinese patients.Detection of this pathogenic mutation is bringing the possibility to detect EVAS at an early stage.We investigated the occurrence of two different PDS mutations in 80 IVS7-2A＞G heterozygotes patients.47/80 patients were found to be compound heterozygotes,whereas a single mutation could be found to be responsible for NSHL in 37 patients.The five most common mutations,including H723R,had a frequency 20%.Of the remaining 3 mutations T410M,15+5G＞A和L676Q with a frequency 11%,the N392Y had frequency 3.75%.Exon17 is frequently found in compound heterozygosity with a IVS7-2A＞G mutation.Our study illustrates that If only a single heterozygous SLC26A4 mutation is identified,several explanations should be considered.They include a second,unidentified mutation in regulatory or noncoding regions or another cause for the phenotype.Part four:Molecular epidemiology of mt DNA C1494T mutation associated with non-syndromic hearing lossMtDNAC1494T mutation is a pathogenic mutation associated with aminoglycoside-induced hearing loss.A systematic and extended mtDNA C1494T mutation screening from Deaf and Dumb school of different area in China has been completed by FQ-PCR and sequencing.In result,we found fourteen patients with mtDNA C1494T mutation,giving a carrier rate of 0.45%.The C1494T mutation thus is a rare mutation in our patients. Part five:Phenotypic analysis for GJB2 235delC heterozygotesGenotype-phenotype correlations of GJB2 gene is a multifaceted condition with pathogenesis of deafness,gene diagnosis and genetic counseling ramifications.In this study,we showed a phenotypic picture for GJB2 heterozygotes carrying one 235delc allele.We demonstrated that the hearing of carriers is impaired principally at 4,000 and 8,000 Hz frequencies with age.In fact,carriers might be hypothesized to be a risk group in whom hearing thresholds may deteriorate more frequently than in non-carriers.