Study And Application Of New Diagnostic Techniques On Genetic Deafness Genes And Molecular Genetic Study On A Family With Nonsyndromic Deafness

As the results of the national epidemiological for deafness show, hereditary hearing loss is extremely heterogeneous. Most of non-syndromic hereditary hearing losses, nevertheless, are found out to result from mutations of few genes, such as GJB2,mtDNA,SLC26A4,et al. This fact grounds the establishment of genetic testing and prenatal diagnosis for hearing loss in China. Our study is dedicated to found economic, high-effective, rapid and high-flux technologies for genetic testing of deafness, and standardize and normalize the contents and processes of genetic testing, consulting and prenatal diagnosis, to make the accomplishment of human genome project widely available in diagnosis for deafness and reduction of deaf baby, and to eventually promote genetic testing, consulting and prenatal diagnosis in China. Another role of our study is to investigate the clinical character and molecular genetic mechanism of a large family inherited as X-linked or maternally type of nonsyndromic deafness, which supply foundation for function study of genetic deafness genes. We carried out our research as the following three parts: Part one:Accurate and rapid diagnosis of common NSHL gene mutations by a multiplex primer extension and denaturing high-performance liquid chromatography techniqueObjectives:Nonsyndromic hearing loss (NSHL) is a common inherited disorder of the nervous system worldwide, and rapid and accurate genotyping methodologies for specific NSHL-causing gene mutations are needed for the diagnosis of the disease in different ethnic populations. Methods:In this study, we performed a novel multiplex primer extension (PE) reaction in combination with denaturing high-performance liquid chromatography (DHPLC) to simultaneously detect and genotype the 6 most common mutations in NSHL (GJB2-235delC, GJB2-299delAT, PDS-A2168G, PDS IVS7-2A>G, mtDNA-A1555G, and mtDNA-C1494T) in the Chinese population. This method involved the amplification of the target sequence, followed by a purification step, a multiplex PE reaction, and DHPLC analysis performed on the Transgenomic Wave DNA fragment analysis system under fully-denaturing conditions. Results:In a contrast analysis, this technique successfully and accurately genotyped 100%(120/120) of the samples previously detected by direct sequencing. For the four deafness genealogy for genetic consultion, this technique also successfully and accurately genotyped 100% of the members simutaniously detected by sequencing. Conclusion:The present study validated the combined PE/DHPLC approach as a simple, rapid, highly accurate, and cost-effective approach for genotyping common disease-causing mutations, and it strongly suggests that this technique may be successfully used in other genetic diseases.Part two:Application of DNA Chips in Clinical Genetic Testing for Sopratic genetic DeafnessChapter I:Feasibility Investigation of DNA Chips Applied in Clinical Genetic Testing for Sopratic genetic DeafnessObjectives:To investigate the feasibility of DNA Chips, and to found a new genetic detecting technology, we use the Illumina Goldengate gene chips in this part. Methods:In order to test the feasibility of DNA Chips, we detected the GJB2₂35delC mutation in 122 sporadic NSHL individuals through DNA Microarray and DNA sequencing at the same time. Results:The results get from the DNA Chips is the same as the traditional methods of sequencing. The detecting rate is 100%. Conclusion:Illumina GoldenGate DNA Chips appears to have some inherent advantages in genetic diagnosis of NSHL, such as low time consuming, high performance and accuracy, which make it fit to be used in clinic practice.Chapter II:Clinical Application of DNA Chips in Rapid Genetic Testing of Sopratic Non-Syndromic Hearing Loss Objectives:To detect the hot genes in sporadic NSHL patients and the association analysis between these genes and genetic deafness. Methods:389 sporadic patients were collected and detected by Illumina GoldenGate DNA Chips. Results:WFS1_Leu829Pro, GJB3_Arg32Trp, OTOF_IVS5+1, TMIE_IVS2₂, PCDH15_3BP_DEL₅601₅603aac and SLC26A4_Gly497Ser appeared negative correlation with NSHL; While rs480926?rs7421943?MYH14_Arg726Ser?TMPRSS3_PRO404LEU?rs878042?COCH MET512THR?MY03A IVS7 2?TMC1_Tyr259Cys?GJB2_Arg165Trp?rs10515535?rs568619. rs7600176?rs3664?WHRN(DFNB31)_IVS2+?rs4679155?COL9A3_9BP_DEL₅41₅49ggtccccca appeared positive correlation with NSHL Conclusion:The Illumina GoldenGate DNA Chips dependent new technique could be well applied for mutation detecting of deafness genes and confirmed the high genetic heterogeneity of genetic deafness.Part three:Special penetrance and analysis of hearing loss in a five-generation Chinese family with the mitochondrial 12S rRNA A1555G mutationObjectives:Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. However, the variable clinical phenotype and incomplete penetrance of mtDNA 1555A>G-induced hearing loss complicate our understanding of this mutation Methods:Here, we performed clinical, genetic, and X-linkage analysis of a 5-generation Chinese family in which all the affected individuals were male. Results:Using X-linkage analysis and mtDNA sequencing, we detected an identical homoplasmic 1555A>G mutation in 9 individuals, and a previously unreported variant 14163C>T in mtDNA. Conclusions:Analysis of the complete mtDNA genome and x-linkage of this family revealed that the 1555A>G may lead to deafness. The 14163C>T is a never-reported variant and may synergism the development of the deafness.