Optimization And Application Of Genetic Diagnostic System For Hereditary Nonsyndromic Hearing Loss

Backgrounds:Heating loss is one of the most common sensory disorders.Profound heating loss occurs in 1～3:1000 neonates and the cause is hereditary in about half.Non-syndromic hearing loss(NSHL) is highly genetic heterogeneous,in which there are no additional abnormalities(more than 70%).Up to April 2009,137 NSHL gene loci have been mapped,and 48 genes have been identified.Of these GJB2 is the most common NSHL associated gene,which accounts for about 50% hereditary prelingual NSHL among Caucasian population.SLC26A4 comes next,accounts for 5%～10%.Mitochondrial DNA mutations account for about 1%.Other types of NSHL are very rare. The first section:Mutations screening of SLC26A4 gene in patients with nonsyndromic hearing loss by Denaturing High-Performance Liquid ChromatorgraphyPurpose:To study SLC26A4 gene mutations in the patients with nonsyndromic hearing loss and provide the clinical guidance of genetic diagnosis.Methodes:PCR and Denaturing High-Performance Liquid Chromatorgraphy(DHPLC) were used to screen the 21 exons and their flanking regions of SLC26A4 gene.Samples with abnormal DHPLC waveforms were sequenced to identify the variations.Results:Among the 30 unrelated NSHL patients without deafness-causing mutations of GJB2 are identified,10 types of variations were detected,including 7 known mutations,2 novel mutations(F572L and D87Y),and 1 known polymorphism(Ivs11+ 47T>C).Ivs7-2A>G is the most common type of variation,accounting for 40%of all the mutations.Conclusion:Mutations screening of SLC26A4 gene in patients with nonsyndromic hearing loss by Denaturing High-Performance Liquid Chromatorgraphy is low and easy.SLC26A4 mutation is a major cause of NSHL,just next to GJB2.Among the NSHL patients without deafness-causing mutations of GJB2,the SLC26A4 detection rate is 23.3%and Ivs7-2A>G is the most common mutation.The second section:Fast detection hot spots of patients with nonsyndromic hearing loss mutations of china by PCR-RFLPPurpose:To develop a quick genetic diagnosis for the patients with hereditary hearing loss by screening hot spots of mutations,namely 235delC of GJB2,IVS7-2A>G of SLC26A4 and 1555A>G of mitochondrial 12S rRNA.Methodes:Multiple PCR amplification of the three fragments covering the expected mutations in GJB2,SLC26A4 and 12S and analysis by restrictive fragment length polymorphism(RFLP).If needed,The following approach is associating DHPLC with sequencing.Results:Eighteen homozygous and 18 heterozygous 235delC,2 homozygous and 13 heterozygousⅣS7-2A>G,and 8 homogeneous 1555A>G were detected in 200 hearing loss patients.All the results were confirmed by sequencing.The detection rate of the three mutant alleles was 21.7%(71/400+8/200=0.217) and the genetic diagnosis rate was 14%((18+2+8)/200=0.14).After applying DHPLC and sequencing,the total genetic diagnosis rate was 18.5%(37/200)Conclusion:It is a convenient,efficient and economical method to screen the hot spots of mutation in the patient with hereditary hearing loss by using PCR-RFLP.