Molecular Epidemiological Investigation Of Common Deafness Genes In Hearing Loss In Xinjiang And The Prenatal Diagnosis And Assessment Of Functinal Assessment Of Mutation Of PAX3 Associated With WS

Objective:To assessment the molecular epidemiology of GJB2,SLC26A4,mt DNA 、GJB3 gene in Nonsyndromic hearing loss in Xinjiang by using the deafness gene kit.To assessment of functinal of mutation in PAX3 associated with one case WS1;SNPscan method for 22 cases of deafness in small families deafness disease gene identification,and 2 cases of GJB2 gene,SLC26A4 proband family prenatal diagnosis practice.Methods:1)To investigate molecular epidemiological of GJB2,SLC26A4,mitochondrial 12 SrRNA gene mutations in non-syndromic severe and sensorineural deafness patients in Urumqi,Changji,Kashi,Kuche,Kashgar,Moyu and Aksu,Xinjiang,we detected them with DNA microarray which is able to perform mutation of 10 hot-spot mutations in four most common pathologic genes,including GJB2(c.235 delC,35del G,176del16 db,299-300delAT),SLC26A4(IVS7-2,2168 A>G),GJB3(c.538C>T,547G>A),mitochondrial 12SrRNA(m.1555A>G,1494C>T)simultaneously;2)To further study the molecular mechanism of WS,we performed aseries of in vitro studies on the one mutation of the PAX3 gene,including generation of eukaryotic expression plasmids of mutation gene,Western blot assays to detect mutant proteins expression,luciferase activity assays to detect mutant proteins activities,immunofluorescence assays to observe mutant proteins subcellular localization,co-immunprecipitation assays to detect interaction between mutant proteins and wild type(WT)proteins as well as mutant protein binding to DNA,in order to understand functional consequences of these mutations and its impact on WT proteins at the level of transcription and Opost-transcription;3)With the SNPscan technology to diagnose the patient in 22 simple families and performe prenatal diagnosis,including 115 mutations of GJB2,SLC26A4,mt DNA.Results:1)Of 3147 patients,530 were found out to carriers of less than one pathogeneic gene mutation.Among them,7.31% have GJB2 mutations,4.61% have SLC26A4 mutations,5.34% have 12 SrRNA mutations;2)The mutant of PAX3,p.G24 fs was loss of function and was not able to transactivate the transcriptional activities of target genes in Luciferase activity assays.The mutant failed to affect the activities of WT proteins in a dose-depandent manner.In immunofluorescence assays,the mutant was localized both in the nucleus and cytoplasm in NIH3T3 cells.3)Of 22 deafness families,3 were found carriers GJB2 mutations,6 have SLC26A4 mutations;2 cases of deafness patients had prenatal diagnosis with amniotic fluid.Conclusion:1)The GJB2 gene,SLC26A4 and mitochondrial gene 12 SrRNA are common pathogeneses of non-syndromic hearing loss in Xinjiang.There are other pathogenic genes in heterozygous mutant deafness patients;2)This study confirmed that PAX3 p.G24 fs mutation can not effectively activate MITF transcription;3)The prevention of deafness can be performed by prenatal diagnosis.