Mutation Analysis Of Hereditary Deafness Family Deafness Genes And Phenotypic Prediction Of Related NsSNPs

Part one: Genetic Analysis for the Predigrees with Hereditary Hearing LossObjective: To investigate the pathogenicity and the molecular mechanism of hearing loss in three affected Chinese pedigrees?JSNY-021,JSNY-022 and JSNY-058?.Methods: For identifying the candidate genes,first,we analyzed the DNA of the three probands from three Chinese pedigrees?JSNY-021,JSNY-022 and JSNY-058?with DNA microarray which is able to perform mutation detection of 9 hot-spot mutations in four most common pathologic genes,including GJB2?35del G,176del16 bp,235del C and 299-300 del AT?,GJB3?C538T?,SLC26A4?IVS7-2A>G,A2168G?and mitochondrial 12 S r RNA?A1555G,C1494T?simultaneously.If the result is negative,we develop a method for the simultaneous detection of 104 genes associated with hereditary hearing loss and 3 micro RNA regions using massively parallel sequencing?MPS?.Subsquently,Sanger sequencing and PCR were used on other members of the three families.To investigate the the molecular mechanism of these three predigrees,we performed conservation analysis with Clustal X2,pathogenic analysis with SIFT,PROVEAN and Mutation taster and molecular dynamics simulation with Amber12 and Amber Tools13.Results: 1)With the DNA microarray,the common mutation IVS7-2A>G of SLC26A4 was identified from the proband of the family JSNY-058,and a rare mutation c.2167C>G of SLC26A4 was obtained by the Sanger sequencing to the coding sequence and splice sites of SLC26A4.2)Using molecular dynamics simulation,we found that the stability of the backbone atoms of the mutant protein c.2167C>G?p.H723D?were reduced as compared with the wild type.3)The genetic diagnostic kit of hereditary deafness based on DNA microarray to the family JSNY-021 and the family JSNY-022 were negative,and then we used the targeted genomic capture and MPS,finally we get the novel candidate deaf-causative mutations c.113G>A?p.G38D?of COCH gene from the proband of the family JSNY-021 and c.2036₂038del AGG?p.E680del?of WFS1 obtained from the proband of the family JSNY-022;4)Genetic analysis revealed that the mutations were cosegregated in the three pedigrees.5)The animo acids encoded by three mutations were all highly conserved and pathogenic by bioinformatic analysis.Conclusions: In our research,combining the targeted genomic capture,massively parallel sequencing and DNA microarray,we identified three candidate causative mutations of COCH,WFS1 and SLC26A4 which enriched the pathogenic mutations of the deafness-related genes.And we predicted the molecular mechanism by bioinformatic analysis which was helpful to the screening of the hereditary heaing loss.Part two: Phenotype Prediction of Pathogenic Nonsynonymous Single Nucleotide Polymorphisms in COCH and WFS1Objective: To study the relationship between SNP genotype and phenotype in other ns SNPs of the COCH and WFS1 gene.Methods: We collected SNPs of COCH and WFS1 gene from the db SNP,HGMD,Deafness Variation databases,and used multiple softwares and tools?including SIFT,Poly Phen-2,Mut Pred,Ph D-SNP,Swiss Model and TMHMM?to filter the SNPs step by step.For verifying the accuracy of the prediction results,we inspected the reference sources and the structural domain of COCH and WFS1 gene,and constructed the structures of colchlin and wolframin.Results: 1)After fitering,we finally identified 8?I176T,R180 Q,G265E,V269 L,I368N,I372 T,R416C and Y424D?and 20?P607L,R732 C,G736D,C742 R,F439C,R375 H,S353C,R832 C,R868C,R732 H,F329I,R375 C,L594R,A874 T,Y739D,R859 W,E394K,S662 P,R517P and T665I?pathogenic ns SNPs,respectively;2)The eight payhogenic ns SNPs of COCH gene were all located in the v WFA domain of cochlin;3)By constructing the transmembrane domain of the walframin,we found most predicted ns SNPs in our result were in the C-terminal domain.Conclusions: With multiple bioinformatic tools,the prediction of pathogenic mutations for the ns SNPs of the COCH and WFS1 gene will provide a blueprint for screening pathogenic mutations,and it will be beneficial to the research of genotype and the phenotype of the ns SNPs in COCH gene and WFS1 gene as well as their functional research.