| OBJECTIVE: Hereditary hearing loss is genetically heterogeneous.This study used targeted next generation sequencing technology to systematically detect the rare deafness-causing genes in different types of hearing loss populations,ranging from non-syndromic hearing loss sporadic cases,dominant families to syndromic hearing loss family.Meanwhile,various research means were used to improve the workflow of data analysis of rare deafness-causing gene mutations and enhance the accuracy of results and clinical application values,including parental genotyping,intra-familial co-segregation,gene function investigation and establishment of benign polymorphism databases.METHODS: Based on excluding GJB2,SLC26A4 and MT-RNR1 common deafness-causing genes by Sanger sequencing,the targeted next generation sequencing was used to detect the rare deafness-causing gene mutations in non-syndromic hearing loss sporadic cases(44 cases),dominant families(9 cases)and syndromic hearing loss family(1 case).Meanwhile,to verify the candidate mutations by parental genotyping,and intra-familial co-segregation respectively;To investigate the gene function,audiology and inner ear pathology of transgenic mice by establishing the mutant yeast and transgenic mice models of rare deafness-causing gene KARS;To establish the database of benign polymorphisms of recessive deafness-causing genes by re-sequencing corresponding genes of normal carrier(29 cases)with known pathogenic mutations.RESULTS:(1)18 out of 44 sporadic cases with non-syndromic hearing loss carried the candidate allelic mutations of autosomal recessive genes and the mutations in 4 cases were false positive results by parental genotyping.Moreover,the candidate mutations of autosomal dominant genes in 16 cases were also ruled out as de novo by parental genotyping;(2)Two mutations p.L311 P and c.120+1G>C of gene POU4F3 were found in 2 out of 9 autosomal dominant non-syndromic hearing loss(ADNSHL)families respectively,confirmed by intra-familial genotype-phenotype co-segregation.Combined with previous studies,POU4F3 mutations caused ADNSHL with variability in the onset age and degree;(3)One syndromic hearing loss family with leukoencephalopathy carried the compound heterozygous mutations p.R477 H and P505 S of gene KARS.Yeast genetic complement experiments showed p.R477 H and p.P505 S mutant products could rescue the defect type krs1 homologous genes.However,compared with wild type mice,the mutant mice with three mutant genotypes(p.R477 H homozygous mutations,p.P505 S homozygous mutations and compound heterozygous mutations)showed remarkable hearing loss.At the age of 8 weeks,cochlear fluorescence staining showed the outer hair cells in basilar membrane bottom(high frequency)of mutant mice reduced in different extents.(4)By re-sequencing the corresponding genes of 29 cases with known pathogenic mutations in autosomal recessive deafness-causing genes,a total of 31 non-synonymous variants were found in the alleles of a known pathogenic mutation and judged as benign polymorphism,including four rare missense variants in controversial or false results judged by previously reports or universal means of next generation sequencing.CONCLUSIONS:(1)In the molecular etiology of sporadic non-syndromic hearing loss populations,mutations in rare autosomal recessive deafness-causing genes account for a prominent role,up to 32%(14/44).In general,mutations in rare dominant deafness-causing genes are not the main genetic etiology;(2)Mutation of gene POU4F3 is a relatively common cause in Chinese Han autosomal dominant non-syndromic hearing loss populations(18%,3/16)and can result in various hearing impairments;(3)KARS gene mutations p.R477H/p.P505 S can lead to hearing loss in both human and transgenic mice,possibly associated with outer hair cell loss.(4)Re-sequencing the corresponding genes of carriers,with known pathogenic mutations in autosomal recessive deafness-causing genes,can establish a database of benign polymorphisms to reduce false positive results. |
PDF Full Text Request