Molecular Mechanism Of Late-onset Nonsyndromic Hearing Loss And Age-related, Aminoglycoside-induced Hearing Loss

Hearing impairment is the most common perception disorder in the world. It is caused by both environmental factors and genetic factors.The hearing loss attributable to genetic factors include not only single gene genetic deafness(such as non-syndromic hearing loss),but also multi-gene genetic deafness(such as age-related heating loss),as well as mitochondrion-related deafness(such as aminoglycoside-induced hearing loss).The research of over all aspects of genetic hearing loss has provided a solid foundation for prevention and treatment of hearing loss.With the gradual completion of the Human Genome Project(Human genome project,HGP),implementation and constant improvement of the International Human Genome HapMap Project(HapMap Project),in the post-genomic era with the rapid development of the genetics,the research of the genetic hearing loss is faced with unprecedented opportunities and challenges.In recent years,the third generation of genetic markers,single nucleotide polymorphisms(SNPs),which came into being because of demand with the development of modern molecular genetics technology platform,were applied in many fields and played an important role in many aspects.It will be the new concerns and fashion trends to apply the SNP in the mapping and cloning pathogenic gene or susceptibility gene of genetic heating loss.In our research,by taking single nucleotide polymorphisms as the genetic marker,we successfully mapped and identified a novel heating loss locus and gene in an autosomal dominant non-syndromic deafness family,completed the screening of common deafness gene GJB2,12Sr RNA and GSTT1/GSTM1 for the susceptibility of presbyacusis,and traced the origin of mitochondrial 12SrRNA C 1494T mutation.Part 1:Mapping,screening and identification of pathogenic gene in one autosomal-dominant nonsyndromic hearing loss familyIn this study,by taking both SNP and STR as the genetic markers,and making use of the linkage analysis,we successfully mapped,screened and identified the pathogenic gene in one autosomal-dominant nonsyndromic hearing loss family step by step.Family HN-W078 was a large Chinese family with hearing loss transmitted through 5 generations.The clinical evaluation of audiology revealed that the hearing loss was late-onset and gradual sensorineural hearing loss which first developed in high frequencies.First of all,we mapped the locus on chromosome 12q24.31-q24.32 region(two-point lod-score of 5.656) with application of the Affymetrix 5.0 SNP Genechip,and then by refined mapping and analysis of haplotype,we mapped the same locus to the region between D12S86 and D 12S 1612(two-point lod-score of 5.35) with application of 16 microsatellite markers.By direct sequencing of candidate genes in mapping region,we found a missense mutation c.377C＞T(p.S126L) in EXON4 of DFNA60 gene,which was totally segregated with the disease phenotype.We confirmed that only the patients in HN-W078 carried this mutation by failing in detecting this mutation in 500 normal subjects.With the method of immunohistochemistry,we found out that the DFNA60 widely expressed in the cochlear,including the organ of Corti,stria vascularis,and spiral ligament,and so on.It had laid the foundation for pathogenic mechanism study of deafness caused by DFNA60 gene mutation.(The locus and gene is novel and has not been published anywhere,it is represented by DFNA60 temporarily).Part 2:Genetic screening for susceptibility genes of presbyacusisPresbyacusis,also refers to age-related hearing loss,is gradual hearing loss with age.With the environmental impact of modern life,the process of aging is being in advance,no exception of the hearing organ.In our study,662 elder people with different age(40-90years old) and different hearing threshold levels through census were collected.According to audiological data,all elder were divided into four groups.Through the direct sequencing and multi-polymerase chain reaction,we had investigated the relationship of GJB2,12S rRNA and GSTT1/GSTM1 genes and susceptibility of presbyacusis and initially explored the hereditary agent of susceptibility for presbyacusis.The findings indicated that 109G＞A and 608T＞C heterozygotic mutations of GJB2 gene,and homoplasmy A827G mutation of mitochondrial 12S rRNA may increase susceptibility of presbyacusis.No relationship was deteced between GSTT1/GSTM1 gene and presbyacusis.In summary,this study not only collected the large scale of subjects with presbyacusis for large-scale,multi-center,multi-disciplinary joint research of presbyacusis' genetic susceptibility,but also provided foundation of analysis for relationship of common deafness genes and genetic susceptibility of presbyacusis.Part 3:Genetics and origin analysis of mitochondrial 12S rRNA C1494T mutationMitochondrial 12S rRNA has been shown to be the hot spot for mutations associated with both aminoglycoside-induced and nonsyndromic hearing loss.The 12S rRNA A1555G and C1494T mutations in the highly conserved A-site of the 12SrRNA have been associated with both aminoglycoside-induced and nonsyndromic hearing loss in many families worldwid.Previous investigations revealed the highly variable penetrance and expressivity of hearing loss in 8 Han Chinese families carrying the C1494T mutation.Matrilineal relatives within and among families carrying the C1494T mutation exhibited a wide range of penetrance,severity,and age-of-onset in hearing loss.These results indicated that the mtDNA mutation 12S rRNA C1494T itself is insufficient to produce the clinical phenotype.Therefore,other modifiers including aminoglycosides,nuclear modifier genes,second mutation,and mitochondrial haplotypes may modulate the phenotypic manifestations of the C1494T mutation.In this study,we reported the clinical,genetic,and molecular characteristics of 13 Han Chinese families with aminoglycoside-induced and nonsyndromic bilateral hearing loss collected by screening of nationwide molecular epidemic.Through the whole mitochondrial genome DNA sequence analysis,we precluded the possibility that the second mutations might significantly modify the manifestation of the C1494T mutation. Through analysis of haplotypes of the probands,13 families come from 7 different provience were classified into 10 haplogroups by the distinct sets of mtDNA polymorphisms belonged to different haplogroups,including haplogroups A,B,D, D4,D4b2,F1,M,M7c,N9al,and H2b.This result suggested that the C1494T mutation occurred sporadically and might be multi-origin mutation through evolution of the mtDNA in China,and these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the C1494T mutation in these Chinese families with different penetrance of hearing loss. Therefore,aminoglycoside-antibiotics is solo well-established factor to contribute to the deafness expression of the C 1494T mutation.So prevention by avoiding the administration of aminoglycoside in individuals carrying C1494T mutation is the most effective way to protect their vulnerable hearing,and a systematic and extended mutational screening of the 12S rRNA C1494T mutation not only for hearing loss patients but also for normal Chinese subjects is necessary before the use of aminoglycosides.