Study On The Etiological Factors,Molecular Pathogenesis And Racial Diversity Of Hearing Loss In Profound Hearing Loss Children

Part I Preoperative evaluation of cochlear implant and etiological study on deaf children with profound hearing lossObjective To preoperative evaluate comprehensively in prelingually deaf children with cochlear implant to ensure the correct indications and good rehabilitative effects of implant. To study the etiologic factors in deaf children applying for cochlear implant, in search of a better way of early detecting the severe deafness.Method Retrospective analyses with preoperative tests were performed in386children applying for cochlear implant. According to the cochlear implant guide and the postoperative analyses with effect of auditory rehabilitation, high-risk factors were studied by a high-risk scale for the parents. Stepwise logistic regression was used to identify variables associated with childhood hearing loss.Result The diagnosis of profound hearing loss was late and Neonatal Audition Screening was not effectively implemented. Hearing aid was not popularization enough. There were162risk factors founded in114cases, including11prenatal factors,88perinatal factors,45postnatal factors and21inheritances. Malformations of inner ear were proved to be a significant high risk for profound deafness in Hunan.Conclusions The preoperative measures, including imaging examination, auditory analysis and otoscopy were significant to the success of operation, the avoidance of complications and the effect of auditory rehabilitations. It is necessary to conduct health education among public, with a view to improving their sense of health and the ability of self-health care, avoided some drugs for their harmful aftereffects. Diagnosis, acoustion and language recovery in early stage played major roles of social communication of deaf children. Part II Study of the molecular epidemiology and pathogenesis of hearing loss in CI recipients or applicants with profound hearing lossObjective Our objective is to analyze the molecular pathogenesis of deafness in CI recipients or applicants and study the genotype and molecular epidemiologic characteristic of severe to profound deafness with Enlarged vestibular aqueduct syndrome (EVAS).Methods236CI recipients or applicant,30cases of EVAS from our department and100control individuals with normal hearing were included in this study. The molecule pathogenesis was analyzed with the DNA microarray which is able to perform mutation detection of9hot-spot mutations in three most common genes, including GJB2(35delG,176del16bp,235de1C and299delAT),GJB3(538C>T),SLC26A4(IVS7-2A>G,2168A>G) and mtDNA12SrRNA(1494C>T及1555A>G). Meanwhile, the results were confirmed with the traditional methods of DNA sequencing. We stopped the examining process if mutations homozygous or composite heterozygous were detected through DNA microarray, or examined the rest exons until a gene mutation was found if no mutation or only pure heterozygosity was detected.Results Among236CI recipients or applicants,93cases were found to have GJB2mutation, and especially40.55%(88/217) of the nonsyndromic deafness patents, only13.16%(5/38) inner ear malfoanatnn patient was detected with GJB2mutations. Two novel mutation, GJB2235de1C/511-512insAACGC and109G>A/52A>C was identified in this study.7patients were found to carry mtDNA A1555G mutation and one found to carry1494C>T among236CI recipients or applicant.21cases were found to have SLC26A4mutation Among236CI recipients or applicant, while among30patients with large vestibular aqueduct,28cases were found to have mutation. A total of16mutations were identified in the study, including5novel mutations(A227P,G368X,IVS8-1G＞T IVS13+9C＞T and Q696X). In all mutations, IVS7-2A>G was the most common mutation; H723R and T410M were another common mutations in Chinese. The GJB3(538C＞T) was the only gene without detecting any mutation.Conclusions DNA microarray can be used to generally screen hot site mutation of common genes in CI recipients or applicants with severe to profound hearing loss. But DNA sequencing is necessary if no mutation or only heterozygosity mutation was detected by DNA microarray. Objective Genetic deafness is highly genetic heterogeneity, and the rate of mutation frequency of the deafness gene is great difference in different area and crowd. GJB2, SLC26A4and DNA12SrRNA have the high mutation frequency in population from different ethnic origins. In recent years, the research about the prevalence of these common deafness genes in Chinese Minority Groups population had the progress and the discovery, but these studies were mainly made in the Chinese Minority Groups population of northwest China. So we study the prevalence of these common deafness genes mutations in the patients in Hunan where occupied by the Miao and Tujia ethnic minority. Our objective is to study Molecular epidemiology of GJB2, SLC26A4and DNA12SrRNA mutations in deafness patients from Miao and Tujia ethnic minority population and Han ethnic origin in Hunan. Methods we detected mutations in gene GJB2, SLC26A4and DNA12SrRNA by the method of DNA micro-array and sequencing, to study the difference in the rate of mutation frequency between Miao and Tujia ethnic minority population and Han ethnic origin.Results GJB2mutations were detected in three nationalities but either of them has a rather high mutation rate of GJB2.235de1C was the most common mutation in three groups. The highest carrier frequency of235de1C is22.81%(13/57) in Miao patients, and the lowest is3.8%in Tujia patients. V37I mutations were found in three groups, and the carrier frequency is high. Novel mutations were found in Tujia patients. The homoplasmic mtDNA A1555G mutation was found in Miao and Tujia patients, and the carrier frequency of A1555G was23.68%(9/38),13.04%(6/46), respectively, which were insignificantly higher than those domestic and international.2Tujia patients and1Han patient were found to carry mtDNA1494C＞T mutation in this study. The IVS7-2A>G mutation of SLC26A4were detected in three groups, and there is no different in the carrier frequency between three groups. None case of2168A>G (H723R) was detected in this study. IVS7-2A>G was the common mutation of SLC26A4in Han patients, and more comprehensive examination of SLC26A4mutations were needed to undergo, and the mutations of deafness gene were detected in more samples in order to determine the characteristic of the molecular epidemiology of deafness genes in northwest China.Conclusions The characteristics of gene mutations of Miao and Tujia patients were basically consistent with one of Han group, but a few differences, reflecting the genetic heterogeneity and national characteristics. Part IV Preliminary study of the relationship between nuclear genes GJB2and drug-induced deafnessChapter1Connexin26Gene (GJB2) Mutation Modulates the Severity of Hearing Loss Associated With the1555A>G Mitochondrial MutationObjective Megadoses of aminoglycosides have been long recognized as ototoxic, but even therapeutic doses of aminoglycosides in1555A>G patients can cause unexpected and permanent hearing loss. Although there are common features among patients, the severity of hearing loss varies greatly. This suggests that other factors, such as nuclear genes, may play a synergistic role in aggravating the hearing loss. Mutations in the GJB2gene have been of particular interest because they have been found to account for about50%of all recessive hearing loss. We had the opportunity to screen for GJB2mutations in21independent Japanese families bearing the1555A>G mutation in which affected members had severe hearing loss.Methods And Results We report a high prevalence of GJB2heterozygous mutations in patients bearing the1555A>G mitochondrial mutation, and describe a family in which potential interaction between GJB2and a mitochondrial gene appears to be the cause of hearing impairment. Patients who are heterozygous for the GJB2mutant allele show hearing loss more severe than that seen in sibs lacking a mutant GJB2allele, suggesting that heterozygous GJB2mutations may synergistically cause hearing loss when in the presence of a1555A>G mutation.Conclusions The present findings indicate that GJB2mutations may sometimes be an aggravating factor, in addition to aminoglycoside antibiotics, in the phenotypic expression of the non-syndromic hearing loss associated with the1555A>G mitochondrial mutation. Chapter2Acute effects of gentamicin on the expression of connexin26in the cochlear lateral wallObjectives/Hypothesis:Aminoglycosides may decrease the expression of some proteins participating in ion-exchange in the lateral wall. Connexin26in cochlear lateral wall may play a role in acquired hearing loss by maintaining endocochlear potential and potassium concentration in endolymph. The effects of aminoglycosides on the expression of connexin26are poorly understood.Methods:We detect changes of connexin26expression in the cochlea lateral wall in protein levels and mRNA levels by means of immunohistochemistry staining, Western blotting and real-time PCR, in the rats after the administration of a single dose of gentamicin.Results:The expression of connexin26was increased overtime in type III fibrocytes after gentamicin administration. The elevated protein level was detected3h after single injection of gentamicin, but the increased mRNA level was only observed after24h.Conclusion:The systematically administration of a single dose of gentamicin can influence distribution and expression of Cx26protein rapidly. We concluded that connexin26played an important role in the acute effect of high-dose gentamicin and probably involved in the pathogenesis of ototoxic deafness. The Cx26protein levels increased transiently but did not accompany the mRNA levels. The Cx26mRNA also increased but failed to translate to protein finally, which suggests an unclear regulation in posttranscriptional level and needs further study.