【作者】 赖若沙；

【导师】 谢鼎华；

【作者基本信息】 中南大学 ， 耳鼻咽喉头颈外科学， 2011， 博士

【摘要】 第一部分极重度感音神经性聋儿童的人工耳蜗植入术前评估及致聋危险因素分析研究目的：对极重度感音神经性聋患儿进行术前全面评估以确保人工耳蜗植入适应证的正确选择及其术后康复效果；了解环境和遗传因素在我国接受人工耳蜗植入群体中的比例。初步探讨湖南地区极重度感音神经性聋患儿的病因,为制定耳聋的预防计划提供理论依据。研究方法：本研究对2005年11月至2011年4月到我院及湖南郴州湘南学院申请行人工耳蜗植入的386例极重度感音神经性耳聋患者进行了回顾性调查,并进行系统的全身体格检查,听力学检查及影像学检查等,进行术前全面评估。对已知的耳聋的危险因素在聋儿的分布情况进行初步的探讨,并对较常见的一些因素进行多因素分析。研究结果：1、我们的研究对象确诊时间较晚,聋儿的平均疑诊时间接近2岁左右,确诊时间接近3岁。本研究中,386例耳聋儿童均存在言语障碍,仅有1例为语后聋。经听力筛查发现仅43例,因家人发现小儿听力差而就诊343例,说明尚有较多小儿未接受听力筛查。在接受人工耳蜗植入术前,386例聋儿中有231例验配助听器。2、386例聋儿含已知的危险因素共162人次,涉及114个聋儿,272人未查及已知的危险因素,占70.5%：有耳聋家族史21人次；围产期危险因素共88人次；产前危险因素共11人次；产后获得性危险因素共45人次。经统计分析可知耳毒性药物、家族史、缺氧、发育畸形、发热抽搐及中耳炎可能引起和促进极重度感音神经性耳聋的发生。而对危险因素的多因素分析表明,内耳畸形(大前庭水管综合症,Mondini畸形,小耳蜗畸形及共同腔畸形)是最主要的危险因素。结论：1、在整个人工耳蜗植入过程中,术前患者的综合评估是至关重要和必不可少的环节,其主要目的是从医学、听力学等多方面综合评价和决定患者是否适合实施人工耳蜗植入手术。2、本研究证实了遗传因素为极重度感音神经性聋儿的主要危险因素。对广大人群普及自我保健及防聋治聋知识,加强孕前、产前致畸因素的预防,预防及早期根治婴幼儿期的中耳炎及发热疾病；尽可能排除存在基因突变的遗传学因素。进行新生儿或儿童高危因素登记,对高危人群进行听力学筛查和随诊,并尽早干预,规范药物使用,将有助于减少极重度耳聋的发生。研究目的：对我国接受人工耳蜗植入群体进行分子病因学研究；重点探讨在接受人工耳蜗植入的病人中,GJB2, SLC26A4、线粒体12S rRNA基因突变发生的概率、突变形式和突变热点,探讨适合中国人极重度感音神经性聋的基因诊断策略。研究大前庭水管综合征(Enlarged vestibular aqueduct syndrome, EVAS)在中国人极重度感音神经性聋患者中的基因型和分子流行病学特点。比较基因芯片法和传统测序法在基因诊断中的优势及差异。研究方法：采集236名接受和申请人工耳蜗植入的重度及极重度感音神经性耳聋患者,30例EVAS患者和100例听力检测正常者的外周血。提取基因组DNA,用耳聋基因芯片检测四个国人中常见的耳聋相关基因中的9个热点突变,包括GJB2(35delG,176del16bp,235delC及299delAT),GJB3(538C＞T),SLC26A4(IVS7-2A>G、2168A>G)和线粒体12S rRNA(1494C>T及1555A>G)。然后用传统测序法对基因芯片检测结果行验证,即对GJB2的编码区,DNA12S rRNA (1494C＞T及1555A>G)和SLC26A4基因的第7和8外显子,19号外显子进行测序。SLC26A4基因有21个外显子,完全检测较困难。为了进一步明确致聋原因,我们对基因芯片检测未发现突变或发现单杂合突变的大前庭水管综合征患者进一步行SLC26A4基因的DNA测序,先筛查第10、17、15、3外显子,然后筛查剩余外显子,直至发现另一个突变或筛查完全部SLC26A4外显子。研究结果：经测序,发现236例患者中93例携带有GJB2基因突变,发生率约35.36%。除3例纯合235deIC被判读为杂合外,测序方法同应用基因芯片检测的结果的结果一致,基因芯片的阳性突变检出率为21.19%(50例)。两种方法在阳性病人检出率间有统计学差异。而两种方法对GJB2基因相关性耳聋(纯合或复合杂合)的确诊率为23.31%(55/236)和16.10%(38/236),也有显著统计学意义。236例患者GJB2致病等位基因频率合计为31.36%(148/472),共发现15种突变类型：5种常见多态性；8种已报道的致病突变；和2种新突变类型。而其中235delC是最常见的致病突变类型,等位基因频率为15.5%(75/472),其次为109G>A,等位基因频率为10.8%(51/475)。新突变52A>C,511-512insAACGC,各有一例以复合杂合的形式出现。在非综合症耳聋患者检出一例223C>T——之前被认为引起综合症性耳聋的突变。非综合征型耳聋组40.55%(88/217)患者发现GJB2基因致病突变。而内耳畸形组的GJB2基因等位基因突变率13.16%(5/38),与正常听力对照无显著差异,说明GJB2基因突变不是内耳畸形的主要病因,但也可能是重要致病影响因素,对其他的致病基因产生协同或叠加的作用。本研究发现236例患者12S rRNA致病基因1494C>T及1555A>G检出率为分别为0.4%(1/236)及3.0%(7/236)。未发现GJB3(538C>T)患者。而在236名极重度感音神经性聋患者中,有21例(8.9%)检测出SLC26A4基因异常,其基因型包括IVS7-2A>G纯合1例,IVS7-2A>G/2168A>G (H723R)复合杂合4例,IVS7-2A>G单纯杂合16例。30例EVAS患者组中,基因芯片方法共检出SLC26A4基因突变25例,检出率为83.33%。而传统测序法中有28例患者检测出了SLC26A4基因突变,检出率为93.33%,与基因芯片法比较,检出率的差异无统计学意义。在本实验中(包括CI组及EVAS组),我们共发现了16种突变类型,其中5种为新的突变类型(A227P, G368X, IVS8-1G> T, IVS13+9C＞T和Q696X)。在所有的突变中,IVS7-2A>G突变的发生率最高,其次为H723R和T410M。结论：耳聋基因芯片对GJB2(35delG,176del16bp,235delC及299delAT) SLC26A4(IVS7-2A>G、2168A>G)和线粒体12S rRNA1555A>G检出率高,能够适用于一般的基因筛查。但对12S rRNA1494C＞T,GJB3(538C＞T)检出率低,与适用人群有关。根据基因芯片快速检测后结果,并结合患者的临床资料,并对相应基因进行测序验证或全序列分析才能很好地将基因芯片的快速、经济、高通量的优势与测序的高准确性和高检出率的优势相结合。第三部分湖南苗族、土家族和汉族常见致聋基因的遗传流行病学调查研究目的：遗传性耳聋具有高度的遗传异质性,不同的地区和人群中耳聋基因的突变频率、突变方式和突变热点有很大的差异。目前GJB2基因、SLC26A4基因、线粒体DNA12SrRNA基因等常见耳聋基因在我国人群中的流行分布情况被广泛研究,并取得了很大进展,研究主要在汉族人群中进行的,在少数民族耳聋人群中的耳聋基因分子流行病学研究较少。在本研究中,我们对湖南湘西少数民族地区的三个民族进行常见耳聋基因的分子流行病学研究,以了解耳聋基因在我国少数民族地区和少数民族人群的流行分布情况,明确相应突变谱和热点突变,为聋病基因的大规模筛查提供依据。研究方法：本研究用基因芯片与传统测序方法联合,在湖南湘西自治区汉族、苗族和土家族141例感音神经性耳聋患者中进行GJB2基因,线粒体DNA12S rRNA A1555G和SLC26A4基因突变分子流行病学调查,主要研究三个常见致聋突变在不同民族中携带率的差异。研究结果：在湖南地区正常听力人群中检测表明,苗族,土家族及汉族的GIB2基因突变等位基因频率为苗族最高,汉族最低,土家族与汉族、苗族与汉族均具有显著差别。三个民族间GJB2基因突变携带率无统计学差异。三个民族的GJB2基因突变均以V37I,235de1C为主。未检测出35de1G。提示湖南土家族和苗族的GJB2基因多态性分布与我国汉族相似,再次证实了三个民族有共同的人类种族起源。但在苗族和土家族正常听力人群中V37I突变的等位基因频率与携带率均较235de1C高,与汉族正好相反。GJB2基因突变在各民族耳聋患者中有较高的携带率,土家族GIB2基因突变携带率及等位基因突变频率均显著性低于汉族患者。235de1C突变是每个民族最常见的突变方式,其次为V37I。235de1C与V37I突变携带率及等位基因突变频率在土家族最低,但三民族间无统计学差异。在土家族患者中检测到三种新的突变类型223C＞(R75W),257C＞G(T86R)及475G>A(D159N)。223C＞T (R75W)虽被the connexin deafness homepage网站收录,但在本研究中被认为引起非综合症性耳聋而不是综合性耳聋,因此也归为新的遗传突变。线粒体DNA12S rRNA A1555G突变是该地区苗族和土家族人群常见的耳聋致病基因,均高于文献报道的汉族中的A1555G突变率。这种现象主要与环境因素有关,特别是与湘西地区对氨基糖苷类抗生素的大量使用有关。苗族、土家族和汉族耳聋患者的SLC26A4IVS7-2A>G,无统计学差异。未检测出2168A>G (H723R)突变。可见IVS7-2A>G也是湘西苗族,土家族耳聋患者常见的SLC26A4基因致病突变。结论：GJB2基因突变在不同人群的分布相似性及高发突变类型及携带率的差异性,既反映了湖南地区三个少数民族人群的遗传同源性也显示了种族的遗传异质性,与其起源和历史原因等导致的长期人群聚集通婚及种族间的交流有关。研究目的：有研究者发现存在核遗传背景可能影响A1555G突变相关听力丧失能否发生。而A1555G点突变的耳聋患者中,GJB2基因突变的携带率较高。在本研究中我们就核基因GJB2是否与药毒性耳聋相关,与A1555G是否存在关联性,其表达是否会受到氨基糖苷类抗生素的影响进行初步的探讨,为药物中毒性耳聋机制的探索以及防治寻求一个新的方向。研究方法：1、我们在携带mtDNA12S rRNA1555A>G点突变的21例散发个家系中检测GJB2基因突变的携带率,以明确GJB2突变与该类患者表型的关系。2、我们应用免疫组化染色、western blot和real-time PCR的方法观察单次大剂量的庆大霉素注射的大鼠耳蜗外侧壁不同时段Cx26表达的影响以及药物中毒性聋大鼠耳蜗外侧壁Cx26的表达,从而从分子水平探讨药毒性耳聋的发病机制。研究结果:1、我们在携带mtDNA12S rRNA1555A>G点突变的多个家系中检测GJB2基因突变的携带率,检测到两种GJB2基因突变109G>A及79G>A+341A>G,其携带率较听力正常并携带1555A>G的对照组家系成员高。同时携带GJB2突变及1555A>G突变的先证者其耳聋程度较无GJB2突变严重,这意味着杂合的GJB2突变可能促使1555A>G突变携带者其耳聋的发生。2、单次大剂量的静脉注射庆大霉素可以快速的影响Cx26蛋白的表达和分布。但是CX26蛋白水平的增加是一过性,而且与mRNA水平变化不一致。庆大霉素也引起Cx26mRNA增加,但是增加的nRNA不能成功的翻译成蛋白质。结论：在1555A>G携带者中GJB2基因突变携带率高,且携带GJB2突变及1555A>G突变的患者耳聋程度较重,GJB2基因也可能是氨基糖苷类抗生素的促进因素。Cx26是庆大霉素作用后,尚未出现任何毒性反应和耳蜗功能改变前最先发生改变的生物标志物。在耳毒性聋的急性阶段,Cx26的在耳蜗的重新分布和高表达可能增加内淋巴钾离子浓度及提高EP,很可能发挥了重要的拮抗机制,从而发挥一定的保护作用。本研究为氨基糖苷类抗生素引起的耳毒性聋的病理机制的研究和预防提供了一个新的方向。氨基糖苷类如何调节Cx26基因转录和翻译尚待研究,本研究是Cx26在氨基糖苷类抗生素的耳毒性作用机制中所发挥作用的初步探讨,还需要更深入的调查研究和实验证明。更多还原

【Abstract】 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.更多还原