【作者】 赵飞帆；

【导师】 王秋菊； 韩东一；

【作者基本信息】 中国人民解放军军医进修学院 ， 耳鼻咽喉科学， 2012， 博士

【摘要】 遗传性耳聋严重影响人类学习交流和社会活动，是亟待解决的日益严重的健康问题。预防耳聋出生缺陷，提高人口素质是关系国计民生的重大问题，而预防的根本，就是深入了解遗传基因及其突变谱，发现中国人群的耳聋基因分布和热点突变，从而能够在人们的婚育指导上进行预测，提早进行预防。目前中国大部分聋人表现为隐性遗传性耳聋，而其中60-70%尚无法明确致聋基因。本研究利用了当前蓬勃发展的新一代测序技术，针对非综合征遗传性耳聋的隐性遗传小家系进行了病因研究，发现了在国际上发病率居2-3位的CDH23基因为负责基因，并发现了新突变。同时，在当前很多耳聋基因得到鉴定的背景下，本研究针对常见隐性遗传性耳聋基因GJB2和SLC26A4，对致聋基因型与临床表型进行了关联分析，在一定程度上阐释了基因型对表型的影响及对临床诊疗的指导意义。第一部分利用外显子测序技术发现隐性遗传性耳聋致聋基因CDH23一、外显子测序技术在3个隐性遗传性耳聋家系中的应用本研究选择了3个隐性遗传性耳聋的核心家系，家系中仅第三代有两例耳聋患者。首先进行了常见隐性耳聋基因和突变的筛查（GJB2、SLC26A4和线粒体DNA1555A>G），未发现携带突变。然后，在784和845家系中，分别选择了两个患者和父母，在855家系中，因父亲亡故，选了两个患者和同胞弟弟，叔叔，及母亲，进行了全外显子测序。测序结果的质控报告显示，测序深度达到50X，外显子长度达到3千万碱基左右，达到了人类全部外显子99%以上覆盖率。每个样本经过筛选，高可信度的数据如下：95000个左右的单核苷酸变异（SNP），2500个左右的剪接位点突变（splice site）,7000左右的插入缺失突变（indel），58000左右的在外显子临近10bp以内的内含子突变（intron）。根据目前已经公开的数据库进行了SNPs过库筛选分析，所采用的数据库包括dbSNP数据库、Hapmap-8数据库、1000Genomes、YanHuang （YH）。首先针对每个家系中每个样本的变异与各个数据库分别比对，筛选出所有数据库均没有报道的罕见变异；然后再把家系的正常人作为对照进行比对，根据疾病表型和基因型共分离的原理，筛选患者中纯合，而在父母或同胞杂合的变异，即得到候选基因的位点。对候选基因位点，选择在多物种间（大鼠、小鼠、狗、猴、鸡、象、X_嗜热病毒、斑马鱼）保守区域的5个碱基以内的插入缺失突变、剪接区、无义突变、经SIFT判定为具有蛋白质功能损害性的（damaging）的错义突变作为首选。在784家系中，未发现符合上述条件的候选基因位点。在845家系中，发现了已知致聋基因CDH23的73553127位置发生的G>A突变（c.6442G>A，p.D2148N），是已在多个人种中报道过的致聋错义突变。在855家系中，发现已知致聋基因CDH23的73270943位置发生C>T突变（c.403C>T, p.Q135*），为终止突变，据多物种间序列保守性预测，可对蛋白质功能产生巨大影响。因此，845和855家系均是已知致聋基因CDH23突变导致的，其中c.6442G>A为已知突变，c.403C>T为新突变。二、CDH23基因新突变验证及分子流行病学研究855号样本经过眼科学检查，发现该患者伴发严重的视网膜色素变性，符合Usher1D综合征的诊断，CDH23基因正是该疾病的负责基因。在100例正常人群对照中，筛查c.403C>T，未发现该突变。本课题组收集到来自中国北方地区的108余例隐性遗传聋核心家系（包括855家系），进行常见隐性耳聋基因GJB2、SLC26A4的全测序研究，筛查了线粒体DNA A1555G，未发现携带已知突变。鉴于CDH23基因为非综合征型遗传性耳聋（NSHL）较为常见的致病基因，经文献复习，查找到与NSHL中隐性耳聋DFNB12相关的CDH23基因29个突变，广泛分布于美国、德国、荷兰、日本、印度、巴基斯坦和阿尔及利亚等世界范围内的高加索人、非洲人和蒙古人种，人种间突变谱差异较大。利用Sequenom公司的基于质谱原理的MassARRAY时间飞行质谱生物芯片系统，在108个家系的患者中进行了CDH23基因与DFNB12相关的已知突变的筛查，并在855号样本中验证了新突变的存在。结果发现：3例单等位基因突变携带者，再结合外显子测序发现的1例纯合子，则CDH23基因在该样本中的NSHL相关等位基因突变率为2.31%（5/216）。第二部分隐性遗传性耳聋患者中常见致聋基因GJB2和SLC26A4基因型与表型关联分析一、295例GJB2双等位基因突变耳聋基因型表型分析缝隙连接蛋白编码基因GJB2是非综合征型感音神经性耳聋（NSSHI）最常见的致病基因。该基因已有超过100种以上的突变被报道与耳聋相关，耳聋级别表现从轻中度到极重度均有覆盖。为评估中国人群中GJB2基因型对耳聋表型的影响，本研究在中国北方NSSHI人群中进行了横断面研究。本研究纳入了295例携带GJB2双等位基因突变的中国北方七省的遗传性耳聋患者。以突变是否导致基因功能缺失将突变分为截短型（T）和非截短型（NT），将双等位基因型分为截短组（T+T），复合杂合截短组（T+NT），和非截短组（NT+NT）。每个基因型组的耳聋纯音测听平均听阈（PTA）进行了χ2卡方检验。发现截短组的耳聋程度显著超过非截短组。同时发现具有基因型c.[79G>A;341A>G]+[79G>A;341A>G]或c.[109G>A]+[79G>A;341A>G]的患者其耳聋程度显著轻于c.235delC纯合子组，而具有c.[235delC]+[176₁91del16]基因型的患者则显著重于c.235delC纯合子组。二、272例儿童大前庭水管患者的基因型表型分析本研究拟初步绘制儿童大前庭水管综合征患者的基因突变谱，并阐释其基因型对耳聋及内耳结构的影响。12岁以下的272名大前庭水管综合征患儿纳入研究，进行了SLC26A4基因测序。其中266例接受了全套听力学检查，可获得平均听阈的数据，这其中，有152例患儿的高分辨率颞骨CT进行了详细测量。根据患者携带突变数量，分为双等位基因型，单等位基因型和无突变型，其中双等位基因型，再根据突变性质，分为双等位基因截短组，双等位基因复合杂合截短组，双等位基因非截短组。临床表型数据包括PTA，每个族群患者数量，前庭水管中段和外口直径等。使用统计学软件SPSS15.0，将基因型数据和表型数据进行了关联分析，使用方法为计量资料t检验和计数资料卡方检验。本组研究人群中，共发现SLC26A4基因的69种突变，其中2种为新突变（c.665G>T和c.1639A>G）。207例患者为双等位基因突变，56例为单等位基因突变，8例未发现突变，1例携带3个突变。经过统计学比较，发现单等位基因组的患者耳聋程度更为稳定（P<0.05）。双等位基因非截短组的患者比双等位基因截短组，复合杂合截短组和单等位基因组更容易出现气骨导差（所有P值<0.05）。不同基因型组之间的前庭水管尺寸则没有明显差异（所有P值>0.05）。不同前庭水管尺寸的患者之间耳聋严重程度也没有明显差异（所有P值>0.05）。本研究发现前庭导水管综合征的儿童中98%携带SLC26A4基因突变。虽然本研究发现了基因型与表型之间的一些关系，但是真正揭示基因型对表型的影响还有很远的距离。更多还原

【Abstract】 Hereditary hearing loss will impact the communication and social activities,and is the increasingly severe disease unresolved. Prevention of deafness andimprovement of population is very important for national economy and the people’slivelihood. The root of the prophylaxis was to thoroughly explore the causative geneand mutation spectrum and discover the gene distribution and hot spot, in order toinstruct the marriage and procreation among deafness to avoid the incidence ofdeafness. Most of Chinese hereditary deafness were in recessive mode, andcausative genes in60-70%population were unknown. This study applied the nextgeneration sequencing technique-exome sequencing in autosomal recessivehereditary hearing loss families, and identified CDH23gene to be the causative gene.CDH23gene was the common deafness gene, and occupied the NO.2-3innonsyndromic hearing loss genes. We then srcreened the known mutations ofCDH23related to DFNB12in108recessive deafness families. In the background ofmultiple causative genes identified, this study performed the genotype phenotyperelation analysis about GJB2and SLC26A4genes, and clarified the inpact ofgenotype to phenotype to some extent.PART1: CDH23was identified as causative gene in autosomalrecessive hereditary hearing loss and known mutations screening in108probands of recessive familiesTo identify the causative gene in3families with autosomal recessive hereditary hearing loss, we performed the common causative gene screening （GJB2、SLC26A4and m.1555A>G）, and no mutation was found out. Then, from family784and845,2patients and their parents were recrued respectively, and from family855,2patients,1sibling, their mother and uncle were recrued for the father was dead. Thewhole exome sequencing was applied in the13subjects.The quality control report of the sequencing showed: sequencing depth was50X,and about95000SNPs, about2500splice site mutations, about7000indels, about58000introns in10bp near the flanking part of exons.Based on the databases, including dbSNP, Hapmap-8,1000Genomes andYanHuang （YH）, the raw data were filtered as follows:Firstly, the raw data from each subject was filtered by such databasesrespectively, and rare mutations were selected. Then the data from patients werefiltered by the data from normal controls. In the third step, according to theco-segregating principle in autosomal recessive hereditary mode, the mutationsappeared homozygous state in patients and heterozygous state in controls were takenas the candidate genes.In the candidate genes, preferred mutations were the indels less than5bp in theconserved region in multiple species, splice site mutations, nonsense mutations, anddamaging missense mutations predicted by SIFT software.In family784, no preferred mutations and genes were identified. In family845,preferred mutation was the mutation of G>A（c.6442G>A，p.D2148N）on73553127of CDH23gene. In family855, a novel mutation（c.403C>T, p.Q135*）in CDH23gene was preferred to be the causative mutation. CDH23gene was the knowncausative gene, and the mutation c.6442G>A was the known mutation while thenovel mutation c.403C>T was thought to be damaging by SIFT software. By theophthalmologic examination, the patient with c.403C>T mutation was found withsevere rentitis pigmentosa （RP）, and was diagnosed with Usher1D syndrome.By literature reviews, about29mutations in CDH23gene related with DFNB12were found out, which were scattering in Caucasians, Africans and Mongolians over the world including America, German, Holland, Japan, India, Pakistan and Algeria.c.403C>T was not found in100geographically matched controls.108families with autosomal recessive hereditary hearing loss were performedthe common causative gene screening （GJB2、SLC26A4and m.1555A>G）, and nomutation was found out. Known mutations screening was applied in the108familiesby MassARRAY system.3monoallelic mutation carriers were found out.Together with2homozygotes found by exome sequencing, and the mutationfrequency was3.24%（7/216）.PART2: Genotype-phenotype correlation analysis in295deafnesscarrying GJB2biallelic mutationsConnexin26coding gene （GJB2） is the primary causative gene fornonsyndromic sensorineural hearing impairment （NSSHI）. More than100mutationsin this gene have been reported to be linked to hearing impairment, from mild toprofound hearing loss. In order to precisely estimate the impact of GJB2mutationsin Chinese population, a cross-sectional study was performed to analyze the auditorydata of Chinese NSSHI patients.295unrelated NSSHI patients with biallelic mutations in GJB2were recruitedfrom7provinces in Northern China from2004to2008. The levels of hearingimpairment （HI） and average pure tone audiometry （PTA） were compared acrossdifferent genotypes by χ2testing. The subjects with the genotypes of combinedtruncating mutations had more cases of severe HI than the subjects with genotype ofnontruncating mutations combination. It was also revealed that subjects carryingeither c.[79G>A;341A>G]+[79G>A;341A>G] or c.[109G>A]+[79G>A;341A>G]had significantly less cases of severe HI than the reference group of homozygousc.235delC, while the subjects carrying c.[235delC]+[176₁91del16] had more casesof severe HI than the homozygous c.235delC group.This is the first study to clarify the correlations between different GJB2 biallelic genotypes and NSSHI phenotype in Chinese population. The Chinesesubjects with two truncating mutations in GJB2were shown to correlate with moresevere hearing impairmentPART3: Genotype-phenotype correlation analysis in272childrendeafness with large vestibular aqueductThis study was designed to draw a spectrum of SLC26A4gene mutations inchildren with large vestibular aqueduct （LVA） and to study the phenotype genotyperelationship.272subjects under age12years with nonsyndromic sensory neural hearing loss（NSSHL） and LVA, received SLC26A4gene screening.266received completeauditory examinations. High-resolution computed tomography （HR-CT） of temporalbone were measured in152subjects. According to genotypes, the phenotypesincluding PTA （pure tone average）, distribution of subjects, and diameter of externalaperture and middle portion of vestibular aqueduct, were compared by t test or chisquare tests by SPSS13.0. Further, divided by dilated level of vestibular aqueduct,subject distribution in different hearing loss levels were compared by chi squaretests by SPSS13.0.69types of mutations were identified, in which2were novel mutations（c.665G>T and c.1639A>G）.170subjects were found with biallelic mutations inSLC26A4gene,56with monoallelic mutation,8with no mutation, and one withthree mutations. The hearing loss was more stable in the subjects with monoallelicmutation than in other genotype groups （P<0.05）. The air-bone gap was morefrequently seen in subjects with biallelic missense mutations than those withbiallelic truncating, heterozygous truncating, or monoallelic mutation （all Pvalues<0.05）. There is no difference for distribution of diameters distribution indifferent genotypes. And there is no dominant correlation between hearing loss leveland dilated levels of vestibule aqueduct. This is the first study to clarify the correlations between different SLC26A4genotypes and phenotype in Chinese children population with LVAS. In childrenwith LVAS,98%carry mutations in SLC26A4gene. Though we found somecorrelation between genotype and phenotype, it is far away from erecting directcorrelation between the mutation genotype and hearing loss level or LVA diameter.更多还原