【作者】 郭胜利；

【导师】 李守霞；

【作者基本信息】 河北医科大学 ， 临床检验诊断学， 2015， 硕士

【摘要】 目的:中国每年新增约90万出生缺陷新生儿,其中耳聋位居出生缺陷首位;约半数耳聋属于遗传性耳聋,以其高发病率、低治愈率造成患儿心理、社会交往能力发育障碍,给家庭、社会带来沉重负担。随着遗传性耳聋相关基因的发现及其致聋机理的深入研究,使耳聋分子诊断、预防及治疗三级体系得以丰富。本研究通过检测邢台市聋哑学校非综合征性耳聋(non-syndromic hearing loss,NSHL)患者常见耳聋相关基因的突变情况,初步掌握本地区NSHL患者常见基因的分布及突变特点,明确本地区病因以制定个性化的基因诊断策略,为耳聋基因筛查、临床分子诊断提供切实的理论依据。方法:1研究对象及信息采集选取邢台市特教学校的非综合征性耳聋学生110例,所有入选者均进行全身体格、纯音测听和声音导抗检查。对NSHL学生采取耳鼻喉专科医生询问学生家长的形式完整填写《常见遗传病信息登记表》及《耳聋病人信息登记表》,并要求全部签署《常见耳聋基因诊断知情同意书》,未满18周岁均由其监护人代签。2样本采集采集入选对象EDTA-K2抗凝的静脉全血,分离血浆和血细胞,-80℃超低温冰箱冷冻保存,建立邢台地区耳聋患者样本库;其中血细胞用于耳聋4个常见突变基因(GJB2、GJB3、SLC26A4、线粒体DNA 12S r RNA基因)的检测。3抽提外周血细胞基因组DNA,采用聚合酶链反应(polymerase chain reaction,PCR)扩增GJB2、GJB3、SLC26A4、线粒体DNA 12S r RNA基因片段,并应用琼脂糖凝胶电泳成像系统进行鉴定。4采用直接测序法对线粒体DNA 12S r RNA基因的2个点突变片段(A1555G、C1494T)所在区域、GJB2基因的2个基因片段、GJB3基因的整个编码区和SLC26A4基因的20个外显子区域进行检测;应用Mutation surveyor软件对所有基因的测序结果进行比对分析。采用Gene Tool Lite 1.0软件将DNA测序得到的序列结果与NCBI网站检索到的GJB2标准序列(NC-000013.9)、GJB3标准序列(NT-004511)、SLC26A4标准序列(NC-000007-13)及mt DNA标准序列(NC-001807)进行比对分析,以筛查是否有突变位点的存在。结果:被检测的110例邢台市特教学校耳聋学生均被诊断为重度以上双侧耳聋且均为非综合征型耳聋。其中有8例SLC26A4突变导致的内耳最常见的畸形前庭水管扩大(enlarged vestibular agueduet,EVA);聋前或其母亲孕期明确应用过氨基糖甙类抗生素者20例(庆大霉素12例、丁胺卡那霉素2例、小诺霉素3例、联合用药患者中庆大霉素加链霉素3例)用药的时间主要集中在研究对象婴幼儿时期或其母亲妊娠期,用药原因多为感冒、发烧和腹泻,具体的用药剂量不详。被检测的110名聋哑学校非综合征型耳聋患者中,携带线粒体DNA 12S r RNA基因A1555G点突变的2例(占1.8%);携带线粒体DNA 12S r RNA基因C1494T点突变的2例(占1.8%);携带GJB2基因235del C纯合突变的11例(占10%),携带GJB2基因复合杂合突变的11例(占10%);携带SLC26A4基因IVS7-2A>G纯合突变的5例(占4.5%),携带SLC26A4基因IVS7-2A>G杂合突变的7例(占6.4%);3例患者携带2个未分类的GJB3的突变基因。所有入选患者共包含38个突变体变异,另外新发现的5个异常的在SLC26A4基因突变:4个错义替换(p.G222S,p.A456D,p.N457I,p.F667L)和一个无义突变(p.W472X)。结论:邢台地区为遗传性NSHL耳聋的高发区,最常见致聋基因为GJB2基因突变,其次为SLC26A4基因突变。虽然线粒体DNA 12S r RNA基因突变不是该地区主要致聋原因,但其突变的筛查能够预测个体对氨基糖甙类抗生素(Aminoglycoside antibiotics,Am An)的遗传易感性,为临床合理用药提供指导,有效降低药物性耳聋的发生率。因此,将GJB2、GJB3、SLC26A4和线粒体DNA 12S r RNA基因作为邢台地区临床常规筛查项目,同时为进一步开展遗传咨询、产前诊断以及基因诊断提供了重要的理论依据。更多还原

【Abstract】 Objective: China increase about 900000 birth defects in newborns each year. Deafness is one of the most common genetic disorder. Previous studies show that there are many reasons for causing deafness, among which are genetic factors, about half of deafness is caused by the genetic defect. Which bring heavy burden to family and society of its high incidence and low cure rate. With the discovery of hereditary deafness related genes and in-depth study of mechanism of deafness, make deaf molecular diagnosis, prevention and treatment of tertiary system are rich. This study focused on analyzing mutations of coding sequence of common deafness syndrome deaf, the rapid genetic diagnosis lay the foundation for deaf.Methods:1 The object of study and information acquisition: 110 non-syndromic hearing loss(NSHL) patients from xingtai special-education schools.Which all entrants to the whole body physique, pure tone audiometry and sound immittance test. 110 non-syndromic hearing loss(NSHL) patients were interviewed for medical histories of hearing loss, use of aminoglycosides, and other clinical abnormalities using questionnaires.2 Samples: Collect 110 cases non- syndromic hearing loss(NSHL) patients the syndrome in patients with type deafness blood specimens, store in-80 refrigerator, establish blood specimens library.℃3 The expression of mitochondrial DNA 12 S r RNA, GJB2, GJB3 and SLC26A4 gene expression were detected by using polymerase chain reaction(PCR) methods.4 Application directly sequenced to detect the mutations of GJB2 gene sequencing method, mitochondrial DNA 12 S r RNA gene, GJB3, and SLC26A4 gene. Using Gene Tool Lite 1.0 software will get the sequence of DNA sequencing results and the NCBI site search to the standard of GJB2 sequence(NC-000013.9) and GJB3 standard(NT-004511), standard of SLC26A4 sequences(NC-000007-13) and the standard of mt DNA sequences(NC-001807) compare analysis, in order to screening the presence of mutations.Results: The hearing loss in the classification standard assess 110 cases of Xingtai city in hebei province deaf students school, 110 cases were diagnosed as bilateral deafness and are based on syndrome type deafness. In 110 an example research object, 8 of the inner ear SLC26A4 mutations in the most common deformity vestibular conduit expand(enlarged vestibular agueduet, EVA). 110 cases of research object, the respondents before or the deaf pregnancy used clear aminoglucoside the class antibiotic 20 cases, of which the most used alone gentamycin with 12 cases, used amikacin with 2 cases, small’s amikacin with 3 cases, combination in patients with streptomycin gentamycin in 3 cases. The use of drug time mainly focus on the research object infants or its mother during pregnancy, used more for a cold, fever and diarrhea, concrete the dosage is unknown. 110 patients were diagnosed with non-syndromic hearing loss(NSHL), 2 causes(1.8%) carried mitochondrial A1555 G mutation, and 2 causes(1.8%) carried mitochondrial C1494 T mutation; 11 causes(10%) showed homozygous GJB2 235 del C mutation, and 11 causes(10%) showed compound heterozygous GJB2 mutation; 5(4.5%) carried homozygous SLC26A4 IVS7-2A>G mutation, and 7(6.4%) had heterozygous SLC26A4 IVS7-2A>G mutation. Three patients carried two unclassified mutations in GJB3 genes. Overall 38 mutant variants were detected in this cohort of patients, including in dittion also found 5 novel mutations in SLC26A4. The 5 novel variants were 4 missense substitutions(p.G222 S, p.A456 D, p.N457 I, p.F667L) and one nonsense mutation(p.W472X).Conclusions: The most common deafness gene of GJB2 gene mutations, followed by SLC26A4 gene mutations for the high incidence of hereditary NSHL deafness in Xingtai area. Although mt DNA 12 S r RNA gene mutation is not deafness main reason in the region, but its mutation screening can predict individual genetic susceptibility to Am An, provide guidance for clinical rational drug use, effectively reduce the incidence of drug-induced deafness. As a result, the GJB2, GJB3, SLC26A4 and mt DNA 12 S r RNA gene as xingtai area clinical routine screening project, at the same time in order to further carry out genetic counseling and prenatal diagnosis and genetic diagnosis provides an important theoretical basis.更多还原