基因芯片技术在非综合征性耳聋快速基因诊断中的应用研究  

Application of DNA microarray in rapid genetic diagnosis of non-syndromic hearing loss

作　　者：王国建[1] 戴朴[1] 韩东一[1] 李彩霞[2] 张地[3] 韩冰[1] 康东洋[1] 张昕[1] 

机构地区：[1]解放军总医院耳鼻咽喉头颈外科,北京100853 [2]清华大学医学院医学系统生物学研究中心,北京100084 [3]博奥生物有限公司暨生物芯片北京国家工程研究中心,北京102206

出　　处：《中华耳科学杂志》2008年第1期61-66,共6页Chinese Journal of Otology

基　　金：国家自然科学基金面上项目(No.30572015);北京市自然基金面上项目(7062062);首都医学发展科研基因北京医学卫生科技联合攻关项目(2005-1032)

摘　　要：目的应用耳聋基因芯片对非综合征性感音神经性耳聋患者进行分子病因学研究,评估其在快速耳聋基因诊断中的可行性。方法采集158名来自北京第三聋哑学校的非综合征性耳聋学生的外周血,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299_300delAT),GJB3(538C>T及547G>A),SLC26A4(IVS7-2A>G、2168A>G)和线粒体DNA 12S rRNA(A1555G)。同时,应用酶切法或直接测序法分别对线粒体12SrRNA A1555G突变,以及GJB2、SLC26A4基因编码区序列进行检测,以验证基因芯片结果的准确性。结果在158名耳聋患者中,基因芯片方法共检出67例携带致聋突变(42.41%)。其中,线粒体DNA 12S rRNA A1555G突变5例(3.16%);GJB2基因突变39例(24.68%),包括235delC纯合突变24例,235delC和299_300delAT复合杂合突变7例,235delC单杂合突变5例,299_300delAT单杂合突变2例,35delG单杂合突变1例;SLC26A4基因突变22例(13.92%),包括IVS7-2A>G纯合突变5例,IVS7-2A>G和2168A>G复合杂合突变5例,IVS7-2A>G单杂合突变11例,2168A>G单杂合1例;另外还有1例患者检出299_300delAT和IVS7-2A>G双杂合突变。未检出GJB3基因突变。除两例纯合235delC被判读为杂合外,基因芯片的结果同酶切及测序方法结果一致,后者的阳性患者检出率为70例(44.3%),与芯片检测结果相比无统计学差异(P=0.250)。经探针优化后,上述两例误判的235delC样品的芯片检测结果均与测序方法一致,同时经测序证实的其他22例235delC纯合突变样品验证,基因芯片的结果均与测序结果一致。结论针对国人常见耳聋相关基因热点突变设计的耳聋基因芯片对非综合征性重度和极重度耳聋患者的突变检出率高(42.41%)。与传统方法相比,它具有快速、高通量、高准确性、低成本等特点,能够满足临床耳聋基因检测的要求;且其操作简单,易于标准化,适于推广,显示出广阔的临床Objectives To investigate the feasibility of a DNA microarray, designed according to the genetic background of China, in rapid genetic diagnosis of non-syndromic sensorineural hearing loss （NSHL）. Methods One hundred and fifty-eight moderate to profound NSHL patients were included in this study. Their genomic DNA samples were extracted from peripheral blood, and detected with the DNA microarray which is able to perform mutation detection of 9 hot-spot mutations in four most common pathologic genes, including GJB2 （35delG, 176del16bp, 235delC, 299_300delAT）, GJB3（538C〉T, 547G〉A）, SLC26A4（IVS7-2A〉G, 2168A〉G） and mitochondrial 12S rRNA（A1555G） simultaneously. At the same time, the results were confirmed with the traditional methods of sequencing and restriction enzyme digestion. Results Of 158 patients, 67 were found out to be carriers of at least one pathogenic gene mutation. Among them, 5 （3.16%） have A1555G mutations, 39 （24.68%） have GJB2 mutations （235delC homozygous mutation in 24 cases, 235delC and 299_300delAT compound heterozygous mutation in 7 cases, 235delC heterozygous mutation in 5 cases, 299_300delAT heterozygous mutation in 2 cases, 35delG heterozygous mutation in 1 case）, 22 （13.92%） have SLC26A4 gene mutations （IVS7-2 A〉G homozygous mutation in 5 cases, IVS7-2 A〉G and 2168A〉G compound heterozygous mutation in 5 cases, IVS7-2 heterozygous mutation in 11 cases, 2168A〉G heterozygous mutation in 1 case）, and 299_300delAT and IVS7-2 A〉G double heterozygous mutation was revealed in one additional patient. No one carries GJB3 mutation. Except that two homozygous 235delC mutation cases were misjudged to be heterozygous mutation, the detection results of patients with microarray are identical to those with traditional methods, of which the positive ratio is 44.3% （70 cases）. After probe optimization, the detection results of that two previously misjudged homozygous 235delC mutation samples are consistent with the sequencing results. The new optimi

关 键 词：耳聋 GJB2基因 SLC26A4基因 GJB3基因 线粒体DNA 突变 基因芯片 

分 类 号：R764.04[医药卫生—耳鼻咽喉科] R349.8[医药卫生—临床医学]