基因芯片联合 Sanger 测序技术在非综合征型聋患者基因诊断中的应用

摘要

Objective To study genotypes in nonsyndromic hearing loss (NSHL ) patients from Guangxi Zhuang Autonomous Region hearing speech rehabilitation center using DNA microarray in combination with Sanger sequencing .Methods Deaf patients received routine physical and otorhinolaryngoloical examinations as well as pure tone autiometry .Brainstem auditory evoked potential test was performed in uncooperative children .Blood samples were obtained from a total of 136 patients ,male 81 ,female 55 ,age from one year five month to seventeen ,having nonsyndromic hearing loss .Genomic DNA was extracted and then 9 hot mutation spots in 4 susceptibility genes were detected by DNA microarray .GJB2 and SLC26A was further detected by Sanger sequencing in the patients with negative results and heterozygotes .Results Among the 136 patients with nonsyndromic hearing loss ,20 cases were positive for GJB2 gene ,SLC26A4 gene or mitochondrial 12SrRNA gene mutations .There were 14 .71% (20/136)patients were positive for hot mutation spots in the deafness related genes ,25% (34/136)patients carried muta‐tions of deafness related genes using DNA microarray in combination with Sanger sequencing .Six SLC26A4 rare mutations (c .259G> T ,c .754C> T ,c .1229C> T ,c .1548_1549insC ,c .1705+5A>G and c .2086C> T) were de‐tected by Sanger sequencing .c .235delC was the most common mutation in GJB2 gene .c .919-2A>G ,c .754C> T and c .1229C> T were the common mutations in SLC26A4 gene .The mutation rate of GJB2 and SLC26A4 was 38 . 24% .and 58 .82% ,respectively .Conclusion Prevalent deafness-associated gene mutations in the nine loci studied were less frequently detected in nonsyndromic hearing loss patients from Guangxi Zhuang Autonomous Region hear‐ing speech rehabilitation center .It can improve the detection rate of deafness gene mutations by using gene microar‐ray in combination with Sanger sequencing .GJB2 and SLC26A4 are the common causative genes .%目的：应用基因芯片联合Sanger测序技术检测广西壮族自治区听力言语康复中心非综合征型聋患者的基因突变谱系及基因型，为基因诊断、遗传咨询及防聋治聋提供参考。方法对广西壮族自治区听力言语康复中心136例双侧重度及以上程度感音神经性非综合征型聋患儿采集外周血并提取基因组DNA ，用遗传性聋基因芯片对4个耳聋易感基因的9个突变位点（GJB2基因：c ．35delG、c ．235delC、c ．176del16、c ．299delAT ；GJB3基因：c ．538C＞ T ；SLC26A4基因：c ．919－2A＞ G、c ．2168A＞ G ；mtDNA 12SrRNA 基因：m ．1494C＞ T、m ．1555A＞G）进行分析。基因芯片技术未确诊的样本采用Sanger测序法进一步检测。结果通过基因芯片技术在20例聋儿中检出GJB2、SLC26A4和线粒体12SrRNA基因突变，总检出率为14．71％（20／136）；结合Sanger测序技术，34例聋儿检出GJB2、SLC26A4和12SrRNA基因突变，总检出率为25％（34／136），发现6种基因芯片技术未检出的SLC26A4突变位点（c ．259G＞ T、c ．754C＞ T、c ．1229C＞ T、c ．1548＿1549insC、c ．1705＋5A＞ G和c ．2086C＞ T ）。GJB2基因以c ．235delC突变为主，SLC26A4基因以c ．919－2A＞ G、c ．754C＞ T 和c ．1229C＞ T 为主；GJB2和SLC26A4基因突变者占本组病例基因突变总例数的38．24％（13／34）和58．82％（20／34）。结论基因芯片联合Sanger测序技术可以提高非综合征型聋患者基因突变的检出率；广西壮族自治区听力言语康复中心非综合征型聋患者热点突变基因以GJB2和SLC26A4基因为常见。