运用二代测序的方法对非综合征性迟发性聋患者进行易感基因检测分析

Non-syndromic deafness genes analyzed with the next-generation sequencing methods

目的通过研究上海地区79例患者迟发性感音神经性聋的致病基因,探讨常见耳聋相关基因的在患者中的点突变特征。方法对79例门诊散发感音神经性聋患者进行听力学检测并排除全身性疾病。DNA样本提取于患者外周血白细胞。利用标准的Illumina二代测序平台对79个临床散发病例基因组DNA在杂交的水平上进行80个常见耳聋基因全外显子的捕获。通过将二代测序分析分析结果与标准的基因组序列进行比对,从而明确DNA序列的改变。检测结果采取PCR扩增和Sanger测序的方法进行验证。结果79例临床散发感音神经性聋患者均为语后聋。利用二代测序技术,在79例患者的中共检测出有1 508个点突变。将点突变共分为9类,划分第Ⅰ、Ⅲ、Ⅴ、Ⅶ类的突变为纯合突变,并被归类为明确或可疑的致病突变。其中有23个DNA样本检测出此类突变。按照检测出的点突变发生数目的多少将常见耳聋基因进行排序,分别为GJB2、DSPP、SLC26A4、COCH、ESRRB、MYO6、SOX2、TMPRSS3及WFS1。结论二代测序法在遗传性聋患者基因突变位点的检测中的应用效果确切,能够有效对耳聋基因进行筛查,弥补了以往测序方法不能准确发现长片段插入性突变的缺陷。

Objective To detect the pathogenic genes of 79 patients with late onset sensorineural hearing loss in in Shanghai.We explored the point mutation characteristics of common deafness-related genes in patients,and provided a scientific basis for screening or diagnosis of deafness genes.Methods Audiometry detections and physical examinations were done for the 79 sporatic patients with sensorineural hearing loss.DNA was extracted from their peripheral blood.Genomic DNA for the coding regions of 80 deafness genes was captured by a hybridization-based method.Captured DNA fragments were processed through the standard next-generation sequencing(NGS)method using the Illumina platform.After aligning the sequenced DNA fragments to the reference human genome,we identified all the sequence variations.The validation was also made for the results by PCR and Sanger sequencing.Results In the 79 sporatic samples,1 508 sequencing mutations were identified,and were divided into 9 categories.TheⅠ,Ⅲ,Ⅴ,Ⅶspecie are homozygotic mutations,which are all definite or suspected disease-causing mutations.These homozygotic mutations were detected in 23 patients.Based on the numbers of the sequencing mutations,the deafness genes were ranked as:GJB2,DSPP,SLC26 A4,COCH,ESRRB,MYO6,SOX2,TMPRSS3 and WFS1.Conclusion The second-generation sequencing method has a definite effect on the detection of genetic mutation sites in patients with sensorineural hearing loss.It can effectively screen the deafness genes and make up for the defects that the previous sequencing methods can not accurately detect the insertion mutation of long fragments.