飞行时间质谱技术在中国人常见耳聋基因检测中的应用  

Screening of common mutations in deaf gene in Chinese patients using MALDI-TOF MS technology

作　　者：金孝华 黄莎莎[3] 庞珊珊[1] 戴朴 高华方[1,2] 马旭 JIN Xiao-hua;HUANG Sha-sha;PANG Shan-shan;DAI Pu;GAO Hua-fang;MA Xu(National Research Institute for Family Planning,Beijing 100081;Graduate School of Peking Union Medical College,Beijing 100005;College of Otolaryngology Head&Neck Surgery,Chinese PLA General Hospital,Chinese PLA Medical School,Beijing 100853)

机构地区：[1]国家卫生健康委科学技术研究所,北京100081 [2]北京协和医学院研究生院,北京100005 [3]中国人民解放军总医院耳鼻咽喉头颈外科医学部,北京100853

出　　处：《生殖医学杂志》2022年第10期1373-1379,共7页Journal of Reproductive Medicine

基　　金：国家重点研发计划项目(2016YFC1000307)。

摘　　要：目的建立基于飞行时间质谱技术(MALDI-TOF MS)的中国人群常见耳聋致病基因热点突变高通量检测方法。方法选取2016年8月至2017年2月于中国人民解放军总医院就诊的700例(327例已知突变位点,373例未进行基因检测)非综合征型耳聋患者作为研究对象,利用327例已知突变位点的非综合征型耳聋样本建立检测耳聋基因(GJB2、SLC26A4和线粒体12S rRNA)30个热点突变位点单一反应MALDI-TOF MS检测方法;利用新建立的单一反应MALDI-TOF MS检测方法对373例未进行基因检测的非综合征型耳聋样本的耳聋致病基因GJB2、SLC26A4和线粒体12S rRNA的30个热点突变进行检测;采取Sanger测序对部分阳性结果样本进行验证。结果建立了基于MALDI-TOF MS的单一反应检测GJB2、SLC26A4和线粒体12S rRNA的30个突变位点的检测方法,其检测结果与临床基因诊断结果一致,符合率为100%;采用建立的单一反应MALDI-TOF MS检测方法检测373例未知基因突变位点的非综合征型耳聋患者样本发现,56.30%(210/373)的患者携带至少1个突变,其中携带复合或纯合突变可以明确基因诊断的患者为155例,占受检人群的41.55%(155/373),210例阳性突变携带者共涉及55种基因型,其中有5例存在多个基因突变;Sanger测序法检测验证发现,56例阳性结果样本的一代测序结果与MALDI-TOF MS检测结果一致,符合率为100%。结论基于MALDI-TOF MS建立的单一反应检测耳聋基因GJB2、SLC26A4和线粒体12S rRNA的30个热点突变的方法具有准确、高通量、低成本等特点,有望成为遗传性耳聋病因学检查和预防性筛查的有效手段。Objective:To establish and verify a high-throughput genotyping method based on MALDI-TOF MS technology to detect common mutations in deaf gene in Chinese patients.Methods:A total of 700 patients with non-syndromic hearing loss(327 patients with known mutation sites and 373 patients without genetic testing)visited Chinese PLA General Hospital from August 2016 to February 2017 were selected as the subjects.A single reaction MALDI-TOF MS method for detecting 30 hotspot mutation sites of deafness genes(GJB2,SLC26A4 and mitochondrial 12S rRNA)was established using 327 non-syndromic deafness samples with known mutation sites.The newly established single reaction MALDI-TOF MS assay was used to detect 30 hotspot mutations in the deafness causing genes GJB2,SLC26A4 and mitochondrial 12S rRNA in 373 non-syndromic deafness samples without genetic testing.Some positive samples were verified by Sanger sequencing.Results:A single reaction method on MALDI-TOF MS for the detection of 30 mutation sites in GJB2,SLC26A4 and mitochondrial 12S rRNA was established.The detection results were 100%consistent with clinical diagnosis.Samples from 373 undiagnosed NSHL patients were tested using this method.The results showed that 56.30%(210/373)of the patients carried at least one mutation.Among them,155 patients with compound or homozygous mutations could be clearly diagnosed,accounting for 41.55%of the tested population;210 positive mutation carriers involved 55 genotypes,of which 5 had multiple gene mutations.The Sanger sequencing results showed that the first-generation sequencing results of 56 samples with positive results were 100%consistent with the MALDI-TOF MS detection results.Conclusions:The single reaction method based on MALDI-TOF MS to detect 30 hotspot mutations in deaf genes GJB2,SLC26A4 and mitochondrial 12S rRNA is accuracy,high throughput and low cost.It is expected to become a new effective means for the etiological examination and preventive screening of hereditary hearing loss.

关 键 词：非综合征型耳聋 基因检测 飞行时间质谱 

分 类 号：R394.3[医药卫生—医学遗传学]