二代测序法用于先天性长QT综合征临床基因检测的假阴性分析  

False-negative Possibility in Genetic Test of Congenital Long QT Syndrome by Next-generation Sequencing

作　　者：李新[1] 刘念[1] 白融[1] 冯莉[1] 阮燕菲[1] 马长生[1] 

机构地区：[1]首都医科大学附属北京安贞医院心内科国家心血管病临床医学研究中心,北京市100029

出　　处：《中国循环杂志》2017年第8期771-775,共5页Chinese Circulation Journal

基　　金：国家自然科学基金(81500246;81470465);北京市自然科学基金(7161003);北京市医管局临床医学发展专项(ZYLX201302)

摘　　要：目的:探讨二代测序法在先天性长QT综合征(LQTS)临床基因检测中的假阴性问题。方法:选取2个商业医学检验实验室(Lab1和Lab2,Hi Seq2000测序平台)、1个商业科研服务实验室(Lab3,Ion Torrnet测序平台)和1个学术机构实验室(Lab 4,Hi Seq2000测序平台)产生的共28例样本数据(Lab1:6例;Lab2:8例;Lab3:8例;Lab4:6例),定量分析LQTS的三个主要致病基因KCNQ1、KCNH2和SCN5A外显子区域测序覆盖度以及可能漏检的致病变异数目。结果:采用Hi Seq2000测序平台的3个实验室(Lab1、Lab2和Lab4)中,三个致病基因外显子区域覆盖度>10倍的比例均高于98%,覆盖度>30倍的区域介于90%~95%。KCNQ1在两个商业医学检验实验室的14例样本中,低于10倍和30倍覆盖的外显子区域比例平均为3.63%和9.84%;低于10倍覆盖区域集中在第一外显子,平均包含约2%的已知致病或疑似致病变异。KCNH2在两个商业医学检验实验室14个样本中,低于10倍和30倍覆盖的区域分别为2.64%和15.76%,低覆盖区分布在多个外显子中。Lab1的数据中,KCNH2低于30倍覆盖区域最高达28.56%,其内包含已知致病或疑似致病变异113个(19.79%)。SCN5A的整体覆盖度最好,四个实验室的数据都不存在低于10倍覆盖的区域,其中两个商业医学检验实验室也不存在低于30倍覆盖的区域。结论:当前的LQTS基因二代测序检测中,KCNQ1和KCNH2都存在一定程度的低覆盖区,因此普遍存在漏检致病变异的可能,假阴性问题值得高度重视。Objective： To explore the false-negative possibility in genetic test of congenital long QT syndrome（LQTS） by nextgeneration sequencing（NGS）.Methods： A total of 28 genomic DNA samples were collected from 4 laboratories including 2 commercial medical laboratories using Hi Seq2000 platform as Lab1, n=6 and Lab2, n=8; 1 commercial research service laboratory using Iontorrent platform as Lab3, n=8 and 1 academic laboratory using Hi Seq2000 platform as Lab 4, n=6. Sequencing coverage in the exons of protein-coding region in 3 main LQTS pathogenic genes as KCNQ1, KCNH2, SCN5 A and possible pathogenic variants were quantitatively analyzed. Results： In Lab1, Lab 2 and Lab 4 with Hi Seq2000 platform, above 98% protein coding regions in 3 pathogenic genes were covered with10-fold reads and 90%-95% were covered with30-fold reads. In 2 commercial medical laboratories, 3.63% and 9.84% protein coding regions of KCNQ1 gene in 14 samples were covered with10-fold reads and with30-fold reads; lower than 10-fold covering region was focused in the 1st exon including about 2% known or likely pathogenic variants. In 2 commercial medical laboratories, 2.64% and 15.76% protein coding regions of KCNH2 gene in 14 samples were covered with〈10-fold reads and with〈30-fold reads; low covering region was located in multiple exons. For the data from Lab 1, ashigh as 28.56% protein coding regions of KCNH2 gene were covered with30-fold reads including 113（19.79%） known or likely pathogenic variants. SCN5 A gene had the best coverage of protein coding region, with no10-fold reads in all 4 Labs and no30-fold reads in 2 commercial medical laboratories.Conclusion： Currently, NGS has low coverage region in both KCNQ1 and KCNH2 genes, pathogenic variants could be missed and false-negative possibility should be highly alert.

关 键 词：QT延长综合征 高通量核苷酸测序 基因 假阴性反应 

分 类 号：R54[医药卫生—心血管疾病]