基于靶基因文库的高通量测序平台建立肥厚型心肌病患者致病基因突变检测方法

目的基于靶基因文库,应用下一代半导体高通量测序平台,建立快速、准确的肥厚型心肌病(hypertrophic cardiomyopathy,HCM)常见致病基因突变检测方法,有利于HCM患者的早期预防及临床分子诊断。方法选择国内外公认的与HCM致病相关的常见基因(MYH7,MYBPC3,TNNT2,TNNI3,ACTC1,PRKAG2,MYL2和MYL3),应用Primer5及Primerselect引物设计软件对靶基因编码区进行引物设计。基于下一代半导体高通量测序平台,对2019年3月至8月在大理大学第一附属医院采集的4例HCM患者外周静脉血液标本,提取基因组,构建靶基因文库,进行上机测序,测序质量评估及变异位点注释。最后对注释分析得到的与HCM致病相关的突变位点进行Sanger测序验证。结果通过建立的高通量测序方法,检出了4个与HCM致病相关的突变位点MYBPC3-c.478 C>T,MYH7-c.161 G>A,TNNI3-c.370 G>C和MYL3-c.337 A>G,并通过金标准的Sanger测序验证,结果完全一致。结论基于靶基因文库及高通量测序平台建立的HCM患者常见致病基因突变检测方法,能为临床分子诊断实验室工作者开展遗传基因突变检测提供参考.

Detection Methods for Gene Mutations in Patients with Hypertrophic Cardiomyopathy by Using the Target Gene Library and Highthroughput Sequencing Platform

Objective This study aimed to establish a rapid and accurate detection method for the mutations of common pathogenic genes in hypertrophic cardiomyopathy(HCM)by using the next-generation high-throughput sequencing platform based on the target gene library,which is con-ducive to the early prevention and clinical molecular diagnosis of HCM patients..Methods The candidate common genes(MYH7,MYBPC3,TNNT2,TNNI3,ACTC1,PRKAG2,MYL2 and MYL3)that were reported to cause HCM were selected.Whole coding exons of the eight HCM path-ogenic genes were designed by using Primer5 and PrimerSelect software.Peripheral blood samples of four HCM patients were harvested from the Affiliated Hospital of Dali University during March to Au-gust 2019.The genome was extracted to construct a target gene library.High-throughput sequencing was performed,the quality of the sequencing evaluated,and the mutation sites annotated.Finally,the pathogenic gene mutations of HCM were verified by Sanger sequencing.Results Four mutations MYBPC3-C.478 C>T,MYHl-c.161 G>A,TIVNB-c.370 G>C and MYL3-C.377 A>G were de-tected using high-throughput sequencing technology.The result was consistent with that of the gold standard Sanger sequencing.Conclusion The detection method for common pathogenic gene muta-tions in HCM patients based on the target gene library and high-throughput sequencing platform can provide references to carry out genetic mutation detection in clinical molecular diagnostic laboratory.