利用目标基因捕获结合第二代测序技术发现腹主动脉瘤致病基因突变  

Targeted Gene Capture and Next-generation Sequencing in Abdominal Aortic Aneurysm Identifies Mutations in Disease-causing Gene

作　　者：郭俊[1,2] 蔡伦[1,2] 李小燕[1,2] 王绿娅 杜杰[1,2] 

机构地区：[1]首都医科大学附属北京安贞医院 [2]北京市心肺血管疾病研究所心血管重塑相关疾病教育部重点实验室,北京市100029

出　　处：《中国动脉硬化杂志》2015年第2期153-155,共3页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金资助项目(81200074);科研基地建设-心血管重大疾病防治协同创新中心资助项目(PXM2013_014226_07_000088)

摘　　要：目的建立目标基因捕获结合第二代测序技术对腹主动脉瘤患者原纤维蛋白1(FBN1)基因进行突变筛查,探讨腹主动脉瘤与FBN1基因突变的关系。方法提取4例腹主动脉瘤患者外周血全基因组DNA,利用Gen Cap目标基因捕获技术,设计FBN1的65个外显子区域特异性捕获探针,与基因组DNA文库进行杂交,将目标基因组区域的DNA片段进行富集后,再利用Illumina Hi Seq2000第二代测序仪进行测序,通过数据分析,确定突变位点,用Sanger测序法对突变位点进行验证。结果设计合成的目标基因特异性捕获探针可有效地捕捉并富集基因组DNA的目标靶片段。4例患者目标区域平均测序深度为448.15~536.61,99.5%~99.7%目标区域覆盖度。经过数据分析及Sanger测序验证发现1个新的错义突变c.2753 C>G(p.Pro918Arg),db SNP137数据库、千人基因组及内部800名正常汉族人数据库均无此突变。经SIFT预测为有害突变。结论本研究所建立的Gen Cap目标基因捕获技术结合Illumina Hi Seq2000第二代测序技术成功地发现了FBN1的新突变。该方法快速而有效,对腹主动脉瘤分子病因学有更好的认识。Aim To develop an approach based on targeted gene capture and next-generation sequencing to determine the genetic defects in patients with abdominal aortic aneurysm precisely and effectively and confirm the role of fibrillin-1( FBN1) in the pathogenesis of abdominal aortic aneurysm. Methods We analyzed four abdominal aortic aneurysm patients. Peripheral blood was collected and genomic DNA was isolated. FBN1 were selected by a gene capture strategy,using Gen Cap custom enrichment kit. The enrichment libraries were sequenced on Illumina Hi Seq 2000 sequencer for paired read 90 bp to determine the mutation frequency in FBN1. Variants which have been reported in db SNP137 or 1000 genome and in-house database were filtered. Selected variants were further validated by PCR and Sanger sequencing. Results For the samples subjected to targeted next-generation sequencing,the average sequencing depths on the targeted regions were yielded from 448. 15 to 536. 61. Meanwhile,coverage of targeted exons were ranged from 99. 5% to 99. 7%. We identified one missense mutation in FBN1: c. 2753 C > G( p. Pro918Arg),which was validated by Sanger sequencing. Conclusions Our results confirm the role of FBN1 in the pathogenesis of abdominal aortic aneurysm and demonstrated the robustness of targeted next-generation sequencing to precisely and rapidly determine genetic defects.腹主动脉瘤( abdominal aortic aneurysm,AAA)

关 键 词：腹主动脉瘤 原纤维蛋白1 目标基因捕获 第二代测序 

分 类 号：R543.1[医药卫生—心血管疾病]