目标基因捕获结合第2代测序技术检测原发性高血压致病基因突变  

Identification of mutations of disease-causing gene in essential hypertension based on targeted gene capture and the second generation sequencing technology

作　　者：郭俊[1,2] 蔡伦[1,2] 李小燕[1,2] 王绿娅[1,2] 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教育部心血管重塑相关疾病重点实验室,北京100029

出　　处：《新乡医学院学报》2014年第6期414-416,419,共4页Journal of Xinxiang Medical University

基　　金：国家自然科学基金资助项目(编号:81200074)

摘　　要：目的建立目标基因捕获结合第2代测序技术,对原发性高血压（EH）患者的13个已知致病基因进行突变筛查,探讨EH与致病基因突变的关系。方法提取5例EH患者外周血全基因组DNA,利用GenCap目标基因捕获技术,设计内皮素转化酶1（ECE1）、G蛋白调节信号5（RGS5）、钠钾三磷腺苷酶通道β1多肽（ATP1β1）、选择素E（SELE）、血管紧张素原（AGT）、血管紧张素受体1（AGTR1）、α-内收蛋白（ADD1）、细胞色素P450 3A亚家族多肽5（CYP3A5）、一氧化氮合酶3（NOS3）、鸟嘌呤核苷酸结合蛋白β3（GNβ3）、一氧化氮合酶2A（NOS2A）、苯基乙醇胺N-甲基转移酶（PNMT）和前列腺素I2合成酶（PTGIS）基因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 HiSeq 2000第2代测序仪进行测序,通过数据分析,确定突变位点,对选定的突变位点用Sanger测序法验证。结果设计合成的目标基因特异性捕获探针可有效捕捉并富集基因组DNA的目标靶片段。目标区域平均测序深度为301.12~431.92,99.30%~99.70%目标区域覆盖度。5例患者中ADD1基因发现3个错义突变;AGT基因发现1个错义突变;SELE基因发现2个错义突变;PNMT基因发现1个剪切位点突变;PTGIS基因发现1个错义突变。结论 GenCap目标基因捕获技术结合Illumina HiSeq 2000第2代测序技术成功发现了EH患者的致病基因突变。该方法快速有效,对深入研究EH的分子病因学有重要意义。Objective To develop an approach based on targeted gene capture and the second generation sequencing in order to determine the genetic defects in patients with essential hypertension（ EH） precisely and effectively and confirm the role of 13 known genes in the pathogenesis of essential hypertension. Methods Peripheral blood was collected and genomic DNA was isolated from 5 EH patients. A total of 13 known genes of EH were selected for deep exome resequencing. Endothelinconverting enzyme 1（ ECE1）,regulator of G protein signaling 5（ RGS5）,adenosine triphosphatase（ ATPase）,Na＋/ K＋transporting,beta-1 polypeptide（ ATP1β1）,selectin E（ SELE）,angiotensinogen（ AGT）,angiotensin receptor 1（ AGTR1）,adducin 1（ ADD1）,cytochrome P450,subfamily ⅢA,polypeptide 5（ CYP3A5）,nitric oxide synthase 3（ NOS3）,guanine nucleotidebinding protein,beta-3（ GNβ3）,nitric oxide synthase 2A（ NOS2A）,phenylethanolamine N-methyltransferase（ PNMT） and prostaglandin I2 synthase（ PTGIS） were selected by a gene capture strategy,using GenCap targeted gene capture technology. The enrichment libraries were sequenced on Illumina HiSeq 2000 sequencer to determine the mutation in these genes. Selected variants were further validated by Sanger sequencing. Results For the samples subjected to targeted gene capture and the second generation sequencing,the average sequencing depths on the targeted regions were yielded from 301. 12- 431. 92. Meanwhile,coverage of targeted exons was ranged from 99. 30% to 99. 70%. Three missense mutations in ADD1,one missense mutations in AGT,two missense mutations in SELE,one splice site mutation in PNMT and one missense mutation in PTGIS were identified. Conclusions Disease-causing of EH patients is determined by the robustness of GenCap targeted gene capture and Illumina HiSeq 2000 sequencer precisely and rapidly. The method provides a reliable strategy for routine gene diagnosis of EH.

关 键 词：原发性高血压 致病基因 目标基因捕获 第2代测序 

分 类 号：R544.1[医药卫生—心血管疾病]