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目的应用第二代测序技术结合DNA pooling策略和生物信息学分析技术寻找原发性高血压的易感基因。方法选择2014年6月至2015年6月深圳市人民医院心内科及重症医学院科门诊和住院的患者,以100例原发性高血压患者和100名健康人分别建立DNA pooling,应用第二代测序技术进行全基因组重测序。使用BWA软件对测序结果进行比对,分别对单核苷酸多态性(SNP)、插入缺失(InDel)、基因拷贝数变异(CNV)、单核苷酸变异(SNV)、结构变异(SV)等变异进行检测,应用ANNOVAR软件对所有的变异序列进行注释。针对检测到的基因变异位点数据,采用Fisher精确检验进行检验分析,检测达到统计学意义(P<0.01)的位点即推测为与疾病相关的变异位点,然后利用公共数据库进行过滤,统计突变类型,并且将其在全基因组水平上进行分布分析,利用基因本体功能注释、京都基因和基因组百科全书(KEGG)通路分析、突变基因相互作用分析对筛选出的变异位点进行生物信息学检验,推测可能参与原发性高血压发病的易感基因。结果分组检验分析得到33 919个SNP位点、18 594个插入缺失位点、352个结构变异位点和88 707个CNV位点。纤维连接蛋白1(FN1)、蛋白激酶N1(PKN1)、融合蛋白19(CD19)、细胞分裂周期蛋白5样蛋白抗体(CDC5L)这4个位点是互相作用网络通路流经数据量最大的几个节点。结论通过全基因组重测序技术获得了全基因组水平上原发性高血压多种基因突变,而且发现尚未报道过的FN1、PKN1、CD19、CDC5L可能是原发性高血压发病过程中的关键节点基因。
Objective To search for the susceptibility genes of essential hypertension by using the second generation sequencing technology combined with DNA pooling strategy and bioinformatics analysis. Methods From June 2014 to June 2015, patients with outpatient and inpatient department of Cardiology and Critical Care Medicine in Shenzhen People’s Hospital were enrolled. DNA pooling was established in 100 patients with essential hypertension and 100 healthy individuals, respectively. The second Genome-wide Sequencing by Genome Sequencing Technique. BWA software was used to compare the sequencing results. SNPs, InDel, CNV, SNV, SV ) And other mutations were detected, the application of ANNOVAR software annotation of all the mutation sequences. According to the data of gene mutation loci detected, the Fisher exact test was used to test and analyze. The loci that reached statistical significance (P <0.01) were presumed to be disease-related variation loci, and then filtered by using public database The genotypes of the mutants were genotyped and analyzed at genome-wide level. Bioinformatics tests were performed on the selected mutated sites using gene ontology functional annotation, KEGG pathway analysis and mutational gene interaction analysis , Speculated that may be involved in the pathogenesis of essential hypertension susceptibility genes. Results A total of 33 919 SNPs, 18 594 insertions, 352 structural variants and 88 707 CNV sites were obtained by group analysis. FN1, PKN1, fusion protein 19 (CD19), and CDC5L are the four sites at which the largest amount of data flows through the interacting network pathways Node. Conclusion Genome-wide multiple gene mutations of essential hypertension have been obtained through genome-wide resequencing. FN1, PKN1, CD19 and CDC5L, which have not been reported, may be the key node genes in the pathogenesis of essential hypertension .