GPR91基因结构/功能及其与藏族原发性高血压的关联

Essential hypertension(EH)is one of the most complex diseases that are thought to result from the interaction of genetic and enviromental factors. To date, the etiology of EH remains unknown. It is considered that the effective samples,such as genetically homo geneous,precise discriminative diagnosis and sufficient quantity, are requested to successfully identiffcation of the susceptibility genes for EH. In this proposal, all participants are Tibetan, who reside on the Qinghai-Tibet plateau, more than 3600 meters above sea level. Genetically, they are a relatively homogeneous isolate. This unique genetic background and altitude-hypoxia natural environment as well as the highest prevalence of EH(＞25％) in China make Tibetan an ideal ethnic group for study on the genetic factors and the interaction of genetic and environmental factors, which could contribute to the etiology and pathogenesis of EH . In the GWAS study , We have found that GPR91(SUCNR1) gene was positive association with EH susceptibility in Tibetan （OR=2.310, 95% CI=1.233~4.056）. In the present study , to identify the functional variants in GPR91 gene, the strategies of case-control association analysis and high-throughput next-generation sequencing technology were conducted, because of genome-wide significant SNPs offer morely Taq but do not provide direct information on the causal variants . Functional region of GPR91 gene was sequenced in cases-controls for each 100 using Ion Torrent (PGM), respectively. Variants mapping will be completed by bioinformation procedure. The difference of variant allele frequencies between cases and controls will be evaluated by P value and functional variants will be confirmed by the loss of function analysis(LOF). Statistical calculations use SAS solfware 13.0 version, interaction analysis of gene and gene will be implemented in the open-source MDR solfware package(v.1.00). We expect to find functional variant in GPR91 gene (including SNP, genotype, mini insertion/deletion, haplotype) and the interaction of gene and gene, and to clarify those variants pathway. The ultimate goal of this work is the application of information obtained on molecular level of essential hypertension mechanisms to the prevention, diagnosis and individual treatment as well as to development of new drug for hypertension.

原发性高血压（EH）是遗传与环境因素相互作用引起的复杂疾病，病因未明。遗传背景单一、诊断准确、足够数量的世居高原低氧环境的藏族人群样本与高通量测序技术是成功鉴定EH相关基因的决定因素。GWAS已发现GPR91基因与藏族EH相关，本项目以世居海拔3680~5000公尺的藏族为研究对象，应用ION Torrent技术，完成病例-对照各100例的GPR91基因功能区扩增子高通量测序。利用生物信息学程序绘制突变图谱；通过组间对比、功能丢失分析、群体频率分布及家系连锁分析，发现并证实功能性突变的真实性，经统计分析鉴定出与EH致病相关的SNPs、基因型、单倍型、多位点交互作用模型。用体外表达观察含不同启动子单倍型片段的表达载体在常氧/低氧条件下报告基因活性变化，以期阐明遗传与低氧因素相互作用对EH发病的影响，将分子水平获得的信息用于EH基因分型，为EH个体化治疗提供科学依据，为新药研发提供新靶点。

原发性高血压（EH）被认为是遗传与环境因素相互作用导致的复杂多基因病，病因未明。历年高血压流行病学调查资料表明，藏族EH具有遗传度高（＞77%，λs=2.4）和舒张压高的特点。藏族是我国主要少数民族之一，世代居住在海拔3000公尺以上的青藏高原，由于文化背景、宗教信仰、历史及地理环境的原因，使其形成遗传背景较单一的相对隔离人群。唯一的遗传背景和极端低氧自然环境，是研究EH遗传与环境因素相互作用引发高血压病因学的理想人群。. 为鉴定藏族EH易感基因，我们采用两步全基因组相关研究策略。第一步，应用Affymetrix SNP6.0芯片，完成基于高血压家系内无血缘关系病例（300例）/对照（300例）pooling DNA GWAS分析。根据GWAS质控标准，选择591,912个常染色体rs位点纳入分析。经Z检验和Bonfferroni校正后，发现600多个与EH相关（P＜5×10-5﹚rs位点，分布在1-22对染色体上，rs出现频数最多的依次为2号染色体（70rs），1号、3号、17号、11号、12号、5号、4号染色体。. 为提高GWAS结果的真实性，减少假阳性，我们应用SpectroCHip11-G384芯片，在病例/对照各1000例大样本验证108个rs（P＜5×10-5）位点的基因型及其频率分布，结果显示108个rs位点中21个位点与高血压明显相关（P＜0.05），其中12个rs位点与高血压易感相关，OR在1.3-6.8，P＜0.05；9个位点为EH保护性基因，OR＜0.810-0.045，P＜0.05。特别是rs9865108C/T位点的CT杂合基因型与藏族EH易感相关（OR=1.57）而与汉、蒙族EH无关联。且OR值随海拔高度升高而增加，在海拔4000公尺和4500-5010公尺，OR值分别为1.47和1.57。. Rs9865108定位于3号染色体3p25.1区，琥珀酸受体基因（SUCNR1）下游18.6kb，编码GPR91G-蛋白偶联受体。推测在低氧环境下该位点可能间接影响琥珀酸受体/琥珀酸信号通路介导肾素分泌而参与高血压发病。