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。世居高原低氧环境的藏族样本与高通量测序和Sequenom MassARRY技术相结合是成功鉴定EH相关基因的决定因素。本项目以世居海拔3680-5000公尺的藏族为研究对象，应用ION Torrent测序，完成病例-对照各100例的GPR91基因功能区高通量测序。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，发现功能性突变，应用spectroCHip11-G384芯片完成病例-对照各500例的大人群验证，证实功能性相关突变的真实性，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，以阐明遗传与低氧因素相互作用致EH的发病机理，为EH预防、个体化治疗和新药研发提供科学依据。

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, high-throughput next-generation sequencing technologies, and spectroCHip11-G384 were conducted. Because of genome-wide significant SNPs offer morely Tag 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.

原发性高血压是遗传与环境相互作用引起的复杂疾病，迄今为止它的遗传学病因，特别是功能性致病基因尚不知晓。本项目以世局海拔3680-5000公尺青藏高原遗传背景较单一的藏族为研究对象，应用ION TORRENT测序技术，通过病例/对照相关分析，鉴定与藏族原发性高血压表型相关的琥珀酸受体91基因功能性变异位点。前期全基因组相关研究已发现rs9865108位点与藏族原发性高血压易感相关，OR=2.310（95%CI=1.233-4.056，p<0.05）。该位点定位于3号染色体25.1区的琥珀酸受体（SUCNR1，即GPR91）基因下游（约1.7kb）。我们假设GPR91基因多态性可能与藏族原发性高血压表型相关。因为，GWAS显著差异常常发生在少数Taq位点，并未提供直接具有致病作用的功能性变异信息。为了获得GPR91基因中可能与高血压表型相关功能性变异，我们完成了基于pooling DNA的病例/对照组分别混合DNA的功能区二代测序。结果未发现病例/对照组间GPR91基因外显子序列中存在具有统计学差异的变异位点。但在5'-非翻译区存在具有接近统计学差异变异位点，有待完成大样本的群体验证和功能研究。