P2RY12基因多态性及启动子区DNA甲基化对氯吡格雷抵抗的影响机制与定量研究

Introduction: Clopidogrel resistance (CR) is a major cause of recurrent ischemic events in ischemic cerebrovascular disease patients after intervention. Genetic factors are directly related to CR. We previously found that there was no statistical difference in the concentrations of clopidogrel active metabolite between ischemic event patients and non-ischemic event patients. Scientific question: what is the relationship between the drug target P2RY12 gene and CR? Scientific hypothesis: P2RY12 gene polymorphisms and the promoter DNA methylation through regulation of gene expressions prevent clopidogrel platelet inhibition leading to ischemic events. Research contents: 1) selection of P2RY12 Tag SNP genetic and functional SNP loci based on Chinese population; 2) study the correlations among site variations and gene expression, platelet activity and ischemic events; 3) study the correlations among P2RY12 gene promoter DNA methylation and gene expression, platelet activity and ischemic events; 4) build clopidogrel’s population pharmacokinetic/pharmacodynamic model based on gene regulatory mechanisms to quantify the impact of P2RY12 gene on platelet activity and ischemic events. Goal: to clarify the mechanisms of P2RY12 gene polymorphisms and promoter DNA methylation affecting CR and to establish a quantitative relationship. Significance: to achieve an accurate screening of CR in patients and individualized clopidogrel dosing.

研究背景：氯吡格雷抵抗是导致缺血性脑血管病介入术后患者缺血事件再发的重要原因，遗传因素与药物抵抗直接相关。我们前期发现：出现与未出现缺血事件患者的活性代谢物浓度无统计学差异。科学问题：药物靶点基因P2RY12与药物抵抗的关系?科学假说：P2RY12基因多态性和启动子区DNA甲基化通过基因表达的调控机制，抵抗药物的抗血小板作用，导致了缺血事件再发。研究内容：选择基于中国人群的P2RY12基因Tag SNP和功能性SNP位点，考察位点变异与基因表达、血小板活性和缺血事件的相关性；考察P2RY12基因启动子区DNA甲基化水平对基因表达、血小板活性和缺血事件的影响；构建基于基因调控机制的氯吡格雷群体药动/药效模型，定量描述基因对血小板活性和缺血事件的影响。目标：阐明P2RY12基因多态性和启动子区DNA甲基化对药物抵抗的影响机制并建立定量关系。意义：实现对药物抵抗患者的准确筛查和个体化给药。

项目背景：氯吡格雷抵抗是导致缺血性脑血管病介入术后患者缺血事件再发的重要原因，遗传因素与药物抵抗直接相关。我们前期发现：出现与未出现缺血事件患者的活性代谢物浓度并无统计学的差异。因此，药物靶点基因P2RY12与药物抵抗的关系成为一个重要的科学问题。我们推测，P2RY12基因多态性和启动子区DNA甲基化通过基因表达的调控机制，抵抗药物的抗血小板作用，导致了缺血事件再发。.研究内容：筛选缺血性脑血管病介入术后患者，采集其生理病理相关信息，介入术后一年内进行跟踪随访，记录其临床事件；选择基于中国人群的P2RY12基因Tag SNP和功能性SNP位点，考察位点变异与患者的血小板活性和缺血事件的相关性；考察P2RY12基因启动子区DNA甲基化水平对血小板活性和缺血事件的影响；通过一系列的统计分析，准确描述该基因对血小板活性和缺血事件的影响。.重要结果：我们检测了P2RY12基因的12个CpG位点，发现其中的三个位点（CpG11 and CpG12 + 13）在病例与对照组患者中存在显著差异，即低甲基化导致了血小板功能较强，临床事件发生率较高。此外，我们还发现P2Y12基因的两个变异位点（rs9859538和rs10935842）与增加的临床事件相关，而且两位点存在着强连锁关系。.关键数据：P2RY12基因的甲基化与SNP位点检测可提供患者抗血小板临床疗效以及临床事件预后信息。.科研意义：阐明了P2RY12基因多态性和启动子区DNA甲基化对药物抵抗的影响，可实现对药物抵抗患者的准确筛查和个体化给药。