代谢酶基因组eQTL对氯吡格雷活性代谢物生成的影响及其功能评价

The individual difference in the formation of clopidogrel active metabolites (AM) is the major source for the individual difference in efficacy of clopidogrel antiplatelet treatment. Published data and our primary data indicated that current known genetic variations were far from sufficient to explain the individual difference in the formation of clopidogrel AM. One of the reasons is that many functional variations were not included, even more than half of expression quantitative trait loci (eQTL) were missed in previous studies. We hypothesize that the discovery of eQTL will improve the predictivity of the individual difference in the formation of clopidogrel AM and occurrence of adverse clinical endpoints. In the proposal, based on previous results, targeted next-generation sequencing and the Network Edge Orienting (NEO) method will be applied to discover causal eQTLs for the formation of clopidogrel AM. The effect of eQTLs in hotspot with the most strong causal relationship with the formation of clopidogrel AM on the pharmacokinetics and antiplatelet efficacy of clopidogrel in patients with coronary artery disease will be evaluated. A risk predictive model with eQTL genotypes will thereafter be developed. Several techniques, including allelic expression imbalance analysis and luciferase Reporter Gene Assays, will further be applied to illustrate the regulation mechanism of eQTL on gene expression. The results of this study will give us insight into the function of genetic variation in metabolic enzyme genes and will help clinic optimize the antiplatelet therapy strategy of clopidogrel in patients with coronary artery disease after percutaneous coronary intervention.

氯吡格雷活性代谢物（AM）生成的个体差异是氯吡格雷抗血小板疗效个体差异的关键来源。我们前期研究及发表数据显示，已知的基因变异不足以解释氯吡格雷AM生成的个体差异，这与以往的研究纳入的功能性基因变异数量不完全有关，甚至有一半以上基因表达数量性状位点（eQTL）没有被纳入。我们推测发现原因性eQTL可提高对氯吡格雷AM生成个体差异及临床不良终点事件的预测。本研究拟在以往研究的基础上，针对代谢酶基因组eQTL富集区域，用目标区域捕获测序和贯穿分析等方法发现影响氯吡格雷AM生成的eQTL；然后在冠心病患者中评价因果关系最强的热点区域eQTL对氯吡格雷AM药动学和疗效中间指标的影响，及对临床终点事件的预测作用；进一步用等位基因不平衡表达分析和荧光素酶报告基因技术等评价eQTL对基因表达的调控作用。本研究将有助于深入了解代谢酶基因变异功能，并有助于危险分层，优化抗血小板给药方案，减少临床终点事件。

氯吡格雷活性代谢物H4生成的个体差异是氯吡格雷抗血小板疗效个体差异的关键来源。本项目分析了中国人肝组织代谢酶基因eQTL，验证了CYP2D6和VKORC1等基因存在eQTL，但是这些eQTL对氯吡格雷活性代谢物的生成没有达到全基因组显著性。为了发现影响氯吡格雷药动学和抗血小板效应的新基因位点，我们通过三个阶段的全基因组关联性研究、并通过验证和功能解析，发现分别位于转运体基因SLC14A2和ABCA1的rs12456693和rs2487032，位于代谢酶基因N6AMT1的rs2254638显著影响氯吡格雷药动学和抗血小板效应。多元回归模型显示这些SNP显著提高了对氯吡格雷治疗后血小板集聚率PRU个体差异的解释程度，从原来的约20%增加到37.7%；用独立人群验证了这些新变异位点对氯吡格雷活性代谢物H4药动学的影响，功能研究表明N6AMT1影响氯吡格雷活化；携带N6AMT1 rs2254638基因变异的患者发生MACE风险增加。我们进一步对入选的PCI术后冠心病患者，用全外显子组分析和目标基因捕获测序分析与MACE相关的基因变异。荟萃整合分析显示在MYOM2 基因中rs17064642与MACE有显著相关，P值达到全基因组显著性(P = 2.95 × 10−9)，CC/CT携带者出现MACE风险显著高于不携带者(17.8% vs. 8.1%; HR, 2.76; 95% CI, 1.98–3.87)。根据GTEx数据库，WDR24基因的两个SNP rs11640115和rs763053在心脏组织中都是eQTL (P=1.16 × 10−6)。我们也观察到另外5个SNP趋于显著（P<1.0 × 10−6），包括KRTAP10-4 (rs201441480), WDR24 (rs11640115), ECHS1 (rs140410716), AGAP3 (rs75750968) and NECAB1 (rs74569896)。其中ECHS1在线粒体脂肪酸 β-氧化途径的第二步中起作用，产生水合酶/异构酶家族成员。ECHS1基因对心肌重构的影响，在动物模型中，Echs1基因表达心肌重构组明显比假手术组低。在验证冠心病心衰患者中血浆ECHS1含量显著少于无心衰或轻度心衰患者。总之，本研究首次系统地整合药动学和抗血小板效应进行GWAS，发现了新的影响氯吡格雷药动学和疗效的功能性基