甜菜碱代谢通路基因遗传变异对PCI术后冠心病患者氯吡格雷疗效的影响及机制研究

Clopidogrel is the basic therapy for coronary artery disease (CAD). Remarkable interindividual difference in clopidogrel efficacy is observed, which is an independent risk factor for major cardiovascular event (MACE) for CAD patients after percutaneous coronary intervention (PCI). The known genetic factors only partially account for the interindividual difference in clopidogrel response. In our previously study, we observed that the rs1047891 (Thr1406Asn) polymorphism in the gene encodes carbamoyl phosphate synthase 1 (CPS1) could lead to decreased clopidogrel response and plasma concentration of the clopidogrel active metabolite H4 in CAD patients after PCI, and interference of CPS1 expression in cultured HepG2 cells could upregulated the expression of clopidogrel activation enzymes including CYP3A4 and CYP2C19 and dereased the intracellular content of glycerine, a metabolite of important methyl donor betaine in the body. Treatment of HepG2 cells with betaine and glycerin increased and decreased the expression of both CYP3A4 and CYP2C19, respectively. Based on these preliminary findings, our program aim to make clear the influences of metabolites in the betaine metabolic pathway as well as genes in the metabolic pathway on pharmacokinetics and pharmacodynamics of clopidogrel in CAD patients after PCI, and to explore whether CPS1 and the pathway polymorphisms functions through affecting betaine metabolism and subsequent DNA methylation of genes involved in clopidogrel metabolism and transportation by using technologies including Co-immunoprecipitation (CoIP) and methylation sequencing. The purpose of the study is to identify new pharmacogenomics biomarkers responsible for interindividual difference in clopidogrel response, as well as provide basis for the individualized use of clopidogrel.

氯吡格雷是冠心病治疗的基石，其疗效个体差异是PCI术后主要心血管事件发生的独立风险因素，目前已知的遗传变异仅能部分解释其疗效个体差异现象。本项目在前期2中心临床研究发现氨甲酰磷酸合成酶1（CPS1）基因多态性可导致PCI术后患者氯吡格雷反应性降低、血浆氯吡格雷活性代谢产物浓度下降；干扰CPS1可上调肝细胞内氯吡格雷活化代谢酶CYP2C19/CYP3A4表达、降低细胞内甜菜碱代谢产物甘氨酸的含量，甜菜碱和甘氨酸可分别上调和下调肝细胞内CYP2C19/CYP3A4表达的基础上，拟临床研究、细胞和动物实验相结合，查明甜菜碱代谢通路代谢酶遗传变异及代谢物对氯吡格雷药代和药效的影响，并应用免疫共沉淀、DNA甲基化测序等技术，查明相关基因遗传变异是否通过影响甜菜碱的代谢，进而影响氯吡格雷代谢酶基因的甲基化和表达而实现。该项目旨在发现氯吡格雷反应个体差异新的药物基因组标志物，为该药的个体化用药奠定基础。

本项目从药物基因组学角度出发，临床研究与细胞、动物实验相结合，查明了CPS1Thr1406Asn（rs1047891）多态性与冠心病患者的预后的关系，以及受CPS1影响的血浆代谢物甜菜碱和TMAO与氯吡格雷反应性、冠心病患者不良事件发生及预后的相关性，并进行机制探讨。结果发现，CPS1 rs1047891携带A等位基因的冠心病患者心肌梗死发生率显著升高，且与PCI术后1年内MACE事件发生存在显著关联，而在缺血性脑卒中患者中CPS1 rs1047891对服药前后血小板聚集率MAR及血浆TMAO水平无影响，发现血浆甜菜碱及TMAO水平均可影响冠心病患者MACE事件的发生及氯吡格雷反应性；动物实验发现甜菜碱可能通过影响小鼠肝脏氯吡格雷活化代谢酶的表达而影响氯吡格雷反应性。通过对PCI术后冠心病患者进行TMAO生成关键酶基因FMO3 rs1736557多态性进行分析，发现FMO3 rs1736557多态性影响氯吡格雷的反应性。体外孵育实验及细胞实验显示，TMAO可能通过影响氯吡格雷活性代谢生成酶的活性及血小板P2Y12通路从而影响氯吡格雷反应性。然而，血浆甜菜碱及TMAO水平对氯吡格雷反应性的影响在缺血性脑卒中患者中未得到验证，而发现氯吡格雷及其活性代谢产物可能通过影响血浆TMAO水平发挥作用；细胞实验发现氯吡格雷影响TMAO代谢酶MTARC1 mRNA及蛋白表达，且动物实验发现氯吡格雷给药后可上调大鼠血浆TMAO的水平。该发现为临床心脑血管疾病的个体化治疗奠定了理论基础。此外，项目还发现GAS5 表达及其rs55829688多态性、GAS5-miR-223-P2Y12调控轴均对氯吡格雷反应性产生影响。同时通过对氯吡格雷极端反应个体进行的GWAS初步发现PSMA2基因多态性对氯吡格雷的反应性有影响，为从全基因组水平及代谢物水平探讨心脑血管疾病患者氯吡格雷反应性和预后个体差异机制、构建个体化用药与治疗提供了范例。