氯吡格雷治疗下CYP2C19与P2Y热点突变致血小板高反应性的效应研究

Influence of CYP2C19 and P2Y hotspot mutations on high platelet reactivity in patients with clopidogrel intake

目的 研究氯吡格雷治疗患者CYP2C19和P2Y热点突变导致血小板因药物耐受而出现高反应性的效应关系,探索检出突变相关功能改变的临床适用方法.方法 病例对照研究.选取104例接受抗血小板治疗且冠状动脉介入术前服用负荷剂量（300 mg/d）氯吡格雷的冠状动脉粥样硬化性心脏病（冠心病）患者,在术后12 ～24 h内测定血小板功能,通过血栓弹力图（TEG）检测血小板抑制率,通过流式细胞术检测CD62p和血管扩张刺激磷蛋白（VASP）;焦磷酸测序法测定中国人群药物代谢酶热点突变CYP2C19＊2（681G＞ A）、CYP2C19＊3 （636G＞ A）和血小板膜糖蛋白受体基因P2Y1 （893C ＞T）、P2Y12 （52G＞ T）4个位点基因多态性.以CYP2C19分型结果为依据分为野生组（＊1/＊ 1）、杂合突变组（＊2/＊1）、纯合突变组（＊2/＊2）,结合其他3个位点突变情况,分析各组氯吡格雷作用下的血小板功能差异.应用单因素方差分析进行不同基因型组间比较,两组间比较选用t检验及非参数检验进行统计学分析.结果 野生组血小板反应指数（PRI）为（35.75±23.11）％,杂合突变组为（48.77 ±24.22）％,纯合突变组为（66.73±15.74）％,差异有统计学意义（F=9.170,P＜0.05）;野生组CD62p水平为（9.38±11.16）％,杂合组为（9.45±8.91）％,纯合突变组为（10.87±8.31）％,差异无统计学意义（F=0.087,P＞0.05）;37例受试个体中未检出P2Y1（893C＞ T）位点突变,检出1例P2Y12（52G＞ T）位点突变.结论 CYP2C19＊2突变与氯吡格雷疗效降低密切相关,VASP检测的血小板反应指数可有效反映其改变.

Objective To investigate high platelet reactivity effective and relative strength that induced by the CYP2C19 and P2Y hotspot mutations,and explore the applicable testing methods in clinical settings.Methods A case-control study was conducted.104 patients with coronary heart disease were enrolled who administered antiplatelet agent treatment and a 300 mg loading dose of clopidogrel within 12-24h before a PCI.ADP inhibition rate was tested by TEG and flow cytometry method was used to detect VASP index （PRI） and CD62p.The citrated and heparined venous whole blood collected should be collected within 12-24 h after PCI.Genotyping was carried out by pyrophosphate method for the hotspot mutations of drug metabolism in Chinese population concluded CYP2C19 ＊ 2 （681G 〉 A）,CYP2C19 ＊ 3 （636G 〉 A）and the platelet glycoprotein receptor gene concluded P2Y1 （893C 〉 T） and P2Y12 （52G 〉 T）.According to the CYP2C19 ＊ 2 sequencing results,patients were grouped into three groups,the wild-type group （＊ 1/＊ 1）,the heterozygous group （＊ 1/＊ 2） and the homozygous group （＊ 1/＊ 2）.Combined with other three mutation sites,platelet function was analyzed among different groups.One-Way ANOVA was used for analysis of variance between groups of different genotypes,and t test and non-parametric test were selected for statistical analysis between the two groups.Results The PRI of wild-type group （35.75 ± 23.11） % was significantly lower （F =9.170,P 〈 0.05） than the heterozygous group （48.77 ± 24.22） % and homozygous group （66.73 ± 15.74） %.No significant difference （F =0.087,P 〉 0.05） was existed on CD62p among the wild-type group （9.38 ± 11.16）%,the heterozygous group （9.45 ± 8.91）% and homozygous group （10.87 ±8.31）%.One P2Y12 （52G 〉 T） mutation was found while no P2Y1 （893C 〉 T） mutation was detected among 37 patients.Conclusion The risk of a low response to clopidogrel was related to the carry of CYP2C19 loss-of-function alleles,and the change can be effectively reflected by VASP analysis.