| Clopidogrel,as the first-line antiplatelet therapy of coronary heart disease(CHD),can effectively reduce mortality and recurrent ischemic events in CHD patients after percutaneous coronary intervention(PCI).However,the antiplatelet efficacy of clopidogrel is associated with substantial individual variability which remains unexplained currently.Clopidogrel is metabolized into active metabolite H4 in vivo to play its role in antiplatelet aggregation,so gene polymorphism involved in clopidogrel metabolism may have important influence on its individual differences of antiplatelet effect.Therefore,this study conducted a genome-wide association analysis(GWAS)jointing analysis of antiplatelet and pharmacokinetic response to clopidogrel to systematically identify the new functional genetic variants affecting its efficacy in Chinese patients with CHD,and evaluated the predictable effects of the new gene variants on the risk of major adverse cardiac events(MACE).Firstly,the GWAS of on-treatment P2Y12 reaction unit(PRU)was performed to identify SNPs associated with antiplatelet effects of clopidogrel in 115 patients with CHD.Combination analysis of the plasma concentration of H4,and particularly focusing on the genes involved in ADME,18 candidate SNPs with pharmacokinetic mechanisms associated with antiplatelet response to clopidogrel were selected.To investigate the association between candidate SNPs with the pharmacokinetics of clopidogrel,a replication study in 31 patients with CHD was performed along with pharmacokinetic assessment after a loading dose of clopidogrel and genotype of 18 candidate SNPs.The association of different genotypes with pharmacokinetic parameters of clopidogrel and H4(Cmax,Tmax,AUC0-4h)was analyzed.Besides CYP2C19*2,we discovered that rs2254638 in N6AMT1,rs12456693 in SLC14A2 and rs2487032 in ABCA1 were significantly associated with the pharmacokinetics of clopidogrel or H4.We further confirmed whether these new variants have a potential function on the activation of clopidogrel in 32 individual human liver S9 fractions extracted from noncancerous liver tissues.The incubation condition of clopidogrel in the liver S9 fraction was established and optimized.The formation of metabolites in individual liver S9 fraction was analyzed,and then compared among different genotypes of the 18 SNPs.CYP2C19*2 and N6AMT1 rs2254638 were found to be associated with decreased formation of H4.The results showed that novel variants(rs2254638 in N6AMT1,rs2487032 in ABCA1)were independent predictors for variability of PRU.It dramatically improves the explanation of the variance in PRU to 37.7%compared with the published value of approximately 20%.Rs2254638 in N6AMT1 and rsl2456693 in SLC14A2 were independent predictors for variability of the H4 concentration.Finally,to validate the predictable effects of candidate SNPs on the risk of MACE and evaluate gene polymorphism as a predictor of clopidogrel efficacy,CHD patients who received clopidogrel therapy were sequentially recruited in an prospective cohort and followed up regularly.Among them,91 patients with MACE during the 18-month follow-up period after PCI were selected as case,while 208 patients without MACE randomly selected as control.Genotyping of candidate SNPs was conducted in the selected patients.Logistic regression analysis showed that rs2254638 in N6AMT1,rsl2456693 in SLC14A2 and rs12913988 in ATP10A were associated with the occurrence of MACE.The new variants in N6AMT1 and SLC14A2 were supposed to affect the antiplatelet efficacy by changing pharmacokinetic behavior of clopidogrel and H4 in vivo.In conclusion,this study systematically identified new genetic variants for reduced efficacy of clopidogrel which dramatically improved the predictability of variability in its response,and enhanced the understanding of the absorption and metabolic mechanisms that influence antiplatelet responses to clopidogrel treatment.It can improve the personalized medicine of clopidogrel treatment. |
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