| Background: Dual antiplatelet therapy with aspirin and clopidogrel is currently standard treatment in patients with coronary heart disease (CHD) underwent percutaneous coronary intervention (PCI) to prevent ensuing atherothrombolitic cardiovascular events, whereas a substantial number of incidents continue to occur.Clopidogrel evoked platelet inhibition exhibits a considerable individual variability and about 4% to 30% of patients treated with conventional doses of clopidogrel do not display adequate antiplatelet efficacy. Low responsiveness to clopidogrel in patients significantly enhances the occurrence of cardiovascular events and death. The interindividual variability in clopidogrel efficacy is multifactorial and might be related to gene polymorphisms. Data has shown that variants in genes encoding proteins involved in clopidogrel absorption, metabolic activation, and biological activity contribute to variability in individual responsiveness to clopidogrel and influence cardiovascular outcomes promoting genotype-guided strategy of clopidogrel treatment in Caucasian patients with CHD undergoing PCI. However, robust evidence is unavailable in Chinese. We aimed to determine whether genetic variations were associated with clinical outcomes in Chinese patients in this study.Methods: Firstly, we resequenced 13 genes (ABCB1, CYP2C19, CYP2C9, CYP2B6,CYP3A4, CYP1A2, CYP3A5, PON1,P2RY1,P2RY6, P2RY12, PEAR1 and ITGB3)involving clopidogrel absorption, activation and activity by Ion Torrent semiconductor sequencing in 138 samples. Secondly, according to resequencing results and previously reports, we selected 13 single nucleotide polymorphisms (SNPs)possibly related to clopidogrel efficacy and genotyped by TaqMan SNP allelic discrimination on 7900 HT Fast Real-Time PCR System in a cohort from Wuhan including 3246 patients with CHD after PCI. At the same time, we followed for one and a half years and recorded the incidence of cardiovascular events and other adverse outcomes. Then we prospectively assessed the modulating effects of these variants on ischemic event occurrence within one and a half years after PCI by survival analysis.Finally, we confirmed our results in another cohort from Shijiazhuang including 948 patients.Results: Through resequencing 13 genes in a cohort of 138 samples, we found 203 variations including 56 missense mutations. After one and a half years follow-up, the primary events (CV death, nonfatal myocardial infarction or nonfatal stroke) occurred in 223 patients (6.8%) in cohort 1. For all tested SNPs, primary event rates did not differ significantly among genotypes (p>0.05) in cohort 1. Notably, patients in cohort 1 exhibited no association between rs4244285 (CYP2C19*2) genotype and the primary efficacy end point (6.29% for GG versus 7.74% for GA, 5.54% for AA;p=0.264). We combined CYP2C19 variant alleles (*2,*3,*17) to reveal its relationship with clinical outcomes in Chineses. Surprisingly, we found that the number of loss-of-function alleles didn't positively correlate to the risk of cardiovascular events. Likewise, no significant relationship between genotypes and the key secondary end point was observed. We confirmed the results in cohort 2 and obtained consistent results.Conclusions: Different from observations in Caucasian populations, genetic variants analyzed in this research have no significant influence on clinical outcomes after PCI in Chinese population.Background: Clopidogrel, as a routine antiplatelet drug, is widely used in patients to reduce cardiovascular events following PCI. Because of genetic variation, patients undergoing PCI show differing responses to clopidogrel therapy. Recently, five SNPs within CYP2C19 (rs4244285, rs4986893, rs12248560), ABCB1 (rs1045642), and ITGB3 (rs5918) were identified that contribute prominently to variability in response to clopidogrel. Given that Sanger sequencing is labor intensive and time consuming,rapid genotyping methods for SNP detection are urgently required before clopidogrel therapy.Methods: We developed a high-resolution melting analysis (HRMA) and TaqMan allelic discrimination assay (TaqMan) to genotype those five SNPs, and compared these two assays with Sanger sequencing on accuracy of genotyping as well as operational characteristics.Results: These two assays showed high accuracy (0.995, 95% CI 0.991 to 0.998 for HRMA; 0.997, 95% CI 0.994 to 0.999 for TaqMan, respectively), sensitivity (0.996,95% CI 0.989 to 1.002 for HRMA; 0.998, 95% CI 0.993 to 1.002 for TaqMan,respectively), and specificity (0.995, 95% CI 0.991 to 0.999 for HRMA; 0.996, 95%CI 0.993 to 1.000 for TaqMan, respectively). Our study indicates that HRMA and TaqMan are easier to operate and obviously faster than Sanger sequencing.Conclusions: HRMA and TaqMan are rapid, convenient, and reliable assays for clopidogrel efficacy genotyping. |
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