| BackgroundCoronary atherosclerotic heart disease(CHD)is the most common cardiovascular disease.At present,percutaneous coronary intervention(PCI)is one of the main treatment for CHD,but it may have a high risk of stent thrombosis,re-thrombosis and other cardiovascular events.Clopidogrel and aspirin combination antiplatelet therapy has become a conventional treatment for preventing cardiac events after PCI for CHD.However,studies have revealed that this combination therapy is unlikely to completely prevent the occurrence of cardiovascular events in patients with CHD,which may be associated with individuals' difference in reactions to antiplatelet drugs,especially the difference in reaction to clopidogrel.Clopidogrel is a kind of precursor drugs,which needs to be metabolized by the liver into active products with antiplatelet effect.CYP2C19 enzyme involves in all metabolic processes of clopidogrel,while CYP2C19 genetic variants are considered to be part of the reasons for clopidogrel difference in individuals.CYP2C19*2(C.681G>A)and *3(C.636G>A)are the most common and mostly studied loss of function alleles.Some foreign studies believed that carrying loss of function(LOF)allele could decrease active metabolites of clopidogrel and increase the incidence of major cardiovascular events that include myocardial infarction,cardiac death,stent thrombosis and target vessel revasularization.Thus,FDA in the United States firstly approved to adopt relevant kits to test the CYP2C19 *2,*3 gene variants clinically.According to the numbers of LOF alleles and the metabolic pharmacokinetic characteristics of clopidogrel in populations with different genotypes,they are divided into extensvie metabolism carrying wild-type genes(extensive metabolism,EM,CYP2C19 1/1),intermediated metabolism carrying 1 LOF allele(intermediated metabolism,IM,CYP2C19 1/2 and 1/3)and poor metabolism carrying 2 LOF alleles(poor metabolism,PM,CYP2C19 2/2,2/3 and 3/3).In our country,similar kits are adopted to detect CYP2C19 *2,*3 alleles,and patients are classified into different metabolism types,hereby to guide clopidogrel administration.It does not know whether this kit is suitable for chinese patients.CYP2C19 *2,*3 variants frequency differs in different races,which may exert different effects on the cardiovascular events.It has been found that CYP2C19 genetic variants incidence is significantly higher that in Asian populations,where 70% of which manifest as reduced clopidogrel efficacy.In addition,age,gender,hepatic and renal function,concomitant diseases,combination therapies and other factors may also affect individuals' responses to clopidogrel,hereby influences occurrence of cardiovascular events in CHD patients after receiving clopidogrel treatment.However,compared to the westerners,the effects of clopidogrel in Asians have been relatively less studied,while large-scale studies on relationship between CYP2C19 gene variants and cardiovascular events have been even more rarely reported.Since Chinese people account for the largest proportion of Asian populations,and there is growing number of immigrants,it is especially necessary to carry out studies in Chinese population.Purpose:We aimed to study CYP2C19 gene variants type,frequency of alleles,genotype and metabolism type distribution in Chinese normal populations and CHD patients receiving clopidogrel therapy following stent implantation,and to analyze whether the clinical kits match DNA sequencing and is suitable for chinese patients.Furthermore,we attempted to explore the relationship between CYP2C19 gene variants and cardiovascular events,looking for possible factors related to risk of cardiovascular events,as well as analyze applicability of the current gene assay kits for clinically guiding clopidogrel administration.We disussed the effect of dose of clopidogrel on cardiovascular events.Methods: 1.Clinical cases and data acquisition: This study aimed inpatients in the Vasculocardiology Department,who were diagnosed as CHD with coronarography and underwent stent implantation,followed by clopidogrel and aspirin combination antiplatelet therapy,were enrolled,and their clinical data were collected.2.CYP2C19 gene variants detection: 2.5ml discarded peripheral blood was collected from the patients were tested CYP2C19 2/3 variants by DNA microarray,from which DNA was extracted.CYP2C19 2/ 3 variants were tested using DNA microarray,and all CYP2C19 exons were detected using direct DNA sequencing.Meanwhile,those of the normal control populations were collected in the same period,whom were unrelated healthy individuals and their age,gender,race matched patient.3.Follow-up: Patients were followed-up via telephone interview at 12 months after they were discharged from the hospital.The clinical cardiovascular events included bleeding,myocardial infarction and recurrent angina after PCI.4.End point analysis: The enrolled CHD patients were grouped according to the genotype(1/1,1/2,1/3,2/2,2/3,3/3)and metabolic type(extensive metabolism,intermediated metabolism,poor metabolism),hereby to analyze whether there was difference of event incidence between the two groups.Meanwhile,correlations of metabolic type and other factors with end points were analyzed.5.Statistical analysis: Statistical analyses were performed using SPSS18.0 software.Measurement data was expressed as mean±standard deviation((?)±s).Comparisons between two groups were analyzed using t-test,while comparisons among multiple groups were conducted using analysis of variance.Count data was expressed as percentage,where samples were compared using chi-square test.Meanwhile,correlation analyses were performed using single-factor and stepwise regression analysis.A difference of p<0.05 was considered statistically significant.Results:1.This study enrolled 817 CHD patients receiving clopidogrel antiplatelet therapy following stent implantation among 1250 CHD patients,as well as 824 normal control population.2.Gene microarray results were exactly the same as those of DNA sequencing,where gene microarray displayed a sensitivity and specificity of 100% in detecting CYP2C19 *2,*3 variants,which had a consistency strength index of 1 with the DNA sequencing.3.Frequencies of alleles at *1,*2 and *3 loci did not show differences with statistical significance compared to those of normal control populations(66.46%,29.56%,3.98% vs.62.62%,33.74%,3.64%,p=0.07).4.Although no 3/3 genotype was found in normal control population,constituent ratio of six genotypes 1/1,1/2,1/3,2/2,2/3 and 3/3 in CHD patients did not show statistically significant difference compared to the normal controls(43.21%,41.25%,5.26%,7.83%,2.20%,0.24% vs 37.86%,44.42%,5.10%,10.44%,2.18%,0% p=0.15).5.Constituent ratio of extensive,intermediated and poor metabolism types did not display difference with statistical significance between CHD patients and normal controls(43.21%,46.51%,10.28% vs 37.86%,49.51%,12.62% p=0.06),and both groups were dominant by extensive and intermediated metabolism,of which intermediated metabolism was most prominent and poor metabolism was the least.6.Among the 817 CHD patients,29 cases(3.55%)occurred bleeding.Of these,the incidence of bleeding did not show statistically significant difference among 6 genotypes of CHD patients(p=0.877),where the incidences of bleeding in patients with 1/1,1/2,1/3,2/2,2/3 and 3/3 genotypes were 4.25%,3.26%,2.32%,1.56%,5.56%,0%,respectively.According to the metabolism type grouping,the bleeding incidence of the extensive metabolism group(4.25%)was higher than those of the intermediated metabolism group(3.16%)and poor metabolism group(2.38%).7.In the 817 CHD patients,35 cases(4.28%)appeared myocardial infarction.In terms of genotype grouping,incidence of myocardial infarction of the 2/2 and 2/ 3 groups were higher than those of the 1/ 1,1/ 2,1/ 3 and 3/ 3 genotype groups(10.94%,11.11% vs 3.97%,3.26%,2.32%,0%).In terms of metabolism type grouping,incidence of myocardial infarction in the poor metabolism group(10.71%)was higher than those of the extensive metabolism group(3.97%)and intermediated metabolism group(3.16%),where the differences were statistically significant(p=0.008).8.88(10.77%)out of the 817 CHD patients appeared recurrent angina after PCI.In terms of genotype grouping,the incidence of recurrent angina after PCI of the 2/ 2 and 2/ 3 groups were higher than those of the 1/ 1,1/ 2,1/ 3 and 3/ 3 groups(17.19%,16.67% vs.11.90%,8.90%,4.65%,0%).Meanwhile,in terms of metabolism type grouping,the incidence of recurrent angina after PCI of the poor metabolism group(16.67%)was higher than those of the extensive group(11.90%)and intermediated metabolism group(8.42%).9.CYP2C19 metabolic type or other analysis factors were not found to be linked to bleeding.10.CYP2C19 metabolism type was associated with myocardial infarction after PCI,where the risk of myocardial infarction in poor metabolism CHD patients was 2.56 times of that in the extensive metabolism CHD patients(95%CI=1.05-6.19,p=0.04),while the myocardial infarction difference between the intermediated metabolism group and extensive metabolism group was statistically insignificant(p=0.503).11.Diabetes was related to occurrence of recurrent angina after PCI.The risk of recurrent angina after PCI in CHD patients with diabetes was 1.72 times higher than that in patients without diabetes(95%CI=1.04-2.83,p=0.04).Meanwhile,number of diseased vessels was also associated with recurrent angina after PCI.And the other factors were found to be irrelevant to the occurrence of recurrent angina after PCI.12.In addition to CYP2C19*2,*3 variants,10 single nucleotide polymorphisms(SNP)that have been reported were detected in both CHD patients and normal controls.They are CYP2C19–p.M1 V,CYP2C19–p.P33 P,CYP2C19–p.R132 Q,CYP2C19–p.A161 P,CYP2C19–p.F267 L,CYP2C19–p.V330 V,CYP2C19– p.I331 V,CYP2C19–p.I359 I,CYP2C19–p.K399 K and CYP2C19–p.G417 G.The G/C variation of CYP2C19–p.A161 P was lack in normal controls,The frequence of CYP2C19–p.V330 V variants in Chinese and Asian were significiantly higher than that in African and European(p<0.01),and did not show difference among others.Conclusions: 1.The gene microarry results of CYP2C19*2,*3 variants match that of DNA sequencing.The frequence of CYP2C19*2,*3genetic variants,the genetype and metabolism carrying the CYP2C19*2,*3genetic variants does not display difference with statistical significance and is significantly higher in chinese than that in african and european.2.Genotype of chinese population with coronary heart disease is related to number of diseased vessels,the 2/2 genotype CHD patients are more prone to have multivessel lesion.3.Applying metabolism classification which is based on gene assay test is beneficial for guiding clopidogrel administration in Chinese population and preventing occurrence of myocardial infarction after PCI.The risk of MI is significantl increased in patients with PM and dose increase of clopidogrel cannot benefit patients. |
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