Association Of CYP2C19 Loss-of-function Variants And P2Y12 H2 Haplotype With High On-treatment Platelet Reactivity And Clinical Outcome In Ischemic Stroke

Objective:Stroke is a complex disease.Platelet activation and aggregation plays a key role in atherosclerosis and cerebral arterial thrombosis.Therefore,antiplatelet drugs are core treatment options for the secondary prevention of non-cardiac stroke.Clopidogrel is a P2Y12 receptor inhibitor that irreversibly binding to P2Y12 receptors and blocks ADP-dependent platelet aggregation pathways,thus playing a potent inhibitory role in platelet aggregation and is therefore widely used in the prevention of stroke.However,the therapeutic effect of clopidogrel varies widely.Some patients still show treatment failure despite being given the standard dose of clopidogrel according to the guidelines,causing about 6%-9% of patients to relapse within one year from stroke onset.Moreover,laboratory platelet function tests identified that about 20%-50% of patients treated with standard or high doses of clopidogrel remained high platelets activity,which may be associated with stroke recurrence.The factors that contribute to clopidogrel hypo-responsiveness or High on-treatment platelet reactivity(HTPR)are manifold,especially,the disfunction of cytochromeP450 enzyme activity resulting from genetic variation,which result in pharmacokinetic changes in clopidogrel metabolism,and pharmacodynamic changes due to abnormality in the receptor structure caused by P2Y12 genetic variation.However,the current findings are not consistent,whether CYP2C19 or P2Y12 or other gene polymorphisms could contribute to variations in clopidogrel responsiveness or the HTPR,then affect the clinical outcome of stroke patients is still confusing.On the other hand,due to various shortcomings of different methods of platelet function tests,the prevalence and definition of HTPR is not uniform across different studies,and there is no agreement on whether genotype and or platelet phenotype test could affect the clinical outcome in stroke patients.In addition,the previous studies mainly focused on foreign Caucasian acute coronary syndrome(ACS)or undergoing percutaneous coronary intervention(PCI)patients.Therefore,we performed this study to detect the frequency and distribution of the most common CYP2C19(* 2,* 3)LOF(loss-of-function)alleles and P2Y12 H2 haplotype in a group of stroke patients,which comes from the area of high stroke incidence located in Northeast China,along with platelet function test to determine the prevalence of HTPR and associations with primary clinical outcome at the end of 6 months.Then evaluate the clinical significance of genotype and phenotype test on the outcome of stroke patients Methods: 1.The prospective cohort study was performed in the first hospital of China Medical University and Liaoning JinQiu Hospital,the first-ever or recurrent stroke patients were selected.Large-artery atherosclerosis and small-artery occlusion(Lacunar)were enrolled according to the TOAST classification system.All patients were given a 75mg/d maintenance dose of clopidogrel for more than one week.(2)Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)techniques were used to determine the genotype of CYP2C19*2(rs4244285)?*3(rs4986893)? P2Y12 T744C(rs2046934)loci,and 5% of the samples were sent to Shanghai Sangon Biotech for direct sequencing of DNA to verify the accuracy of PCR-RFLP genotyping results.According to the results,the CYP2C19 metabolic type(Extensive,Intermediate and poor metabolizer)and P2Y12 haplotype(H1 or H2)were determined,to analyze the effect of different genotypes on platelet aggregation rate.HTPR was defined according to the maximum platelet aggregation rate(MPAR)value,and the risk factors for HTPR was detected by univariate analysis and binary logistic regression analysis.4.The platelet function of patients was detected by LTA method,after taking clopidogrel for one week with the standard dose.The primary outcome was a composite endpoint which include recurrent stroke,TIA,myocardial infarction,and death.Clinical events were recorded via telephone or outpatient visit through a 6 months follow-up duration.Kaplan-Meier analysis and Cox regression analysis was used to identify risk factors associated with HTPR and clinical outcomes.Results: A total of 131 patients from the 2 centers completed all the genotyping,platelet function tests and clinical follow-up.According to the TOSAT classification,79 cases of large-artery atherosclerosis and 52 cases of small-artery occlusion stroke patients were analyzed.Of the 131 patients,83 were male patients,accounting for 63.4%.The average age of patients was 61.4 ± 10.9 years old,hypertension and diabetes mellitus were prevalent,the proportion was 66.4% and 42.7%,respectively.The genotype results were identified by PCR-RFLP and confirmed by DNA direct sequencing,of which 35(68.7%)were CYP2C19 * 1 homozygotes(A / A),35(26.7%)were heterozygotes,and 6(4.6%)were *2/*2 homozygotes(A / A).The result of CYP2C19 *3 genotype showed that 115(87.8%)were G/G,16 cases were *1/*3 heterozygote(G / A),and 16 of them were *3/*3 homozygotes(A/A).The frequency of P2Y12 genotypes was 89(67.9%)for T/T type,33(25.2%)for C/T heterozygote and 9(6.9)for C/C type.All allele frequency was in Hardy-Weinberg equilibrium.3.The 5?mol/L ADP induced MPAR measured by LTA method varies greatly,with a maximum of 81%,a minimum of 11% and a median of 45%(IQR,34-59%).According to the definition of HTPR(5?mol/L ADP induced MPAR> 46%),the incidence of HTPR was 48.1%.4.The difference in MPAR between EM & IM group,and EM & PM group was significant(p<0.05,p<0.01,respectively),There was no significant difference between IM group and PM group,neither among groups with different H2 carriers(p = 0.55).The prevalence of HTPR was different between CYP2C19 LOF carriers and noncarriers.Multiple logistic regression analysis showed that carrying of at least one CYP2C19 * 2 or * 3 loss-of-function allele was an independent risk factor for HTPR(OR,5.81;95% CI,2.54-13.28;P <0.001),while P2Y12 H2 haplotype was not a risk factor for HTPR(p = 0.10).COX regression analysis suggested that HTPR was an independent risk factor for the occurrence of ischemic primary composite end point(HR,3.1;95%CI,1.07-8.99;p=0.04).Conclusions: 1.Based on the results of LTA assay,HTPR is prevalent in stroke population in northeastern China,with the incidence of HTPR> 48%.2.carrying of at least 1 of CYP2C19 * 2 or * 3 loss-of-function allele is an independent risk factor for the occurrence of HTPR,however,no significant correlation was found between P2Y12 H2 haplotype and HTPR.3.No significant associations were observed between genotype(CYP2C19 LOF alleles and P2Y12 H2 haplotype)and clinical outcome in stroke patients.4.The phenotype HTPR is associated with higher odds of primary composite endpoints.