Relationship Between The Genetic Polymorphisms And The Recurrence Of Ischemic Stroke

Background and Objectives:In China,cerebrovascular disease has the characteristics of high morbidity,high recurrence rate,high disability rate and high mortality.At the same time,about 30%of stroke cases are recurrent strokes which is associated with poor prognosis compared with the the initial stroke.In China,the recurrence risk of ischemic stroke within one year is about 6.99-21.39%within the one-year.Nevertheless,the factors leading to higher recurrence risk of ischemic stroke are still poorly understood.It is important to predict the recurrence of the stroke.And then,some appropriate preventive measures could be taken in patients at higher risk.This study was aimed to examine the relationships among the genetic polymorphisms and the recurrence of ischemic stroke.1.To investigate the relationship among the 120GPIIIaPIA2(1565T>C),106PEAR1(G>A),168PTGS1(-842A>G)polymorphisms and recurrent ischemic stroke(RIS)at admission by retrospective analysis.2.By a one-year follow-up,examine the relationships 120GPIIIaPIA2(1565T>C),106PEAR1(G>A)and 168PTGS1(-842A>G)polymorphisms and the relapse or death of ischemic stroke within 1 year after taking aspirin in cerebral infarction patients.3.By a one-year follow-up,to investigate the relationships 152PON1(576G>A),CYP2C19*2(9154G>A)and CYP2C19*3(7948G>A)polymorphisms and the relapse or death of ischemic stroke within 1 year after taking clopidogrel in cerebral infarction patients.Methods:Part I:Association of 120GPIIIaPIA2,106PEAR1,168PTGS1 gene polymorphisms in the onset and recurrence of cerebral infarction(A retrospective analysis)This was a single-center,retrospective,case-control study of patients seen in consultation between March 2016 and December 2016 at the Shandong Provincial Hospital.A total of 193 patients with IS were included and divided into the initial stroke group and the RIS group according to admission.The 152PON1(576G>A),CYP2C19*2(9154G>A)and CYP2C19*3(7948G>A)polymorphisms were determined by fluorescence in situ hybridization.Part Ⅱ:Effects of 120GPIIIaPIA2,106PEAR1,and 168PTGS1 gene polymorphisms on recurrence or death within 1 year after taking aspirin in ischemic stroke patients(A follow-up study)In this study,193 patients who were hospitalized in Shandong Provincial Hospital from March 2016 to December 2016 were initially enrolled.Aspirin medication was prescribed after discharge and one-year follow-up was performed.35 patients were not able to complete the follow-up and 158 patients were eventually enrolled.According to the follow-up results,they were divided into poor prognosis group(including recurrence and death)and no recurrence group.The 152PON1(576G>A),CYP2C19*2(9154G>A)and CYP2C19*3(7948G>A)polymorphisms were determined by fluorescence in situ hybridization.Part III:Effect of 152PON1,CYP2C19*2,CYP2C19*3 gene polymorphism on recurrence or death within 1 year after clopidogrel treatment in ischemic stroke patients(A follow-up study)In this study,98 patients who were admitted in Shandong Provincial Hospital from March 2016 to December 2016 were initially enrolled.They take clopidogrel for at least 3 months after discharge and one year follow-up was performed.5 patients were lost to follow-up.93 cases were included in the statistics eventually.According to the follow-up results,they were divided into poor prognosis group(including recurrence and death)and no recurrence group.The 152PON1(576G>A),CYP2C19*2(9154G>A)and CYP2C19*3(7948G>A)polymorphisms were determined by fluorescence in situ hybridization.Statistical analysisThe Hardy-Weinberg equilibrium was tested for the genetic polymorphism using the chi-square test(P>0.05 was considered to meet the H-W equilibrium).Continuous variables were presented as means±standard deviation(SD)and compared using the Student’s t-test.Categorical variables were reported as counts(percentages)and the chi-square test was used to compare the groups.Multivariable logistic regression analysis was used to identify independent predictors for RIS.All statistical analyses were performed using SPSS 20.0(IBM,Armonk,NY,USA).Two-tailed P<0.05 was considered statistically significant.Results:Part I:Association of 120GPIIIaPIA2,106PEAR1,168PTGS1 gene polymorphisms in the onset and recurrence of cerebral infarction(A retrospective analysis)A total of 193 patients with ischemic stroke were included.Which were including 129 males(66.84%)and 64 females(33.16%);aged 35 to 88 years,with an average age of 64.14±10.21 years;137 patients(70.98%)with hypertension;53 patients(27.46%)with diabetes;79 patients(40.93%)with long-term smoking history;79 patients(40.93%)with long-term drinking history.Based on previous history of cerebral infarction at admission,there were 56 patients with RIS(29.0%)and 137 with initial stroke(71.0%).There were no differences between the two groups regarding age,gender,smoker,alcohol use,blood pressure,diabetes,HDL-C,TG,HCY,and CRP.Compared with the initial group,the RIS group showed lower LDL-C levels(P=0.04).120GPIIIaPIA2(1565T>C)and 106PEAR1(G>A)meet Hardy-Weinberg equilibrium(P>0.05),168PTGS1(-842A>G)did not meet the Hardy-Weinberg equilibrium.The 168PTGS1(-842A>G),120GPIIIaPIA2(1565T>C)genotype frequency was not significantly different between the two groups(P=0.319,P=0.522).Moreover,the frequency of 120GPIIIaPIA2(1565T>C)C allele was very low,no CC type was observed,and only one case of CT type(0.5%).The AA genotype of the 106PEAR1(G>A)polymorphism was more frequent in the RIS group(17.9%vs.5.8%,P=0.009).The A allele also showed a higher frequency than the G allele in the RIS group(P=0.02).The multivariable logistic regression analysis(stepwise regression method)showed that 106PEAR1(G>A)(OR=3.24,95%CI:1.04～10,14,P=0.043)was independently associated with RIS.After adjusting the lipid-lowering drugs and LDL,multivariate logistic regression analysis(mandatory regression method)showed that 106PEAR1(G>A)AA type(OR=3.857,95%CI:1.047-14.209,P=0.042)was independently associated with RIS.Part II:Effects of 120GPIIIaPIA2,106PEAR1,and 168PTGS1 gene polymorphisms on recurrence or death within 1 year after taking aspirin in ischemic stroke patients(A follow-up study)158 patients were eventually enrolled.Which were including 104 males(65.82%),54 females(34.18%);aged 35 to 88 years,with an average age of 63.65 ±10.25 years;113 patients(71.58%)with hypertension;44 patients(27.84%)with diabetes;65 patients(41.13%)with long-term smoking history;67 patients(42.41%)with long-term drinking history.According to the follow-up results,they were divided into poor prognosis group including recurrence and death(n=27,17.09%)and no recurrence group(n=131,82.91%),of which 6 patients died(3.80%).There were no significant differences in age,gender,smoker,alcohol consumption,blood pressure,diabetes,cholesterol levels,HDL-C,LDL-C TG,HCY and CRP between the two groups(P>0.05).120GPIIIaPIA2(1565T>C)and 106PEAR1(G>A)meet Hardy-Weinberg equilibrium(P>0.05),168PTGS1(-842A>G)did not meet the Hardy-Weinberg equilibrium.The frequency of 120GPIIIaPIA2(1565T>C)C allele was very low,no CC type was observed,and only one case of CT type(0.6%).The 120GPIIIaPIA2(1565T>C),168PTGS1(-842A>G)genotype frequency was not significantly different between the two groups(P=0.649,P=0.420).106PEAR1(G>A)polymorphism was significantly different between the two groups(P=0.033),of which GG/GA+AA was statistically different between the two groups(P=0.011)After adjusting the baseline factors,Multivariate logistic regression analysis showed that the 106PEAR1(G>A)mutant GA+AA genotype was independently associated with poor prognosis(OR=3.85,95%CI:1.319 to 11.239,P=0.014)Part Ⅲ:Effect of 152PON1,CYP2C19*2,CYP2C19*3 gene polymorphism on recurrence or death within 1 year after clopidogrel treatment in ischemic stroke patients(A follow-up study)93 patients with ischemic stroke were included finally.Which were including 66 males(70.97%),27 females(29.03%);aged 38 to 84 years,with an average age of 61.97±10.54 years;57 patients(61.29%)with hypertension;20 patients(21.51%)with diabetes;45 patients(48.39%)with long-term smoking history;43 patients(46.24%)with long-term drinking history.According to the follow-up results,they were divided into poor prognosis group including recurrence and death(n=19,20.43%)and no recurrence group(n=74,79.57%).of which 6 patients died(3.80%).There were no significant differences in age,gender,smoker,alcohol consumption,blood pressure,diabetes,cholesterol levels,HDL-C,LDL-C TG,HCY and CRP between the two groups(P>0.05).CYP2C19*2(9154G>A)meet Hardy-Weinberg equilibrium(P=0.77),152PON1(576G>A)and CYP2C19*3(7948G>A)did not meet the Hardy-Weinberg equilibrium(P<0.05).CYP2C19*2(9154G>A),CYP2C19*3(7948G>A)genotype frequency was not significantly different between the two groups(P=0.865,P=0.397).Baseline factors and 152PON1(576G>A)were included in multivariate logistic regression analysis,mandatory regression method showed that 152PON1(576G>A)AA genotype was not a risk factor for recurrent or death of ischemic stroke after clopidogrel treatment(OR=4.851,95%CI:0.924-25.470,P=0.062).After adjustment for baseline factor,152PON1(576G>A)AA in the recessive model(OR=0.212,95%CI:0.040～1.114,P=0.067)was independently associated with recurrent or death of ischemic stroke.Conclusion:1.The mutant homozygous AA genotype of 106PEAR1(G>A)might be an independent risk factor for RIS.2.120GPIIIaPIA2(1565T>C),168PTGS1(-842A>G)polymorphisms were not associated with recurrent ischemic stroke.3.120GPIIIaPIA2(1565T>C)gene mutation rate is very low.4.106 PEAR1(G>A)wild homozygous GG genotype is lower risk for recurrent or death in ischemic stroke patients after taking aspirin.Mutant GA+AA genotype may be a risk factor for recurrent or death of cerebral infarction.5.120GPIIIaPIA2(1565T>C),168PTGS1(-842A>G)polymorphisms were not associated with the prognosis of cerebral infarction after aspirin treatment.6.152PON1(576G>A),CYP2C19*2(9154G>A)and CYP2C19*3(7948G>A)gene polymorphisms were not associated with recurrence or death of cerebral infarction within 1 year after clopidogrel treatment in ischemic stroke patients.Research Significance and ProspectThis study consists of three parts.1)By retrospective analysis,to investigate the relationship between the 120GPIIIaPIA2(1565T>C),106PEAR1(G>A),168PTGS1(-842A>G)polymorphisms and recurrent ischemic stroke(RIS)at admission.2)Through the follow-up study,examine the relationships between 120GPIIIaPIA2(1565T>C),106PEAR1(G>A),168PTGS1(-842A>G)polymorphisms and the relapse or death of ischemic stroke within 1 year after taking aspirin in cerebral infarction patients.3)Through the follow-up study,to investigate the relationships 152PON1(576G>A),CYP2C 19*2(9154G>A)and CYP2C19*3(7948G>A)polymorphisms and the relapse or death of ischemic stroke within 1 year after taking clopidogrel in cerebral infarction patients.The study has certain clinical significance and innovation.The assessment of genetic polymorphisms in the prediction of RIS warrants further investigation in order to reasonably use of antiplatelet drugs,improve patient management and prognosis after a first ischemic stroke.However,this study may have certain limitations.First,the results may have possible bias due to the relatively small sample size.Secondly,this study only investigated 120GPIIIaPIA2(1565T>C),106PEAR1(G>A),168PTGS1(-842A>G)genes and 152PON1(576G>A),CYP2C19*2(9154G>A)and CYP2C19*3(7948G>A)gene single nucleotide polymorphism.Several other gene polymorphisms may be associated with RIS or antiplatelet drug resistance.Therefore,future research should involve multiple populations,a larger sample size,and a larger set of genetic variants to study the factors related to the recurrence of ischemic stroke.