Combined Influence Of ABCB1 Genetic Polymorphism And DNA Methylation On Aspirin Resistance In Chinese Ischemic Stroke Patients

Objective1.To determine the frequency of single nucleotide polymorphisms(SNPs)of ABCBl gene in Chinese ischemic stroke patients.2.To determine the methylation status in promoter region of ABCBl gene in Chinese ischemic stroke patients.3.To further evaluate the ABCB1 gene polymorphism and promoter region methylation level on AR in Chinese ischemic stroke patients.Methods1.The distribution frequency of different genotypes in three SNPs between the two groupsOur research was a prospective cohort analysis of eligible stroke patients(n=302,aged 20～93 years)who received aspirin(100mg/day for ≥7days).To detect the platelet function,the thrombelastography5000 platelet mapping system(TEG,Haemoscope Corporation,USA)and platelet aggregation test were used together.According to the result,patients would be divided into two groups: AR group and aspirin sensitive(AS)group.The results of platelet aggregation testing were expressed in aspirin reaction units(ARU).Greater than or equal to 550 ARU was determined as AR.TEG was used to determine the inhibition of platelet aggregation induced by arachidonic acid(AA).AA inhibition(AA%)[8] = 100%-[(thrombin induced clot maximum strength-AA induced clot strength)/(thrombin induced clot maximum strength-residual clot strength)]* 100%.AR is defined when AA% is less than 50%,while AS is defined when it is greater than or equal to 50%.All patients’ blood sampleswere taken 3ml from the elbow vein,and collected in EDTA tubes(-20 ℃ preservation).ABCB1 genetic polymorphism were analyzed using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)and direct sequencing.2.The correlation of promoter methylation status of ABCB1 gene between the two groupsIn this experiment,the methylation of promoter region was analyzed by combined bisulfite restriction analysis(COBRA)as previously described.Bisulfite treatment of genomic DNA was performed using TG DNA Methylation kit.After an initial denaturation,the amplified products which were identified by 2% agarose gel electrophoresis.For COBRA analysis the restriction enzymes TaqI and Hsp 92II(New England Biolabs)were used to examine the methylation status of the amplified regions.This enzyme recognition sequence contains the CG nucleotide sequence.If the cytosine in recognition sequence occurs methylation,the CGCG sequence will be retained after PCR amplification,then recognized and cuted by enzyme.If there no methylation occurred,the measured sequence will be converted into a TGTG.The recognition site of the endonuclease is missing and the sequence cannot be cut.In this way,the percentage of methylation in the original sample can be obtained after the enzyme digestion,electrophoresis separation,and scanning quantification.Methylation level(%)for a specific cytosine was defined by Image J software.All experiments were repeated twice to assess the reproducibility of results.3.Statistical AnalysisAll data were analysed by SPSS software(version 23.0).Continuous variables were analyzed using T test(normal distribution)or Mann-Whitney U test(non-normal distribution).Genotype freguencies for three SNPs between different groups were assessed by using the chi2 test.The Mann–Whitney U test and Kruskal-Wallis test were used to compare the methylation level among different groups.Logistic regression was used to test the interaction of ABCB1 methylation status and AA%.Haplotype analysis was performed using online software SHEsis.Hardy-Weinberg equilibrium test was used.It is determined to be Hardy-Weinberg balanced when P>0.05.And the haplotype frequency <3% should be eliminated.It indicated statistical significance when P < 0.05.Results1.The distribution frequency of different genotypes in three SNPs between the two groupsA total of 302 cases were included in our study and were divided into the AR group(n=77)and AS group(n=225)according to the patients’ clinical data and definition of AR.The genotype distributions of the three SNPsloci in the ABCB1 gene were consistent with the Hardy-Weinberg genetic equilibrium test(P>0.05)and showed good population representativeness.The allele and genotype frequencies of three ABCB1 SNPs(C1236T,G2677T/Aand C3435T)were tested to estimate the association with ischemic stroke patients.The frequencies of the allele T of C3435 T showed a significantly higher occurrence in AR group than AS(OR = 1.765,p=0.018),and the genotype CT+TT of C3435 T also showed a significant higher occurrence in AR groupthan AS(OR =1.912,p=0.034).There were no difference in the frequency and genotype distribution of C1236 T and G2677T/A alleles between the two groups(p >0.05).2.The correlation of promoter methylation status of ABCB1 gene between the two groupsThe methylation level of ABCB1 promoter region in 302 stroke patients was detected by COBRA method.The result shows that the median methylation level of promoter region of ABCB1 gene instroke population was 14.01%(0.14%-29.35%).Subjects in AR group showed a lower methylation level than AS group(p<0.001).Our study revealed a significant correlation of C3435 T polymorphisms with DNA methylation status.we found significant different between the genotype CC,CT and TT of C3435 T individuals DNA methylation status(p=0.006).The result shows that,the methylation degree of ABCB1 promoter region in different genotypes of C3435 T was significantly different.However,there was no significant difference between C1236 T and G2677T/A genotypes(P>0.05).3.Correlation of ABCB1 gene methylation with AA%ABCB1 gene methylation level in whole blood was compared among AA%.In a linear regression model,ABCB1 gene promoter region methylation level was independently related with AA%.And ABCB1 gene promoter region methylation level correlated positively with AA%(R=0.781,P<0.001),suggesting that ABCB1 gene methylation level might link to increased AA%.On the other hand,ABCB1 hypomethylation might lead to decreased AA%,resulting in a decreased response to ASA.ConclusionInconclusion,The level of methylation in the promoter region of ABCB1 gene was highly correlated with the occurrence of AR.These data suggests that routine evaluation of ABCB1 methylation in wholeblood might serve as a biomarker of patients who are at higherrisk of AR and developing ischemic eventswhen ASA is used after ischemic stroke.It alsocan assist in the selection of therapeutic protocols and provide further opportunities for subsequent clinical intervention in these patients.