Establishment And Evaluation Of Molecular Detection Method For Ischemic Stroke Susceptibility Gene

Objectives To establish a molecular detection system(polymerase chain reaction and high resolution melting,PCR-HRM)for the genotyping 14 SNPs(single nucleotide polymorphism)associated with ischemic stroke identified by genome-wide association studies and validate the relationship of 14 loci associated with the genetic susceptibility of ischemic stroke in the Gansu region of China.Methods Using Primer Quest Tool in the online software IDT(https://sg.idtdna.com)to design specific amplification primers for 14 loci in this study.On the basis of PCR,PCR-HRM detection method was established to detect 14 loci in clinical specimens of 449 patients with ischemic stroke.To verify the genetic susceptibility of 14 SNPs in the Gansu patients with ischemic stroke by chi-square test,binomial logistic regression,linkage disequilibrium(LD)and haplotype analysis.The Sanger sequencing method was used as the gold standard to verify the methodology and evaluate its sensitivity,specificity,repeatability and reproducibility.Results(1)Sanger sequencing confirmed that the PCR-HRM method could detect 449 samples genotype with 14 loci successfully.The success rate and accuracy of the PCR-HRM detection system established in this study were 100% and the clinical performance evaluation results showed that the sensitivity and specificity of the established method were good.(2)The results showed that 6 loci had statistically significant differences in genotype frequency and allele frequency(P<0.05).They were rs6825454,rs11957829,rs16896398,rs7859727,rs35436 and rs2229383.(3)Regressive analysis of the risk in the genetic model of disease showed that wild-type TT of rs6825454,homozygous mutant TT and heterozygous mutant CT of rs35436 and homozygous mutant TT and heterozygous mutant GT of rs2229383,wild type AA of rs16896398 all increased the risk of ischemic stroke(OR>1,P<0.05),but homozygous mutant CC of and heterozygous mutant CT rs6825454,homozygous mutant TT of rs7859727,wild-type CC of rs35436,wild-type GG of rs2229383 and homozygous mutant TT and heterozygous mutant AT of rs16896398 both reduced the risk of ischemic stroke(OR<1,P<0.05).(4)Stratified analysis of the case group according to different TG,CHO,HDL and LDL levels showed that there were statistical differences in the genotype frequency or allele frequency of rs12037987,rs6825454,rs35436,rs7304841,rs2229383 and rs17612742 loci(P<0.05).(5)Haplotype analysis showed that haplotype TT of rs17612742 and rs6825454 on chromosome 4 could increase the risk of ischemic stroke(OR>1,P<0.05),haplotype CC and CT were protective factors for the onset of ischemic stroke(OR<1,P<0.05).Haplotype AC of rs7304841 and rs35436 on chromosome 12 is a protective factor for the incidence of ischemic stroke(OR<1,P<0.05),and haplotype AT can increase the risk of ischemic stroke(OR>1,P<0.05).Conclusion The molecular diagnostic method in this work has high sensitivity and specificity,with repeatability of method and good reproducibility of experimental results,which has guiding significance for routine clinical genotyping.rs6825454,rs11957829,rs16896398,rs7859727,rs35436 and rs2229383 were associated with the susceptibility of ischemic stroke in the Gansu region of China.