Influence Of The Polymorphisms In COL1A2 And MIF On Sudden Cardiac Death In Chinese Populations

Part I Association between an indel polymorphism in the 3′UTR of COL1A2 and the risk of sudden cardiac death in Chinese populationsObjective: To evaluate the association between the Insertion/Deletion(Ins/Del,Indel)polymorphism(rs3917)within the 3’ untranslated region(3’UTR)of COL1A2 gene and sudden cardiac death(SCD)in Chinese populations,and investigate the underlying mechanisms and the possible role of rs3917 in the pathogenesis of SCD using the genotype-phenotype correlation analysis as well as bioinformatics approach.Methods:(1)Bioinformatic techniques were applied to screen polymorphisms in the 3’UTR of COL1A2,from which we chose the potential functional polymorphisms(rs3917)as candidate.DNA was extracted from all the blood samples of 79 SCD cases and 328 healthy controls collected,then genotype was done using PCR combined with polyacrylamide gel electrophoresis(PAGE)method.And logistic regression was used to analyze the association between rs3917 and the risk of SCD.(2)Real-time PCR analysis was used to investigate the correlation between the genotypes of rs3917 and the expression level of COL1A2 in human myocardium tissue samples.(3)Bioinformatics method was used to predict micro RNA which may affect COL1A2 transcriptional activity.(4)Constructed reporter plamid vetors with approximately 300 bp fragments from the 3’UTR of COL1A2 including the Ins/Del allele of rs3917(p GL3-COL1A2-WT/MT)were transfected into 293 T cell lines,and Dual Luciferase assay was performed to explore the impact of mi R-296-3p on COL1A2 transcription activity.Results:(1)Statistics of genotyping results revealed that the genotypic and allelic frequency of the rs3917 in the 3’UTR of COL1A2 were in an adequate and balanced distribution,and there were no deviations from the Hardy-Weinberg equilibrium(P>0.05).Logistic regression analysis showed that the deletion allele of rs3917 significantly increased the risk of SCD [odds ratio(OR)= 1.82;95% confidence interval(CI)=1.08-3.06;P = 0.0159].(2)Real-time PCR results demonstrated that the expression level of COL1A2 m RNA with Ins/Ins genotype was 1.83 fold higher(P<0.01)than that with Ins/Del genotype in human myocardium tissue samples.(3)Bioinformatic analysis demonstrated that hsa-mi R-296-3p may affect the transcriptional activity of COL1A2.(4)The luciferase activity analysis in an in vitro reporter gene system suggested that hsa-mi R-296-3p could inhibit the reporter gene expression in both WT and MT groups,and the inhibition in the WT group was more obvious.Conclusion:(1)The rs3917 polymorphism in the 3’UTR of COL1A2 was highly relevant to the risk of SCD in Chinese populations.(2)The genotype-phenotype correlation between rs3917 and the expression level of COL1A2 in myocardium tissues was confirmed.(3)The rs3917 polymorphism was likely to be functional in regulating COL1A2 expression through interfering the binding of hsa-mi R-296-3p to the 3’UTR of COL1A2,then participating in the occurrence of SCD.Part II Influence of functional polymorphism in MIF promoter on sudden cardiac death in Chinese populationsObjective: To evaluate the influence of a functional polymorphism in MIF promoter on sudden cardiac death in Chinese populations,and investigate the underlying mechanisms and the possible role of-794 CATT5-8 in the pathogenesis of SCD using genotype-phenotype correlation analysis as well as bioinformatics approach.Methods:(1)Bioinformatic techniques were applied to screen polymorphisms in the promoter region of MIF,from which we chose the potential functional polymorphisms(-794 CATT5-8)as candidate.DNA was extracted from all the blood samples of 79 SCD cases and 313 healthy controls collected,then genotype was done using PCR combined with polyacrylamide gel electrophoresis(PAGE)method.And logistic regression was used to analyze the association between-794 CATT5-8 and the risk of SCD.(2)Using statistical methods,we analyzed and compared the frequency of allele distribution of-794 CATT5-8 polymorphism in different countries and analyzed the relationship between this polymorphism and different diseases.Results:(1)There was no significant association between the-794 CATT5-8 polymorphism and SCD susceptibility in our research.(2)The allele frequency distributions of the polymorphism in Chinese populations were quite different from that of American and European populations(P = 0.005,P = 0.0001,respectively),but similar to Japanese populations(P = 0.827).Conclusion:(1)There was no significant association between-794 CATT5-8 polymorphism and SCD risk in our research.