Association Between An Indel Polymorphism Within CTH And The Risk Of Sudden Cardiac Death In Chinese Populations

Objective:To evaluate the influence of the Insertion/Deletion(Indel)polymorphism(rs113044851)in the 3’ untranslated region(3’UTR)of CTH gene and sudden cardiac death(SCD)susceptibility in Chinese Han population,and explore the possible role of the polymorphism in the pathogenesis of SCD and the potential molecular mechanism through bioinformatics approach and the analysis of genotype-phenotype correlations.Methods:(1)First,polymorphisms in the 3’UTR of CTH were screened by bioinformatic tools,from which a potential functional 4bp Indel polymorphism(rsl 13044851)was carried out as candidate.A case-control study consisting 112 SCD cases and 417 healthy controls were performed using PCR combined with PAGE method.Logistic regression was applied to evaluate the association between rs113044851 and SCD susceptibility.(2)Western blot(WB)was used to examine the expression of CTH protein in human myocardium tissue samples with different genotypes of rs113044851,and then analyze the genotype-phenotype correlation.(3)Bioinformatics methods were used to predict microRNA(miRNA)that bind to CTH gene and can be affected by rs113044851 polymorphism.(4)Constructed luciferase reporter gene vetors with a length of approximately 300 base pair(bp)from the 3’UTR of CTH,containing rs113044851 polymorphism(pGL3-CTH-WT/MT),were co-transfected into 293T cell line with pRL-SV40 and miR-1324 mimic/miRNA mimic negative control respectively,and Dual Luciferase assay was performed to exame the luciferase activity in different treatment groups,and validate the effection of rsl 13044851 on the transcriptional activity of CTH.Results:(1)Genotyping results illustrated that the Indel polymorphism(rs113044851)had balanced and adequate genotypic and allelic frequency distribution.There were no deviations from the Hardy-Weinberg equilibrium(P>0.05),suggesting that the included samples were representative of the population.Logistic regression analysis demonstrated that after adjustment for age and sex,compared with those who carry Del/Del genotype,individuals with Ins/Del genotype significantly decrease the risk of SCD(OR=0.61,95%CI:0.37-0.99,P<0.05).At the allelic level,compared with the deletion allele,the insertion allele of rs113044851 was correlated with a significant decrease on the risk of SCD(OR=0.58,95%CI:0.38-0.88,P<0.01).(2)WB results revealed that the expression level of CTH protein in samples with Del/Del genotype was significantly higher than those with Ins/Del genotypes.(3)Bioinformation analysis indicated has-miR-1324 may affect the transcriptional activity of CTH.(4)Results of Dual Luciferase assay suggested that has-miR-1324 could inhibit the expression of reporter genes in both WT and MT groups,and the inhibition was more obvious in the MT group.Conclusions:(1)The Indel polymorphism(rs113044851)in the 3’UTR of CTH was significantly correlated with SCD susceptibility in Chinese Han population.(2)The positive genotype-phenotype correlation between different genotypes of rs113044851 and the expression level of CTH protein in myocardium tissues was validated.(3)The Indel polymorphism rs113044851 may regulate the expression of CTH through affecting the binding of hsa-miR-1324 to CTH mRNA,and furtherly participate in the occurrence of SCD.