An Insertion/deletion Polymorphism Within 5'UTR Of STIM1 Modulates Sudden Cardiac Death Risk In Chinese Populations

Objective: To study the correlation between the the insertion/deletion(Indel)polymophism rs147711952 in the 5'UTR of STIM1 gene and SCD risk in Chinese population,and the potential mechanisms in SCD pathogenesis using the genotype-phenotype correlation analysis as well as functional experiments and bioinformatics approach.Methods:(1)First,bioinformatics techniques were used to screen polymorphisms located in 5'UTR of STIM1,and a 5bp Indel polymotphism rs147711952 with potential function was chosen as candidate.Then,collected blood samples from 119 SCD cases and350 healthy controls,then categorized by pathological features.Next,PCR combined with polyacrylamide gel electrophoresis(PAGE)method were used to genotyping,and logistic regression was applied to analyze the association between rs147711952 and the risk of SCD.(2)Second,analysing Genotype-phenotype correlation,Real-time PCR was used to analyze the association between the genotypes of the Indel polymorphism and the expression level of STIM1 m RNA,micro-4687 and loc105376527 in myocardium tissue samples.(4)To study potential mechanisms:(1)Reconstructed reporter plasmid vectors with about 500 bp fragment from 5'UTR overlapping of STIM1 with Ins/Del allele(rs147711952)were constructed and transfected into 293 T cell line,and then Dual Luciferase assay was used to analyze the effect of the transcription activity of STIM1 and mi R-4687.(2)EMSA was used to anaylze the different binding strength between DNA(WT and MT)and nuclear extract.(3)Then,bioinformatics approach was used to predict the target genes of micro-4687,and then the correlation between the Indel polymorphism and expression level of its target gene was anaylzed by Real-time PCR.(4)Finally,pearson's correlation was used to anaylze the correlation between STIM1 and loc105376527.Results:(1)The statistics of genotyping results confirmed that the genotypic and allelic frequency of the Indel polymorphism(rs147711952)in the 5'UTR of STIM1 was in an adequate and balanced distribution.In our control group,the allelic frequencies of Ins and Delwere 0.67 and 0.33,respectively.Thus,there were no deviations from the Hardy-Weinberg equilibrium(P>0.05).Logistic regression analysis indicated that after adjustment for sex and age,individuals with the Ins/Del genotype possess an increased risk of SCD compared with individuals carrying the Del/Del genotype(OR=3.26,95%CI: 1.75-6.07,P<0.01).Statistics on the allelic level also revealed that individuals with the Del allele have a significantly increased risk of SCD compared with that of the individuals carrying the Ins allele only(OR=2.03,95%CI: 1.08-3.77,P<0.05).(2)Real-time PCR results demonstrated that STIM1 expression level has no significant difference among different genotypes,but the mi R-4687 expression level,located at downstream of rs147711952 in human myocardium tissue samples with Del/Del genotype was 0.62 fold lower(P<0.05)than that with Ins/Ins genotype;and the loc105376527 expression level,with Ins/Ins genotype was 8.87 fold higher(P<0.05)than that with Del/Del genotype in human myocardium tissue samples(3)potential mechanisms:(1)Dual-luciferase assay result revealed that luciferase activity of cells transfected with p GL3-STIM1-WT and p GL3-STIM1-MT were higher than controls.Moreover,luciferase activity of cells with p GL3-STIM1-WT was higher than cells with p GL3-STIM1-MT;(2)Result of EMSA shows that nuclear extract binding with Del/Del genotype,but not with Ins/Ins genotype;(3)Bioinformatic analysis combined with Real-time PCR shows that SCN5 A is micro-4687 target gene,and its expression level is significant decrease in Del/Del;(4)through pearson's correlation shows that there was some correlation between loc105376527 and STIM1.Conclusion:(1)rs147711052 in 5'UTR of STIM1 was highly relevant to the risk of SCD in Chinese population;(2)STIM1 expression level have no significant difference in different genotype,but the expression level of micro-4687 and loc105376527 have been affected by rs147711952;(3)we speculate that this Indel polymorphism participate in regulating expression of SCN5 A through micro-4687 change,and this change may due to different binding strength between DNA and nuclear extract;(4)Indel polymorphism may also affect loc105376527 expression leve then induce STIM1 expression level change.(5)rs147711052 may act as a genetic marker and applied into the molecular diagnosis of SCD.