An Insertion/deletion Polymorphism Within 3’UTR Of RYR2 Modulates Sudden Unexplained Death Risk In Chinese Populations

Objective: To evaluate the effects of an Insertion/Deletion(Ins/Del,Indel)polymorphism(rs10692285)within the 3’ untranslated region(3’UTR)of RYR2 gene on modulating sudden death risk in Chinese population,and investigate the underlying mechanisms and the possible role of rs10692285 in the pathogenesis of sudden death using the genotype-phenotype correlation analysis as well as bioinformatics approach.Methods:(1)First,bioinformatic techniques were applied to screen polymorphisms in the 3’UTR of RYR2,from which we chose a potential functional 4bp Indel polymorphism(rs10692285)as candidate.After the blood samples of 78 sudden death cases and 740 healthy controls were collected,case-control studies were performed within the cases categorized by pathological features and their matched controls using PCR combined with polyacrylamide gel electrophoresis(PAGE)method.And logistic regression was used to analyze the association between rs10692285 and the risk of sudden unexplained death(SUD)or sudden cardiac death caused by atherosclerosis(SCD-AS),respectively.(2)Real-time PCR analysis was used to investigate the correlation between the genotypes of the Indel polymorphism and the expression level of RYR2 on transcriptional level in human myocardium tissue samples.(3)WB quantification and IHC were used to verify the genotype-phenotype correlation between the genotypes of the Indel polymorphism and the expression level of RYR2 on translational level in human myocardium tissue samples.(4)Constructed reporter plamid vetors with approximately 300 bp fragments from the 3’UTR of RYR2,including the Ins/Del allele of rs10692285(pGL3-RYR2-WT/MT),were transfected into two cell lines(293T and H9C2)respectively,and Dual Luciferase assay was performed to further explore the impact of rs10692285 on RYR2 transcription activity.(5)The influence of the Indel polymorphism on the binding of RYR2 with specific miRNA,and on RYR2 local conformation,was predicted by bioinformatic approach.Results:(1)Statistics of genotyping results revealed that the genotypic and allelic frequency of the Indel polymorphism(rs10692285)in the 3’UTR of RYR2 was in an adequate and balanced distribution.In our control group,the allelic frequencies of Ins and Del were 0.228 and 0.772,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 SUD compared with individuals carrying the Del/Del genotype(OR=3.22,95%CI: 1.28-8.22,P<0.01).Statistics on the allelic level also revealed that individuals with the Ins allele have a significantly increased risk of SUD compared with individuals carrying the Del allele only(OR=2.03,95%CI: 1.08-3.77,P<0.05,Statistical power=0.743).However,there is no significant association between rs10692285 and SCD-AS susceptibility according to several common genetic models.(2)Real-time PCR results demonstrated that the expression level of RYR2 with Ins/Ins and Ins/Del genotype was 2.34 fold higher(P<0.01)than that with Del/Del genotype in human myocardium tissue samples.(3)Both WB and IHC results suggested that the RYR2 protein expression level in samples with Ins/Ins genotype was significantly higher than those with Ins/Del and Del/Del genotypes.(4)Results of Dual Luciferase assay demonstrated that the luciferase activity of cells transfected with pGL3-RYR2-WT or pGL3-RYR2-MT was both higher than that of cells transfected with control constructs.Furthermore,the luciferase activity of cells transfected with pGL3-RYR2-MT was higher than that of cells transfected with pGL3-RYR2-WT in both of the two cell lines(293T and H9C2).(5)Bioinformatic analysis demonstrated that rs10692285 could interfere the binding of hsa-miR-3614-5p to its target sequence in the 3’UTR of RYR2.In addition,this Indel polymorphism could alter the RYR2 mRNA local folding structures as well.Conclusion:(1)The rs10692285 polymorphism in the 3’UTR of RYR2 was highly relevant to the risk of SUD in Chinese population,but there is no significant association between rs10692285 and SCD-AS risk.(2)The genotype-phenotype correlation between rs10692285 and the expression level of RYR2 in myocardium tissues was confirmed.(3)The rs10692285 polymorphism was likely to be functional in regulating RYR2 expression through interfering the binding of hsa-miR-3614-5p to the 3’UTR of RYR2 or directly affecting the local folding structures of RYR2 mRNA.