Correlation Between Genetic Polymorphisms Of COX10 And HSPA1B Genes And Sudden Cardiac Death And Generation Of Risk Model

Part Ⅰ Association study between Insertion/Deletion polymorphism of COX10 gene and sudden cardiac death in Chinese populationsObjective:To investigate the association between an insertion/deletion(Indel)polymorphism(rs397763766)in 3’UTR of COX10 gene and sudden cardiac death(SCD)susceptibility in Chinese Han populations and the potential mechanism in the pathogeneisis of SCD.Methods:(1)Polymorphism screening:All single nucleotide polymorphisms(SNPs)and Indel polymorphisms with minor allele frequency(MAF)>0.01 in 3’UTR of COX10 gene were selected and their Linkage Disequilibrium(LD)and expression quantitative trait loci(eQTL)were analyzed using data from the 1000 Genomes Project(1000G)database.In the final,the 2-base(2-bp)Indel polymorphism rs397763766 was selected as the candidate variant in our case-control study.(2)Case-control study:Blood samples from 158 SCD cases and 624 matched healthy controls were collected and genotyped.Logistic regression analysis was used to study the association between rs397763766 variant and SCD susceptibility.(3)Genotype-phenotype correlation analysis:Real-time PCR and Western blot(WB)quantification were utilized to investigate the genotype-phenotype correlation between the genotypes of rs397763766 and COX10 expression at mRNA and protein level in cardiac tissues,respectively.(4)Genotype-phenotype correlation verification and bioinformatic analysis:Miranda was used to predict microRNA(miRNA)which may affect COX10 transcriptional activity that could be regulated by rs397763766 variant;Constructed reporter plasmid vectors including the Indel allele of rs397763766 were transfected into 293T cell line and Dual-Luciferase Reporter Assay was performed to further explore the effect of rs397763766 on COX10 transcription activity through interfering binding with miRNA.Results:(1)Polymorphism screening:There are 14 SNPs and Indels with MAF>0.01 in the 3’UTR of COX10 gene.Additional LD analysis suggested that two Indel variants rs200239586 and rs397763766 with other three SNPs were in one LD block.The capillary electrophoresis(CE)coupled with PCR-based short tandem repeat(STR)assays are commonly used in forensic practice.Since the Indel variations have similar length polymorphism characteristic with STR markers,these kind of Indels have the same compatibility with the current forensic CE platform.Since rs200239586 is a variant with duplicated thymine that is difficult in genotyping,we choose rs397763766 as the candidate variant representing the LD block to investigate its association with SCD.(2)Statistical analysis of genotyping results from SCD and healthy controls revealed that the genotypic and allelic frequencies of rs397763766 in the 3’UTR of COX10 gene were in a balanced distribution.Genotypic frequencies observed in the controls were consistent with the HardyWeinberg equilibrium(P=0.554).Logistic regression analysis showed that the del/del genotype of rs397763766 was associated with an increased risk for SCD[OR=2.49,95%CI:1.49-4.16,P=4.01×10-4]susceptibility than ins/ins genotype in codominant model;similar trend was also found in dominant and additive models.(3)Genotype-phenotype correlation analysis:Real-time PCR results showed that the relative COX10 expression at mRNA level in samples with ins/del and del/del genotype was 2.08 fold higher than that with ins/ins genotype(P<0.01),while WB results indicated that COX10 expression at protein level was not significantly different(P>0.05)between different genotypes.(4)Genotype-phenotype correlation verification and bioinformatic analysis:It is demonstrated that hsa-miR-15b could bind with 3’UTR of COX10 gene that could be regulated by rs397763766 variant on Miranda;Dual Luciferase Reporter Assay results demonstrated that the luciferase activities of cells transfected with pGL3-Control-COX10-MT were obviously higher than that with pGL3-Control-COX10-WT.The luciferase activities of cells co-transfected with pGL3Control-COX10-WT and miR-15b-mimic were significantly reduced compared with the control group and reduced more obviously with the concentration of miR-15b-mimic increased,and the luciferase activities of cells with pGL3-Control-COX10-MT and miR15b-mimic were also decreased with the concentration of miR-15b-mimic increased to 100nM.Conclusion:(1)The rs397763766 variant within 3’UTR of COX10 gene was highly correlated with SCD susceptibility in Chinese Han populations.(2)The rs397763766 variant was likely to regulate COX10 expression at mRNA level through interfering binding with hsa-miR-15b,thus conferred the risk of SCD.(3)The rs397763766 variant might be a potential marker for forensic molecular diagnosis and genetic counseling of SCD.Part Ⅱ Genetic correlation study of an Indel polymorphism in HSPA1B gene with the risk of SCD in Chinese populationsObjective:To investigate the correlation between an Indel polymorphism(rs3036297)within 3’UTR of HSPA1B gene and SCD susceptibility in Chinese populations and the possible role of rs3036297variant in the pathogenesis of SCD.Methods:(1)Polymorphism screening:All SNPs and Indel polymorphisms with MAF>0.05 in 3’UTR of HSPA1B gene were selected and based on the forensic practice,the 5-bp Indel variant rs3036297 with potential functions was selected as the candidate variant.(2)Case-control study:Blood samples from 157 SCD cases and 658 matched healthy controls were collected and genotyped.Logistic regression analysis was used to study the correlation between rs3036297 variant and SCD susceptibility.(3)Bioinformatic analysis:Miranda was utilized to predict miRNA which may affect HSPA1B transcriptional activity that could be regulated by rs3036297 variant;Haploreg and ENCODE databases were used to predict the functional relevance of rs3036297;GTEx and 1000G databases were employed to investigate eQTL genes regulated by rs3036297 variant;JASPAR database was utilized to predict the transcriptional factors that could bind with promoters of eQTL genes and be regulated by rs3036297 variant.(4)Genotype-phenotype correlation verification:Constructed reporter plasmid vectors with approximately 320 bp fragments including the Indel allele of rs3036297 were transfected into 293T cell line and Dual-Luciferase Reporter Assay was performed to further explore the effect of rs3036297 on HSPA1B transcription activity through interfering binding with miRNA.Real-time PCR was used to investigate the genotype-phenotype correlation betweet genotypes of rs3036297 and eQTL gene expression at mRNA level.Results:(1)Polymorphism screening:All variants within HSPA1B gene ith MAF>0.05 were selected(rs144223778,rs6457452,rs2763979 and rs3036297).The first three variants were SNPs while the last was Indel polymorphism.Since Indel polymorphisms have similar length polymorphic characteristics with STR markers which are compatible with the current forensic capillary electrophoresis(CE)platform,the rs3036297 variant was chosen as the candidate variant to investigate its correlation with SCD susceptibility and its potential eQTL effect.(2)Statistical analysis of genotyping results from SCD and healthy controls revealed that the genotypic and allelic frequencies of rs3036297 in the 3’UTR of HSPA1B gene were in a balanced distribution.Genotypic frequencies observed in the controls were consistent with the Hardy-Weinberg equilibrium(P>0.05).Logistic regression analysis showed that the ins/ins genotype of rs3036297 was associated with a decreased risk for SCD[OR=0.31,95%CI:0.14-0.70,P=3.24×10-3]susceptibility than del/del genotype in codominant model;similar trend was also found in dominant and additive models.(3)Bioinformatic analysis:It was demonstrated by miRanda that hsa-miR-134-5p could bind with 3’UTR of HSPA1B gene which could be regulated by rs3036297 variant;Functional annotation from Heploreg,ENCODE databases revealed that variant ra3036297 resided in regions that are rich in H3K4me1 and H3K27ac,the markers of cis-regulatory element.;eQTL analysis using data from GTEx and 1000G databases identified that the promoter of HLA-DRB5 gene could interact with rs3036297 variant,with higher expressions observed in samples with del allele;JASPAR database indicated that transcriptional factor HNF1A could bind with the promoter region of HLA-DRB5 gene regulated by rs3036297 variant.(4)Potential regulating mechanisms:Dual-Luciferase Reporter Assay results demonstrated that the luciferase activities of cells transfected with pGL3-Control-HSPA1B-MT were obviously lower than that with pGL3-Control-HSPA1B-WT.The luciferase activities of cells co-transfected with pGL3-Control-HSPA1B-MT and miR-134-5p-mimic were significantly reduced while cells co-transfected with pGL3-Control-HSPA1B-WT and miR134-5p-mimic were not significantly changed;Real-time PCR analysis showed that the relative HLA-DRB5 expression at mRNA level in samples with del/del genotype was 1.78 fold higher than that with ins/ins and ins/del genotype.Conclusion:(1)The rs3036297 variant in 3’UTR of HSPA1B gene was highly correlated with SCD susceptibility in Chinese Han populations.(2)The rs3036297 variant may regulate HSPA1B expression via a mechanism of miR-134-5p binding and HLA-DRB5 expression via a long-range promoter interaction which both contributed to SCD susceptibility.(3)The rs3036297 variant might be a potential marker for forensic molecular diagnosis and genetic counseling of SCD.Part Ⅲ Genetic risk score predicts the risk of SCD in Chinese populationsObjective:Try to establish a risk prediction model for SCD in Chinese populations,based on the susceptibility loci of SCD confirmed in our lab.Methods:Based on genotypes of the SCD susceptibility variants confirmed in our lab,genetic risk score(GRS)and weighted genetic risk score(wGRS)were calculated to evaluate the co-effects of these variants in predicting SCD risk.The receiver operator characteristic(ROC)curve was generated and the area under the curve(AUC)was calculated to determine the discrimination of GRS or wGRS between SCD cases and healthy controls.Results:Logistic regression analysis showed that people with GRS ≥ 6 conferred higher risk of SCD than that with GRS<6[OR=3.67,95%CI(2.42-5.55)]and people with wGRS>3.184 conferred higher risk of SCD than that with wGRS ≤3.184[OR=4.74,95%CI(3.02-7.46)].The AUC base on GRS and wGRS were 0.713 and 0.723,respectively.Conclusion:GRS calculated by the 5 SCD susceptibility variants confirmed in our lab was associated with increased risk of SCD.