Establishment Of CAD Prediction Model Based On SelS Gene Risk Mutation Sites And Study On Promoter Mutation Mechanism

Objective:(1)In this study,detecting the new mutation site of SelS gene in 48 coronary artery disease(CAD)patients in xinjiang,and was verified in 1028 people to clarify the correlation between these sites mutations and CAD.(2)Through molecular cloning,site-specific mutation and other techniques,luciferase reporter gene vectors of SelS gene promoter region of wild type and mutant type were constructed.The luciferase expression activity of each group of vectors in cells,the electrophoretic mobility experiment(EMSA)and EMSA competitive experiment were conducted to further explore the pathogenesis of CAD caused by mutations in SelS gene promoter region.Methods:(1)Using polymerase chain reaction-direct sequencing to select 48 patients who were diagnosed with CAD by coronary angiography;the SelS gene promoter region and coding region were amplified,DNAMan software and Chromas software were used for sequencing results.Compared with the sequence,the SNP loci found were compared in the human genome database.For example,the SNP loci were not found in the Hap Map database,NCBI / SNP database,and the Japanese genome database,so the SNP sites were identified as new SNP site;(2)A case-control study was used to select 576 patients with CAD confirmed by coronary angiography and 452 patients in the control group.Genotyping these new mutations site of SelS gene and tagged SNP,and analysis of the correlation between the new mutation sites of SelS gene and tagged SNPs and CAD;(3)using molecular cloning technology to link the target fragment to the gene containing luciferase the reporter gene pGL3-Basic vector and constructing a wild-type SelS promoter reporter gene vector pGL3-Basic-SelS-WT and Mutant SelS promoter reporter gene vector pGL3-Basic-SelS-Mut;the promoter reporter gene vectors were transfectedinto HEK-293 T cells in vitro using liposome transient transfection technology,and the different luciferase reporter gene detection systems were used to analyze the differences effects of vectors on transcriptional activity of downstream genes.Use SPSS19.0 software package to process the data.Results:(1)We genotyped the SelS genes rs117613208,rs117512970,rs986500879,and rs542989868 at four SNP loci.For the rs117613208 locus,the AA,AT,and TT genotype frequencies of the CAD group and the control group were:0.883,0.146,0.016 and 0.901,0.096,0.002,the genotype distribution was statistically different between the two groups(P = 0.001).There was no significant difference in the frequency of genotype distribution between rs117512970,rs986500879,and rs542989868(P>0.05).In multivariate logistic regression analysis,after adjusting for confounding factors,the TT genotype at rs117613208 locus was still an independent risk factor for CAD.Those with TT genotype at rs117613208 locus were 2.107 times more likely to develop CAD than non-carriers.(2)Agarose gel electrophoresis,double-enzyme digestion,and gene sequencing verified that a luciferase reporter gene vector containing the wild-type and mutant SelS gene promoter region was successfully constructed.The transcriptional activity of the SelS mutant promoter was significantly lower than the transcriptional activity of the SelS wild-type gene promoter,which proved that mutations could cause SelS transcriptional activity to be significantly decreased.The EMSA and competitive EMSA experiments we conducted showed that the SelS gene promoter region had specific binding to nuclear proteins.Conclusion:(1)This study describes for the first time the distribution frequency of SelS gene polymorphisms in Xinjiang population,which enriches the genetic resource pool of SelS gene in Xinjiang population;(2)The rs117613208 site of SelS gene is associated with the occurrence of CAD in Xinjiang population,rs117613208 site TT genotype carriers have a significantly increased risk of CAD;The GASDLY score including risk allele and clinical variables,has a good diagnostic performance for CAD.(3)this study proved SelS gene promoter region mutations can lead to SelS transcription activity significantly decreased,and competitive EMSA experiments show the combination of gene promoter region and nucleoprotein has specificity,prompt SelS gene promoter point mutation may be associated with the pathogenesis of the xinjiang region population CAD.