Genetic Variants And Functional Analyses Of The ATG16L1 Gene Promoter In Acute Myocardial Infarction

Background:Acute myocardial infarction(AMI),a common complex disease caused by an interaction between genetic and environmental factors,is a serious type of coronary artery disease(CAD)and is also a leading cause of death worldwide.Autophagy is a highly conserved lysosome-mediated protein and organelle degradation mechanism,which plays a vital role in maintaining cell homeostasis.Many evidences indicate that autophagy is closely related to the occurrence and development of many diseases,including malignant tumors,neurodegenerative diseases,cardiovascular diseases,immune diseases and so on.In the cardiovascular system,autophagy is essential to preserve the homeostasis and function of the heart and blood vessels,and participate in the occurrence and development of many cardiovascular diseases.Studies have shown that autophagy defects damage cardiomyocytes,vascular endothelial cells and vascular smooth muscle cells,leading to cardiac dysfunction.Autophagy-related 16-like 1(ATG16L1)is a key regulatory factor of autophagy and plays an important role in induced autophagy.Although there are some studies on ATG16L1 in the context of immunity,atherosclerosis and cardiomyocytes,there is no research on the role of ATG16L1 gene in the occurrence and development of CAD or AMI.We speculate that the mutation in the promoter region of the ATG16L1 gene may be involved in the occurrence and development of human AMI as a low-frequency risk factor.Objective:By identifying the sequence variation in the promoter region of the ATG16L1 gene in the AMI and the control group,and performing statistical analysis on the data of DNA sequence variants(DSVs)and single nucleotide polymorphisms(SNPs),the genetic variation of the promoter region of ATG16L1 gene was studied.Through further research on the influence of the variant sequence of the promoter region of the ATG16L1 gene on the transcriptional activity of the ATG16L1 gene,and the changes in the binding of the ATG16L1 gene promoter and transcription factors by the DSVs and SNPs associated with AMI,the functional analysis of the ATG16L1 gene promoter was carried out.By studying the genetic factors and molecular mechanisms of AMI at the genetic level and combining with environmental factors,can provide molecular genetic basis for the evaluation,prevention and treatment of AMI.Methods:1.The ATG16L1 gene promoter was amplified and sequenced by polymerase chain reaction(PCR)in 329 AMI patients and 359 controls using case-control method.Data statistics and analysis were carried out after DNA sequencing and alignment of SNPs database.2.Fisher’s exact probability test was used to perform Hardy-Weinberg equilibrium test on the allele distribution frequency of the two groups of SNPs to analyze whether the population studied in this study reached genetic equilibrium and the reliability of the data in this study.Significant differences in allele and genotype frequency were assessed using the chi-square test.The odds ratio(OR)and 95%confidence interval(CI)of Logistic regression analysis were used to measure the correlation between SNPs and AMI incidence.The association between five genetic models(dominant,dominant,dominant,recessive,and log-additive)and AMI was analyzed by SNP-Stats(https://www.snpstats)after adjusting for age and sex.3.Linkage disequilibrium(LD)among SNPs was analyzed using Haploview software(version 4.2),and haplotype association analysis was performed using SHE-sis online software(http://analysis.bio-x.cn/SHEsisMain.htm).Multifactorial gene-gene and gene-environment interactions are widely present in complex diseases such as cardiovascular disease.Generalized multifactor dimensionality reduction(GMDR)software was used to analyze the interactions between SNP-SNP.4.A recombinant plasmid containing the mutant sequence of the ATG16L1 gene promoter was constructed.The functional analysis of the mutant sequence in the ATG16L1 gene promoter region was performed by transfection of H9C2 and HEK-293 cells with dual luciferase reporter assay(DR)luciferase expression level.5.For the mutation sites of ATG16L1 gene promoter identified only in AMI patients,TRANSFAC database was used to analyze and predict the affected transcription factors in human,and the electrophoretic mobility shift assay(EMSA)was used to detect the interaction between protein and DNA sequence in vitro.Result:1.A total of 12 DSVs,including 10 SNPs,were identified by PCR sequence analysis of the promoter region of the ATG16L1 gene in 690 subjects.Three SNPs(g.233250693 T>C[rs185213911],g.233250946 G>A[rs568956599]and g.233251133 C>G[rs1301744254])were identified in 5 patients with AMI,not in the control group.In addition,six SNPs(g.233250522 G>C[rs75824126],g.233250873 G>A[rsl46693112],g.233250963 T>C[rs1816753],g.233251039 T>C[rs12476635],g.233251112 A>T[rs74599577],and g.233251699 T>G[rs2289477])were identified in both AMI patients and controls at similar frequencies(P>0.05).2.The Hardy-Weinberg equilibrium test indicated that all SNPs(rs75824126,rs185213911,rs146693112,rs568956599,rs1 816753,rs12476635,rs74599577,rs1301744254,and rs2289477)in the two groups showed Hardy-Weinberg equilibrium(control group:P=0.747、1.000、0.380、0.158、0.747、1.000、0.747、1.000、0.380、0.158、0.747.1.000;AMI group:P=0.550、0.959、0.967、0.138、0.398、0.550、0.956)(P>0.05),indicating that the samples were from a population with genetic balance and had good representativeness.The chi-square test evaluation results show that there were no statistically significant differences in the frequency of genotypic distribution among cases and control groups.Unconditional Logistic regression analysis showed that 9 SNPs in the promoter region of ATG16L1 gene had no significant correlation with AMI(P>0.05).There was no significant difference between SNPs in the promoter region of Atg16L1 and AMI among five genetic models(P>0.05).3.LDS and haplotype analysis of 9 SNPs in the promoter region of Atg16L1 gene by Haploview and SHE-sis software showed that rs75824126 and rs74599577 had perfect LD(D=1.000,R2=1.000),rs1816753 and rs2289477 were linked to each other(D’>0.800,r2>0.330);RS1816753 and RS12476635 constitute LD haplotype module.In the haplotype analysis region,there was no significant difference in the frequency distribution between AMI group and control group for the single ploidy of ATG16L1 promoter(P>0.05).There was no statistical difference in the interaction between SNP and SNP(P>0.05).4.The effects of DSVs on the transcriptional activity of the ATG16L1 promoter were detected by constructing a luciferase reporter gene vector containing the following wild type and variant ATG16L1 promoters:wild-type pGL3-WT,pGL3-233250693C,pGL3-233250873A,pGL3-233250946A,pGL3-233251039C,pGL3-233251111T,pGL3-233251133G,pGL3-233251186G,pGL3-233251524C,pGL3-233251563T,and pGL3-233251699G The constructed reporter gene vector and the reference plasmid PRL-TK were co-transfected into H9c2 and HEK-293 cells in vitro to detect the dual luciferase activity.The transcriptional activity of ATG16L1 gene promoter mutants was compared to that of wild type(100%).The results showed that three SNPs(233250693 T>C[rs185213911],233250946 G>A[rs568956599],233251133 C>G[rs 1301744254])significantly increased the transcriptional activity of Atg16L1 gene promoter(P<0.05).5.Through the analysis of the TRANSFAC database to predict the binding sites of transcription factors affected by DSVs,it was found that SNPs identified in AMI patients can create,modify and eliminate putative binding sites of transcription factors.Among them,SNP g.233250693 T>C(rs185213911)can create binding sites for transcription factors NF-kappaB and GKLF,weaken the binding of transcription factor ETS1,and eliminate the binding sites of transcription factors RBP-Jkappa and TCF-1;g.233250946 G>A(rs568956599)Create a binding site for transcription factor PPAR and VDR,enhance the binding of transcription factor T3R-β,weaken the binding of transcription factor AP-2 α and CP2,and eliminate transcription factors Elk-1,Fli1,ETS1 Binding site;g.233251133 C>G(rs1301744254)can create binding sites for transcription factors ZIC3,Zbtb44 and SREBP-1/2,weaken the binding of transcription factor c-MAF,and eliminate transcription factors meisl,HDAC1,ATF-4 And CTCF binding site.The EMSA results showed that g.233250693 T>C(rs185213911)weakened the binding of the transcription factor to the promoter of the ATG16L1 gene,g.233251133 C>G(rs1301744254)eliminated the binding of the transcription factor to the promoter of the ATG16L1 gene,and g.233250946 G>A(rs568956599)did not affect the binding of transcription factors.Therefore,DSVs may alter the activity of ATG16L1 gene promoter by interfering with the binding of transcription factors and ATG16L1 gene promoter,affect the transcription level of ATG16L1 gene,and then change the level and function of autophagy to affect the occurrence and development of AMI.Conclusion:In this study,the genetic variation and function of the ATG16L1 gene promoter in acute myocardial infarction were analyzed.Three DSVs were identified in AMI patients,which significantly changed the transcriptional activity of the promoter of the ATG16L1 gene,and two of them significantly affected the binding of related transcription factors.Therefore,these DSVs may cause dysregulation of ATG16L1 gene expression,as a low-frequency risk factor,promote the occurrence and development of AMI through autophagy and non-autophagy functions.