| Background and Aims:Stroke is one of the highest morbidity, mortality, disability diseases in the current world. It is a complex disease comprised of two clinical manifestations, hemorrhagic or ischemic brain injury according to the different pathogenesis. China has a high incidence of stroke with an increasing rate year by year, and the ischemic stroke(IS)accounts for 45.5%~75.9% of all cases of stroke. Therefore, it is very important theoretical basis for the stroke prevention to study the risk factors and pathogenesis of IS.DeCODE Study Group firstly reported the independent risk gene, 5-lipoxygenase activating protein(ALOX5AP) gene, which may cause myocardial infarction(MI) and ischemic stroke by genome-wide screening in Icelandic population. The5-lipoxygenase activating protein(FLAP), encoded by the ALOX5 AP gene, is a crucial mediator of the initial biosynthesis of leukotrienes from arachidonic acid catalyzed by 5-lipoxygenase(5LO) and causes the accumulation of LTs in fatty deposits on the arterial wall. The subsequent collapse of these deposits by the immune system has been implicated in the development of atherosclerosis and an increased risk of IS.With the development of the association study to a genome-wide association analysis study(GWAS) era, the GWAS of IS has been studied in the European population recently, such as Icelandic population and Japanese population. However,it still exists the genetic heterogeneity among different races. Therefore, how to do the genetic association study of IS in the GWAS era will face a great challenge.Recent studies have found that epigenetic mechanism is involved in the onset and development of cardiovascular disease and gradually being revealed. Epigenetics is stable and heritable patterns of changes in the regulation of gene activity and expression that are not dependent on alterations of gene sequence. DNA methylation is an important epigenetic mechanism which regulates gene expression. It is the covalent modification of cytosine residues in the DNA sequence through the addition of a methyl group that converts cytosine to 5-methyl cytosine. In recent years, there is an increasing evidence that the abnormal gene expression resulting from the alternation of the DNA methylation level. However, the methylation status of the ALOX5 AP gene in the promoter region has not been reported.The introduction or removal of cytosine-phosphate-guanine(CpG) dinucleotides,which are possible sites of DNA methylation in gene promoter Cp G island, has been suggested as a potential mechanism through which SNPs(Cp G-SNPs) can affect gene function via DNA methylation, causing allele specific DNA methylation(ASM)phenomenon. Therefore, Cp G-SNPs is considered to adjust the interaction of the genetic(SNP), and epigenetic(DNA methylation), which affect the underlying mechanisms of gene expression function. At present, Association study of between the promoter Cp G-SNPs and DNA methylation in the ALOX5 AP gene with IS has not yet been reported.Therefore, there produced a hypothesis that the promoter CpG-SNPs in theALOX5AP gene maybe regulate the m RNA expression of gene via allele specific DNA methylation(ASM), and then affect the genetic susceptibility of IS. The aim of this study is to reveal the pathogenesis of IS and its characteristics from the novel respective of genetic, epigenetic, and their interaction, which provides new ideas for IS clinical treatment and prevention.Study population:1.1 Selection of IS patientsIS patients who were selected in departments of neurology in the First Affiliated Hospital of Zhengzhou University and the First People Hospital of Zhengzhou were enrolled from March 2015 to March 2016.Initial population: 40 patients with IS, 26 men, 14 women, mean age of 61.1±13.3years old.Verify the crowd: 90 patients with IS, 51 men, 39 women, mean age of 60.5±12.5years old.IS was diagnosed by the diagnostic criteria of ischemic stroke revised in the fourth session of national conference on cerebrovascular disease. Brain imaging was carried out by computed tomography and/or magnetic resonance imaging, and ancillary diagnostic investigations and standardized blood tests were also performed.1.2 Selection of control groupControl group were selected from the same demographic area and matched with the cases by age, sex and residency. All controls were free of cerebral hemorrhage and cardiovascular disease and so on.Initial population: 40 patients with IS, 18 men, 22 women, mean age of 57.9±14.1years old.Verify the crowd: 96 patients with IS, 42 men, 54 women, mean age of 62.5±13.2years old.All the subjects studies had no blood relationship with each other. And all of them were native Han population in Henan Province. Signed consent form was got from every candidate. The study protocols were authorized by the Ethics Committee on Human Research of Zhengzhou University.Methods:Whole blood was collected using EDTA-Na2 anticoagulation, and genomic DNA and RNA was extracted using the whole blood genomic extraction kit, then reverse transcription. We firstly detected the m RNA expression level with RT-PCR and then the DNA methylation level of ALOX5 AP gene promoter with the bisulfite sequencing PCR(BSP). Six SNPs loci in ALOX5 AP gene promoter Cp G island, rs370679485,rs74043542, rs146576796, rs4073259, rs116205889, rs115053311 were selected and genotyped by Sanger sequencing technology. Finally, the positive correlation Cp G-SNP loci rs4073259 was validated in a larger cohort by SNa Pshot minisequencing technology.All statistical analysis was performed using the SPSS 19.0 package. Differences in quantitative variables between groups were expressed with mean±SD and analyzed using the Student’s t-test and one-way ANOVA analysis. Pearson’s chi-squared test was used to test for differences in qualitative variables and genotype/allele frequencies. Hardy-Weinberg equilibrium(HWE) testing was carried out by using SHEsis software. 95% confidence interval(CI) and Odds ratio(OR) were calculated to test the association between risk factors and IS. P value less than 0.05 was considered statistically significant. Association study of the average DNA methylation of the ALOX5 AP gene promoter and m RNA expression level was carried out by using the Bivariate correlation analysis method.Results:1. The m RNA expression level of ALOX5 AP gene was significantly higher in the IS patients(2.23±0.31) than that in controls(1.03±0.25, P<0.05).2. The DNA methylation level of ALOX5 AP gene promoter in IS group was significantly higher than that of Control group(P<0.05) by detecting 17 Cp Gs loci in the promoter region of ALOX5 AP gene; In the gender stratification analysis,methylation level of IS group was significantly higher than that of control group in male(P<0.05).3. The DNA methylation level of ALOX5 AP gene promoter was significantly positively related with its m RNA expression level(r=0.341, P=0.031).4. The GG genetype and G allele in the Cp G-SNP loci rs4073259 of ALOX5 AP gene were significantly associated with a reduced risk of IS(P=0.038, OR=0.415, 95%CI:0.179~0.960; P=0.043, OR=0.656, 95%CI: 0.435~0.988). And GG genotype was revealed to be associated with a decreased risk of IS with the recessive genetic mode(P=0.039, OR=0.471, 95%CI: 0.228~0.979).5. Compared the average methylation level of Cp G8 among AA 、 AG and GG genotypes of Cp G-SNP loci rs4073259, the methylation level of GG genotype was significantly higher than that of AA genotype in Cp G8(P<0.05). The m RNA expression levels of GG genotype was also significantly lower than that of AG and GG genotypes, the difference was considered statistically significant(P<0.05).Conclusions:1. The overexpression of proinflammatory gene ALOX5 AP maybe mediate the causation of IS by participating in arterial wall inflammation.2. ALOX5 AP gene promoter hypermethylation are significantly associated with IS,and gender differences.3. ALOX5 AP gene CpG-SNP loci rs4073259 is a potential genetic protective factor for IS.4. The G allele specific DNA methylation(ASM) of CpG-SNP loci rs4073259 in the ALOX5 AP gene inhibit the expression of gene, and then affect the genetic susceptibility of IS. |
PDF Full Text Request