SNP Analysis Of Five Candidate Genes And Study Of Their Genetic Susceptibility With Ischemic Cerebrovascular Disease

In post-genomic era, functional genomics has become the emphases and focus of life sciences. Because of their abundance and widespread genomic distributions, single nucletide polymorphisms (SNPs) have become the most useful genome markers in human molecular genetics and are used in gene mapping, disease-associated variants, genome-wide haplotype map, disease susceptibility, population genetics as well as pharmacogenomics and environmental genomics.Ischemic cerebrovascular disease (ICVD) is a multifactorial disease caused by environmental and genetic factors. The etiology of ICVD is complicated and is not understood completely until now. Although a number of conventional risk factors, including hypertension, diabetes, the ages, hyperlipidemia, smoking, fat, and hyperhomocysteinemia, might contribute to the development of ischemic cerebrovascular disease, genetic changes play a crucial role in it. The studies have demonstrated that the variations in genomic DNA sequence, i.e. gene polymorphisms are the most important determinants for disease susceptibility, clinical phenotype diversity and different response to pharmacotherapies. The dominant approach to the association has been to study the polymorphisms within the candidate genes from biological pathways and processes strongly implicated in the pathogenesis of ICVD. There are numerous kinds of genes associated with ICVD, for example, hypertension-associated genes, coagulation genes, atherosclerotic genes and the Hcy metabolism-relative enzyme genes. But these conclusions almost have come from the Occident and Japan. To the best of our knowledge, no large-scale reports are available for the Chinese Han nationality until now. moreover, many candidate gene frequencies have different ethnic background and geographical distribution.So we will take SNP as genetic marker and select 5—lipoxygenase activating protein gene(ALOX5AP), angiotensinogen(AGT)gene, Methylenetetrahydrofolate reductase(MTHFR)gene, Cystathioninep-synthase, (CBS)gene and Methionine synthase(MS)gene as candidate genes of ICVD to search for disease-associated variants and identifying susceptibility genes in the Henan Han nationality in our case-control association study.The main contents including three parts:Part oneA study of the correlation between six SNPs of 5—lipoxygenase activating protein gene and Ischemic cerebrovascular diseaseMethods246 cases as patient group with ischemic cerebrovascular disease in department of neurology in Henan Provincial Hospital were enrolled from December 2004 to July 2005. 145 of them were males and 101 were females, with an average of 59.98 years old. Four subtypes of ICVD were recruited including 99 cerebral thrombosis, 82 cerebral infarction, 37 lacunar infarction and 28 transient ischemic attack. Individuals were excluded when they had brain tumors, chronic inflammatory diseases, and autoimmune diseases. 245 healthy volunteers of control group were recruited in the study. There were 134 males and 121 females with an average of 56.28 years old. All people were unrelated Henan Han Nationality by blood and without cancer, epilepsy and hepatic or renal diseases. Everyone enrolled must take a questionnaire to say his age, sex, smoking, drinking and so on. Consents of all participants were obtained.Six SNPs of SG13S114,SG13S377,SG13S41,SG13S35,SG13S89 and SG13S32 in the ALOX5AP were genotyped by SNaPshot analysis. The material steps were as follows: Genomic DNA was extracted from peripheral white blood cells by using the phenol/chloroform method. Touch-Down PCR amplification was adopted. In order to eliminate the excess of primers and dNTPs, the PCR products were digested by SAP and ExoⅠ. Multiplex PCR SNaPshot reaction was performed. Finally, Mix 9μl HiDi Formamide,0.25μl Liz120 size standard and 1μl purified extension product denatured at 95℃5 minutes and quenched on ice for 2 minutes at minimum, then loading on ABI3730xl and run GeneMapper4.0 to analyse the results.All the data were analyzed by SPSS 12.0 package. The chi-square test was used to compare the distributions of genotypes and allele of SNPs. The linkage disequilibrium(LD), haplotype frequencies were estimated with the program SHEsis. Deviation from the Hardy-Weinberg equilibrium were assessed by the chi-square test. The multivariate Logistic regression model was used to revise of the possible confounding factors on the association of the candidate gene with ICVD. These factors including age, sex, drinking and cigarette smoking. Associations between genotypes and ICVD risk were expressed as odds ratio (OR) and 95% confidence interval (CI). P values were two-tailed, and statistical significance was accepted at P＜0.05.ResultsThe genotype frequencies of SG13S114/AA(17.1%) and allele gene frequencies of SG13S114/A (41.3%) in ICVD patients were significantly higher than that(10.6%, 32.7%) in control (P＜0.05). We found the significant differences between male patients and male control group(P＜0.05). The significant differences were also found in patients with early age of onset and control group(P＜0.05). The persons with SG13S114/A allele genes had higher risk of ICVD by relative risk analysis, OR=1.350, 95%C1(1.041～1.751). Haplotype analysis revealed that SG13S377G/SG13S114A/SG13S41A/SG13S35G and SG13S377T/SG13S114G/SG13S41G/SG13S35A had significant differences between ICVD group and control group (P＜0.05).Pairwise Linkage disequilibrium analyses showed that the polymorphic loci between SG13S114 and SG13S377, SG13S32 and SG13S377 were in strong Linkage disequilibrium (D'＞0.8).The allele gene frequencies of SG13S35 in the Henan Han nationality were evidently different from those in the British.Part twoA study of the relationship between the angiotensinogen gene polymorphism and Ischemic cerebrovascular diseaseMethodsThe patient group and the control group were the same as part one. Four SNPs of G-6A, A-20C, T174M and M235T in the AGT gene were genotyped by PCR-RFLP method. All the data were analyzed by SPSS 12.0 package. The chi-square test was used to compare the distributions of genotypes and allele of SNPs. Hardy-Weinberg equilibrium, linkage disequilibrium(LD), haplotype frequencies were estimated with the program SHEsis. Associations between genotypes and ICVD risk were expressed as odds ratio (OR) and 95% confidence interval (CI). P values were two-tailed, and statistical significance was accepted at P＜0.05.ResultsThere were significant differences in the genotype frequencies of A-20C/AC between the ICVD group(26%) and the control group(18.4%, P＜0.05). The allele gene frequencies of A-20C/C in the ICVD patients(13.8%) were significantly higher than those (9.6%) in the control group(P＜0.05). We found the significant differences between male patients and control(P＜0.05). The significant differences were also found in patients with later age of onset and the control(P＜0.05). The persons with A-20C/C allele gene had higher risk of ICVD by relative risk analysis, OR=1.511, 95% CI(1.018～2.242). Haplotype analysis revealed that—20A/-6G/174C/235T and—20C/-6G/174C/235T had significant differences between the ICVD group and the control group (P＜0.05).Pairwise Linkage disequilibrium analyses showed that the polymorphic loci between G-6A and M235T, G-6A and A-20C, A-20C and T174M, T174M and G-6A were in strong Linkage disequilibrium (D'＞0.8).The allele gene frequencies of G-6A and M235T in the Henan Han nationality were evidently different from those in the Occident of population.Part threeA study of the relationship between polymorphisms of homocysteine metabolism related enzymes and ischemic cerebrovascular diseaseMethods 412 cases as patient group with ischemic cerebrovascular disease were enrolled. The diagnosis criterion of ICVD was just as the part one. 250 of them were males and 162 were females, with an average of 60.58±11.62 years old. Three subtypes of ICVD were recruited including 354 cerebral thrombosis, 23 lacunar infarction and 35 transient ischemic attack. Individuals were excluded when they had brain tumors, chronic inflammatory diseases, and autoimmune diseases. 500 healthy volunteers of the control group were recruited in the study. There were 274 males and 226 females with an average of 54.68±10.44 years old. All people were unrelated Henan Han nationality by blood and without cancer, epilepsy and hepatic or renal diseases. Consents of all participants were obtained.Three key enzyme gene polymorphisms of 5,10-methylenetrahydrofolate reductase (MTHFR), Methionine synthase(MS) and Cystathionine-βsynthase (CBS) in the homocysteine metabolism were genotyped by PCR-RFLP method. All the data were analyzed by SPSS 12.0 package. The chi-square test was used to compare the distributions of genotypes and allele of SNPs. Hardy-Weinberg equilibrium, linkage disequilibrium(LD), haplotype frequencies were estimated with the program SHEsis. The associations between genotypes and ICVD risk were expressed as odds ratio (OR) and 95% confidence interval (CI). P values were two-tailed, and statistical significance was accepted at p＜0.05.ResultsThere were significant differences in the genotype frequencies of MTHFRC677T/TT between the ICVD group (40%) and the control group(32.8%, P＜0.05). The allele gene frequencies of MTHFRC677T/T in ICVD patients were significantly higher than those in the control group(P＜0.05). We also found the significant differences between the male patients and the male control group(P＜0.05). The persons with MTHFR/TT genotype had higher risk of ICVD by relative risk analysis, OR=2.264, 95%CI(1.592～3.218). Haplotype analysis revealed that 677T/2756A and 677C/2756A had significant differences between the ICVD group and the control group (p＜0.0001). Pairwise Linkage disequilibrium analyses showed that the polymorphic loci between MTHFR/C677T and MS/A2756G were not in Linkage disequilibrium (D'=0.012).The allele gene frequencies of MTHFR/C677T and CBS844ins68 in the Henan Han nationality were evidently different from those in the Occident of population.Conelusions1. The polymorphic loci of SG13S114, A-20C and MTHFR/C677T are associated with ischemic cerebrovascular disease and might be risk factors of ischemic cerebrovascular disease of the Henan Han nationality. The association is significantly more prevalent in male patients of ICVD.2. Maybe the polymorphism of SG13S114 is related with young ICVD patients onset in the Henan Han nationality.3. It is likely that the different genotypes of AGT/A-20C are associated with old ICVD patients onset in the Henan Han nationality.4. Maybe the haplotypes of SG13S377G/SG13S114A/SG13S41A/SG13S35G,—20C/-6G/174C/235T and MTHFR677T/MS2756A may increase the risk of ischemic cerebrovascular disease in the Henan Han nationality.5. Maybe the haplotypes of SG13S377T/SG13S114G/SG13S41G/SG13S35A,—20A/-6G/174C/235T and MTHFR677C/MS2756A may have the protective effect against ischemic cerebrovascular disease in the Henan Han nationality.6. The polymorphic loci between SG13S114 and SG13S377, SG13S32 and SG13S377, G-6A and M235T, G-6A and A-20C, A-20C and T174M, T174M and G-6A are in the strong Linkage disequilibrium.7. The gene frequencies of SG13S35, G-6A, M235T and MTHFR/C677T vary remarkably in peoples of different ethnic backgrounds and geographical distributions.