| Background.Epidemiologic studies have shown that hyperlipidemia, hypertension, cigarette smoking, diabetes mellitus, obesity and family history of cardiovascular disease are the risk factors for coronary heart disease (CHD). Although the accumulation of coronary risk factors often leads to CHD, however, there is a population of patients without such classic risk factors who develop coronary artery disease. CHD may arise from interactions between environmental and genetic factors in general, but the precise mechanism by which coronary heart disease occurs remains to be elucidated. This study tested the hypothesis that a missense Glu298Asp variant within exon 7 of the endothelial nitric oxide synthase (eNOS) gene may be a potent risk for CHD in Chinese population. Methods.To examine the distribution of the missense Glu298Asp variant in exon on 7 of the eNOS gene, polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) analysis was performed in the 106 unrelated CHD patients (74 men and 32 women, mean age 65?0 years) and 108 unrelated normal subjects (68 men and 40 women, mean age 64? years). AH patients met thediagnostic criteria for ischemic heart disease by the World Health Organization (WHO), among which 58 ones were confirmed by coronary angiography. There was no relationship between individuals in the study population.Peripheral venous blood samples were collected after a 12-hour overnight fast. Serum total cholesterol (TC) and triglyceride (TG) were measured by conventional methods. Genomic DNA was extracted from whole blood by rapid salting out method. Using the primers described by Hingorani AD,et al ,we performed polymerase chain reaction (PCR) amplification. The 207bp PCR product for exon 7 was digested with direct restriction enzyme, Ban II. A single Ban II site was present in the wild type allele (G at 894), whereas no Ban II site was found in the mutant allele (T at 894). Therefore, treatment of the wild type (GG) with Ban II yielded 125bp and 82bp fragments. In the case of the heterozygous mutant, digestion with Ban II resulted in three fragments of 207,125 and 82bp in size. And a single 207bp fragment was observed upon in the homozygous variant (TT) with Ban II. These products were electrophoresed on 3% agarose gels, and DNA was visualized by ethidium bromide straining.The gene counting method was used to estimate the allele and genotype frequencies in patients group and normal subjects. StatisticVanalyses were performed with the SPSS 10.0. Values were considered statisticallysignificant at PO.05.Results.1. The genotype distribution was compatible with the Hardy-Weinberg equilibrium in the CHD, as well as MI and angina pectoris (AP) subgroups.2. Among the normal subjects, the frequencies of the eNOS/GG, GT and TTgenotypes were 0.907, 0.093 and 0.000, respectively. The G and T allele frequencies were 0.954 and 0.046.3. We assumed the effects of the T allele to be dominant (GT and TT combined vs GG), the eNOS genotypes distribution in CHD patients, as well as MI subgroup were significantly different from the normal subjects (P<0.05). The frequencies of T allele in CHD ,MI and AP subgroups were significantly higher than that in the normal subjects (PO.05), respectively. The eNOS genotypes distribution or the frequencies of T allele had no difference between MI and AP subgroups.4. We found no difference between the eNOS variant (include the distribution of genotypes and the frequencies of T allele ) and the number of diseased vessels in the CHD group (P>0.05).5. There were no differences among genotypes in terms of years, sex, body mass index, systolic blood pressure, diastolic blood pressure, TC, TG. The prevalences of hyperlipidemia, hypertension (HT), cigrette smoking, diabetes mellitus and obesity, were similar in the eNOS genotypes (P>0.05).6. The eNOS genotype distribution has no difference between CHD patients with and without family history of cardiovascular disease (P>0.05).7. We found significant differences... |
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