| BackgroundCoronary heart disease (CHD) is the leading cause of mortality and morbidity in many countries, a number of genetic risk factors for the development of coronary heart disease has been identified in the past. Some of these represent polymorphisms in the genes of proteins which are associated with the process of blood lipid metabolism .. thrombosis and vascular contraction or dilation.High plasma concentration of cholesterol, in particular low-density lipoprotein cholesterol (LDL-C), is one of the principal risk factors in the development of atherosclerosis. In humans, cholesterol exists as free cholesterol and cholesterol esters. Under normal condition, 75% of plasma cholesterol is in the form of cholesterol esters. Esterification of cholesterol in plasma is catalyzed by lecithinxholesterol acyltransferase (LCAT). While esterification of cholesterol in cells is catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT).Two ACAT isoforms (ACAT-1 and ACAT-2) have been reported in humans, their distributions and functional roles are different. ACAT-1 is expressed in almost all cells, and functions in intracellular cholesterol homeostasis and in the foam-cell formation in the development of early atherosclerotic lesions. Unlike ACAT-1, ACAT-2 is mainly found in liver and intestine, plays an important role in intestinal cholesterol absorption and the production and release of ApoB-containing lipoproteins.The ACAT-2 gene maps to chromosome 12 and consists of 21000 base pairs, including 15 exons. Katsuren and He et al identified a total of 11 polymorphisms in the ACAT-2 gene. Study in the Japanese population showed that 734C/T heterozygotes had significantly higher plasma apoC-Ⅲ levels compared to 734CC homozygotes. While study in the Singapore Chinese coronary heart disease population found that the 734T allele frequency was significantly lower in CHD(+) than CHD(-), which suggested T allele may be protective against CHD.Since ACAT2-734C/T polymorphism may play a role in the process of coronary heart disease by affecting the process of blood lipid metabolism. The research on the relationship between this polymorphism and coronary heart disease may be worthy.In this study, we investigated the ACAT-2 -734C/T polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Our objective is to study the distribution of -734C/T gene polymorphism in Chinese people and its relationship with coronary heart disease (CHD).Subject and Methods 1. SubjectsCHD(-) group: 123 CHD(-) subjects(69 men and 54 women, mean aged 59.4±10.4 years) served as a control group. All subjects were consecutively collected form the hospitalized in the department of cardiology, the second affiliated hospital of medical college, Zhejiang University. All patients were confirmed have no coronary heart disease by coronary angiography.CHD(+) group: 151 coronary heart disease patients(118 men and 33 women, mean aged 60.3±10.5 years) were consecutively collected from the hospitalized in the department of cardiology, the second affiliated hospital of medical college, Zhejiang University from March, 2005 to December, 2005. All patients were confirmed by coronary angiography.2. Documentation of CHD severityThe severity of coronary stenosis was determined by the number of significantly stenosed coronary arteries as follows. Angiograms were assessed by two cardiologists who were unaware that the patients were to be included in the study. Each angiogram was classified as revealing either coronary lesion with more than 50% luminal stenosis or one, two, or three major epicardial coronary arteries with more than 50% luminal obstructions.3. Risk for coronary heart diseaseAll patients completed a questionnaire that included demographic data: age, sex, hyperlipidemia. hypertension, diabetes mellitus and smoking, et al.1) Hyperlipidemia was defined as serum total cholesterol (TC) 220mg/dl or more;low-density lipoprotein cholesterol (LDL-C) 140mg/dl or more;triglyceride (TG) 150mg/dl or more;high-density lipoprotein cholesterol (HDL-C) 35mg/dl or less.2) Hypertension was defined as 140/90mmHg or more. Both previous medicalrecords of raised blood pressure and present values were collected.3) Diabetes mellitus (DM) was recorded in accordance with 1997 criteria by the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus(ADA).4) Smokes consists of current smokes and patients who had ceased smoking. Evaluation of the number of cigarettes consumed per day was thought to be unreliable.4. Laboratory methods for ACAT-2 -734C/T gene polymorphism1) DNA was extracted from the peripheral blood leukocytes by standard phenol and chloroform method.2) ACAT-2 -734C/T genotype was determined by PCR-RFLP. The forward primer: S'-CAGATCTTACACTCTGCCTGCCTCT-3';the reverse primer: 5'-TGCACCTGCTGGCTTCATTCAGTCA-3'. PCR was carried out using a 25ul reaction mix containing 10 × Reaction buffer(without MgCl2) 1.5ul, 25 mM MgCl2 1.5ul. 10 mM dNTP 0.75ul, genomic DNA 100ng, Taq DNA polymerase 1.25U and 7.5pmol each primer. The PCR cycles were modified as follows: initial denaturation at 94℃ for 4 minutes followed by 33 cycles at 94℃ for 30 second, at 58 ℃ for 30 second and at 72 ℃ for 60 second, at last 72 ℃ for 4 minutes. PCR products were then digested with BarriH I at 37℃ for 16h and digestion product were separated on 2% agarose gels and visualized with ethidium bromide.5. Statistical analysisStatistic analyses were performed with the SPSS for windows 11.5 statistic program package. All continuous variables were expressed as mean±standard deviation. The gene counting method was used to estimate the allele and genotype frequencies in patients group and control group. The Hardy-Weinberg equilibrium for the frequencies of genotypes was tested by x2 analysis. Clinical characteristicsbetween different groups were tested with one-way ANOVA or nonparametric test. Comparison between groups was performed by t test or one-way ANOVA. Values were considered statistically significant at P<0.05.Results1. The genotype distribution of the control group and CHD group was in the Hardy-Weinberg equilibrium (p>0.05).2. The polymorphism of ACAT-2 -734C/T was screened in the control group and CHD group. TT, CT and CC genotypes and T, C allele were found.3. The distribution of genotype in the control group was 59.35% for CC, 38.21% for CT and 2.44% for TT genotype, and CHD group was 49.67%, 42.38% and 7.95% respectively. There was no significant difference between two groups. No significant difference was found among these three groups between different sexual, either.4. The frequency of the T allele in the control group and CHD group was 21.54% and 29.14%, respectively. There was significant difference between two groups (x2=4.092,p=0.043).5. The genotype distribution and allele frequency of ACAT-2 -734C/T was no difference between single, double and triple vessel disease. No difference was found between myocardial infarction and non-myocardial infarction, either.6. The genotype distribution and allele frequency of ACAT-2 -734C/T was no difference between hypertension, hyperlipidemia, DM, smoking and no hypertension, no hyperlipidemia, no DM, no smoking.7. Fasting serum lipid was no difference between the three genotype of ACAT-2 -734C/T.Conclusions1. There is a polymorphism of ACAT-2 -734C/T in Zhejiang Han population.2. T allele may be a susceptibility gene of coronary heart disease.3. ACAT-2 -734C/T polymorphism is not associated with the degree and type of the CHD.4. ACAT-2 -734C/T polymorphism is not associated with hypertension, hyperlipidemia, DM and smoking.5. ACAT-2 -734C/T polymorphism does not affect the level of fasting serum lipid. |
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