Association Between The Single Nucleotide Polymorphism Of LIPG And Coronary Atherosclerosis Disease

BackgroundCAD has complex genetic traits, with multiple genetic and environmental components contributing to susceptibility. Growing evidence indicates that low plasma high density lipoprotein cholesterol (HDL-C) levels are closely associated with the risk of coronary atherosclerosis disease (CAD) and that high HDL-C levels constitute a major risk-reducing factor for CAD. Besides modified by environmental factors, plasma high density lipoprotein (HDL) cholesterol (HDL-C) concentration is highly heritable. And the heritability may be associated with genetic polymorphisms in the key regulators of HDL metabolism including endothelial lipase (LIPG). Therefore, it will be of great significance to investigate into the susceptive gene of CAD in order to prevent, postpone or treat the CAD.Endothelial lipase (EL) is a newly discovered member of the triglyceride (TG) lipase gene family, which is highly homologous to LPL and HL. Studies in mouse have also shown that, like LPL and HL, EL may play an important regulatory role in lipid metabolism, in particular, HDL-C metabolism. Badellino et al. used a newly developed sandwich enzyme-linked immunosorbent assay to measure the plasma EL concentration and confirmed that there was a highly statistically significant negative association between EL levels and HDL-C levels. Furthermore, small studies in selected populations have reported higher HDL-C levels among carriers of the common C584T/ex3and T42C/in5variant in LIPG, but whether these variants are associated with plasma lipids and risk of coronary heart disease (CHD) in the general population is unclear. In this dissertation, a case-control study has been carried out to assess the associations of the C584T/ex3and T42C/in5variant with plasma lipids and risk of CHD in a Chinese population.MethodsⅠ. Study populationThe study population was composed of941CAD patients and794control subjects. All enrolled subjects were unrelated ethnic Han Chinese residing in or near the city of Beijing. The CAD patients were consecutively recruited from the inpatients who were admitted to Peking Union Medicine College Hospital (PUMCH) due to angina pectoris, acute myocardial infarction, or other symptoms and signs of cardiovascular disease from January2007to April2009. CAD was defined as angiographic evidence of at least one segment of a major coronary artery, including the left anterior descending, left circumflex, and right coronary artery, with>50%organic stenosis. The coronary angiograms were judged by experienced cardiologists who were unaware that the patients were to be included in this study. Among the941CAD patients,392had AMI. The non-CAD control group consisted of794subjects matched by age who were selected during the same period in Physical examination center of PUMCH. Considering that it was unethical to perform coronary angiography to rule out the presence of asymptomatic CAD, the following inclusion criteria were used:no history of atherosclerotic vascular diseases and no symptoms of angina pectoris and with normal ECG. All subjects (CAD patients and control subjects) included in this study had no history of significant concomitant diseases, including cardiomyopathy, systemic autoimmune disease, renal failure, dyshepatia, thyroid disease and malignant diseases. Informed consent was obtained from each subject. This study was approved by the PUMCH Ethics Committee. Hypertension, diabetes mellitus, and hypercholesterolemia are defined as follows. Subjects were considered to be hypertensive if their systolic blood pressure≥140mm Hg and/or their diastolic pressure≥90mm Hg or if they were already being treated with antihypertensive drugs. Diabetes was defined as fasting blood glucose≥7.0mmol/1or a diagnosis of diabetes needing diet therapy or already on antidiabetic drug therapy. Hypercholesterolemia was defined as total cholesterol (TC) level≥5.69mmol/1or already on drugs. One hundred twenty one (12.9%) CAD patients were on lipid-lowering medications. Subjects were defined as smokers if they smoked≥10years and≥=1cigarettes per day.Ⅱ. Laboratory measurementsApproximately2ml fasting blood samples were obtained by venipuncture in the early morning of the day after admission. DNA was extracted by DNA extraction kit from EDTA anticoagulated blood and stored at-20℃. The levels of plasma HDL-C, TC, TG, and low density lipoprotein cholesterol (LDL-C) were accessed using standard methods by chemistry analyzer. Glucose levels were measured by a glucose oxidase method. Genotypes were determined by Taqman allelic discrimination (ABI7500HT, Applied Biosystems) using rs2000813and rs2276269. The case status of the samples was blinded when analysing the DNA in the laboratory. Controls were included in each run and repeated genotyping of a random5%subset by derect sequencing yielded100%identical genotypes. Plasma EL concentration was measured by Sandwich Enzyme-Linked Immunosorbent Assay. Ⅲ. Statistical analysesStatistical analyses were performed with SPSS version13.0. The Kolmogorov-Smirnov test was used to analyze the normality of the distribution of Continuous variables. Differences of continuous variables without skewness between cases and controls were presented as means±standard diviation and calculated by the Student’s t-test or One way ANOVA. The association of EL concentration with continuous variables was examined using Pearson Correlation. Differences of continuous variables departing from the normal distribution between the two groups were presented as medians (25th-75th percentiles) and analyzed by Kruskal-Wallis-H test. The association of EL concentration with continuous variables was examined using Spearman Correlation. Categorical variables were presented using frequency counts and compared by Chi-square test. The Hardy-Weinberg equilibrium was evaluated by Chi-square goodness-of-fit test. Univariate analysis and multiple logistic regression analysis were performed to determine the variables that independently contributed to the presence of CAD. Odds ratio (OR) and95%confidence interval (CI) were calculated. Values of the disequilibrium coefficient D’were computed to assess the extent of disequilibrium across the LIPG gene, and an Expectation-Maximization algorithm was used to estimate haplotype frequencies. The haplotype analysis was calculated by SHESIS on-line software. A value of P<0.05(two-tailed test) was considered statistically significant.ResultsⅠ. Baseline characteristicsThere was no significant difference between the cases and controls in age. However, compared with the control subjects, patients with CAD had higher sex ratio (male/female), body mass index (BMI), prevalence of hypertension and hypercholesterolemia, rate of smoking, and higher levels of TG, but lower TC, LDL-C and HDL-C.Ⅱ. Genotypes and allele frequenciesAs for LIPG-C584T/ex3, CC, CT, TT genotypes can be found in CAD cases and control subjects. The frequencies of C and T allele in control group were72%and28%, respectively, while in CAD group72.5%and27.5%, respectively. The LIPG-C584T/ex3genotype followed Hardy-Weinberg equilibrium both in patient and control groups. There are no association between LIPG-C584T/ex3and CAD (x2=0.131, p=0.717), meanwhile logistic regression analysis also revealed that there was no significant correlation between CAD and LIPG-C584T/ex3(OR=0.877, p=0.755). There was no significant difference between LIPG-C584T/ex3and the level of HDL-C or other lipid.As for LIPG-T42C/in5, TT, TC, CC genotypes can be found in CAD cases and control subjects. The frequencies of T and C allele in control group were70.8%and29.2%respectively, while in CAD group71.4%and28.6%, respectively. The genotype distribution was in accordance with Hardy-Weinberg equilibrium. There are no association between LIPG-T42C/in5and CAD (x2=0.112, p=0.738), meanwhile logistic regression analysis also revealed that there was no significant correlation between CAD and LIPG-T42C/in5(OR=0.742, p=0.742). The T allele carriers (TT, TC) had significantly lower ApoA-I levels compared with CC genotype carriers in women control group. However, there was no significant difference between LIPG-T42C/in5and other lipid levels.The two SNPs were in linkage disequilibrium (r2=0.803). The three haplotypes (CC, CT, TC) were analyzed. And the result showed that there are no significant association between any haplotype and CADⅢ. Association of EL concentrations with plasma lipid and lipoprotein concentrationsData analysis indicated that plasma EL concentration was significantly correlated with HDL-C (r=-0.159, p<0.05), TG (r=0.238, p<0.01), BMI (r=0.149, p<0.01).ConclusionOur analysis among CAD patients and controls from Han ethnic Chinese people dose not support an association between either LIPG-C584T/ex3or T42C/in5and the risk of CAD, HDL-C level, or other plasma lipids. Human EL concentrations are singificaltly associated with HDL-C negatively, and TG、BMI positively.