Associations Of ApoA5 Polymorphisms And Coronary Heart Disease In The Chinese

BACKGROUNDCoronary heart disease(CHD),pathologically featured by anthrosclerosis of coronary arteries ,is caused by the combined effect of various genetic and environmental factors .Genetic factors play a key role in its development .The associations between coronary heart disease and single nucleotide polymorphisms(SNPs) of ApoA5 gene have been attention-getting since it was discovered in 2001.Apolipoprotein is on the surface of lipoprotein. It helps to transport the lipoprotein all over the body. About 20 kinds of apolipoprotein have been found. Apolipoprotein A5(ApoA5),which was found by Pennacchio in 2001,is a new member of the apolipoprotein family. They found the gene ApoA5 by comparing the ApoA1 / C3 / A4 gene clusters of human and mice. It is located on the 11st chromosome,30000 basepairs downstream to ApoA1 / C3 / A4 gene cluster. It has 1889 base pairs, consisting of 4 exons and 3 introns. Its protein product contains 366 amino acids, similar to apoa4.So it was named ApoA5.According to the researches on animal and human genome, it was found that the polymorphisms of ApoA5 gene have great influence on the serum triglyceride(TG) level, and ApoA5 is the first apolipoprotein which leads to decreased serum TG level by over-expression. Accordingly, research on the associations among ApoA5 gene polymorphisms, TG and CHD has become a hotspot.Pennacchio found 4 common SNPs, 1259T>C,476G>A,-1131T>C and -12238T>C, named SNP1,SNP2, SNP3, SNP4 respectively. After that, they found SNPs, such as -3A>G and 56C＞G, which are in linkage disequilibrium with the previous four SNPs. There are 3 common haplotypes: ApoA5*1 {-1131T. -3A. 56C. 476G. 1259T}, ApoA5*2{featured with -1131C}, ApoA5*3{featured with 56G}.Thelatter two haplotypes are in relationship with hypertriglyceridemia. And there was report of 553G>T polymorphism. Till now, more than 20 SNPs have been embodied by NCBI. -1131T>C,56C>G and 553G>T are studied the most among them.In this study, we investigated the distribution of -1131 T>C and c.553G>T polymorphisms in the Chinese. Meanwhile, we studied the relationships among the polymorphisms, serum triglyceride and coronary heart disease.SUBJECT AND METHODS1. SubjectsControl group: All subjects, expelled from coronary heart disease by coronary angiography , were collected from the hospitalized patients in the department of cardiology, the second affiliated hospital, college of medicine, Zhejiang University in 2005.CHD group: All subjects were continuously collected from the patients in the department of cardiology, the second affiliated hospital, college of medicine, Zhejiang University in 2005. All patients were confirmed by coronary angiography.2. Documentation of CHD severityThe severity of coronary heart disease is determined by the number of significantly stenosed coronary arteries as follows. Angiograms were assessed by two experienced intervention cardiologists. 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. The lesions within the left trunk is defined as two branch lesions.3. Risks for coronary heart diseaseAll patients completed a questionnaire that included demographic data: age, sex, hyperlipidemia, hypertension, diabetes mellitus and smoking, et al.Hypertension was defined as 140/90mmHg or more. Both previous medical records of raised blood pressure and present values were collected.Diabetes mellitus (DM) was recorded in accordance with 1997 criteria by theExpert Committee on the Diagnosis and Classification of Diabetes Mellitus(ADA).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.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 methods4.1 DNA was extracted from the peripheral blood leukocytes by standard phenol and chloroform method.4.2 ApoA5-1131T>C genotypes was determined by polymerase chain reaction-restrictive fragment length polymorphism(PCR-RFLP).The forward primer: 5'-GAT TGA TTC AAG ATG CAT TTA GGA C -3', the reverse primer:5'- CCC CAG GAA CTG GAG CGA AAT T -3'[1];PCR was carried out using a 25ul reaction mix containing 10×Reaction buffer(without MgCl₂) 2.5ul, 25 mM MgCl₂ 1.5ul, 10 mM dNTP 0.5ul, genomic DNA 100ng, Taq DNA polymerase0.25U and 5pmol of each primer.The PCR cycles were set as follows: initial denaturation at 94℃ for 5 minutes followed by 35 cycles at 94℃ for 50 seconds, at 58℃ for 50 seconds and at 72℃ for 60 seconds, at last another 72℃ for 10 minutes. PCR products were then digested with TrulI(MBI Fermentas) at 65℃ for 6h and 2.5% agarose gel electrophoresis, which was mixed with ethidium bromide(EB) was used to separate the products. The gel was visualized with the ultraviolet radiation.4.3 ApoA5 c.553G>T genotypes was determined by polymerase chain reaction-restrictive fragment length polymorphism(PCR-RFLP).The forward primer:5'- AGA CAC CAA GGC CCA GTT GCT GGG -3',the reverse primer:5'- ATG CCG CTC ACC AGG CTC TCG GCG -3'[3]; PCR reaction system is the same to the previous one. The PCR cycles were set as follows: initial denaturation at 95℃ for 5 minutes followed by 35 cycles at 95℃ for 30 seconds, at58℃ for 30 seconds and at 72℃ for 60 seconds, at last another 72℃ for 7 minutes. PCR products were then digested with HaeIII(MBI Fermentas) at 37℃ overnight and 3% agarose gel electrophoresis, which was mixed with ethidium bromide(EB),was used to separate the products. The gel was visualized with the ultraviolet radiation. 5. Statistical analysisContinuous variables were expressed as mean ± standard deviation. The counting method was used to estimate the allele and genotype frequencies in CHD group and control group. The Hardy-Weinberg equilibrium for the frequencies of genotypes was tested by X² analysis. Clinical characteristics between 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 when P＜0.05. Statistic analyses were performed with the SPSS 11.5.Results1. ApoA5-1131T>C1.1 We studied 270 cases to analysis the polymorphisms of ApoA5 -1131T>C. Between CHD and control groups, there is difference in smoking rate, but not in sex, age, hypertension history, diabetes history and fasting serum lipid.1.2 The genotype distribution of the control group and CHD group is in Hardy-Weinberg equilibrium. No difference was found in the genotype distribution between the control group and CHD group. ApoA5 -1131 C allele in CHD group was found to be more frequent than that of control group,and there is a statistical difference. (p<0.05).1.3 ApoA5 -1131 C allele was found to be correlated with a higher level of fasting serum triglyceride.2. ApoA5 c.553G>T2.1 We studied 269 cases to analysis the polymorphisms of ApoA5 c.553G>T.Between CHD and control groups, there is on difference in sex. age. smokingrate, hypertension history, diabetes history and fasting serum lipid.2.2 The genotype distribution of the control group and CHD group is in Hardy-Weinberg equilibrium. There was difference in the genotype distribution between the control group and CHD group. ApoA5 c.553 T allele was found to be correlated with the susceptibility to coronary heart disease. (p<0.05).2.3 ApoA5 c.553 T genotype was found to be correlated with a higher level of fasting serum triglyceride. No other differences were found between genotypes.2.4 ApoA5 c.553 T genotype was not found to be correlated with the severity of coronary heart disease.Conclusions1. ApoA5 -1131 C allele is correlated with the susceptibility to coronary artery disease.2. ApoA5 -1131 C allele is correlated with elevated serum fasting triglyceride level.3. ApoA5 c.553 T allele is correlated with the susceptibility to coronary artery disease.4. ApoA5 c.553 T allele is correlated with elevated serum fasting triglyceride level.5. No relationship is found between ApoA5 c.553 T allele and the severity of coronary heart disease.