The Relationship Of The CAG Repeat Polymorphism Of MEF2A Gene And Coronary Artery Disease In Chinese Population

BACKGROUND AND METHODSCardiovascular disease is the most important cause of morbidity and mortality in developed and developing countries. The major cardiovascular diseases, including coronary heart disease (CHD), myocardial infarction (MI), congestive heart failure and common congenital heart disease, are caused by multiple genetic and environmental factors, as well as the interactions between them. The underlying molecular pathogenic mechanisms for these disorders are still largely unknown, but genetic-epidemiological and twin studies have proved that gene expression may play a central role in the development and progression of cardiovascular disease. The highest morbidity and mortality of CHD is in the northeast area of China according to the epidemiological survey from Monica. So it is of great importance to explore the prevention and cure strategy in Northeast HAN population.Great focuses had ever been put on the association between coronary artery disease and high risk factors, such as lipid metabolism, blood coagulation factor,renin-angiotensin system and so on. With the achievement of Human Genome Project and the application of the third generation genetic markers, single nucleotide polymorphism (SNP), some breakthrough have been made on the chromosomal localization and detect of the predisposing locus at present. Linkage disequilibrium SNP locus mainly concentrated on the chromosome segment of No. 1, 3, 6, 9 and 14. But no disease-causing predisposing genes have ever been determined so far.In 2003, a genetic linkage of CHD/MI family with a DNA polymorphism in chromosome 15q26 region (D15S120) was demonstrated with the genome-wide screen research on a large CHD /MI family with autosomal dominant inheritance. The previous found seven-amino acid deletion of human myocyte enhancer factor-2A (MEF2A) gene was reported to be a functional mutation that disrupted nuclear localization of MEF2A, reduced MEF2A-mediated transcription activation, and abolished synergistic activation by a dominant-negative mechanism. In addition, three missense mutations screed from 207 sporadic CHD patients were clustered within the major transcriptional activation domain of MEF2A and significantly reduced the transcriptional activation of MEF2A through a loss-of-function mechanism. These data strongly support that MEF2A is an autosomal dominant form of CHD /MI (adCHD1). MEF2 was originally identified as a muscle-specific factor that binds an A/T-rich consensus sequence associated with a large number of genes expressed in skeletal and cardiac muscle. MEF2 contains four family member: MEF2A, MEF2B, MEF2C, and MEF2D which share homology within a DNA binding and dimerization motif. MEF2A gene is located in 15q26, which consists of 499aa,with a MADS structural domain at N terminal and a phosphorylation site at C terminal. It lies in the downstream of the signaling pathway of many muscle-specific and cardiac-specific genes and can activate the synthesis of multiple structure proteins. Most MEF2A-deficient mice show dilation of the right ventricle, mitochondrial disorganization and experience sudden death in the first week of life. In addition, the normal heart and vascular system fail to form in MEF2C-null mice. In conclusion, MEF2 is playing the key role in modulating the vascular smooth muscle cell proliferation, endothelial cell development and keeping the integrity of vessel wall.While conflicting hypothesis were also put forward in other researches. To clarify this issue, we performed a screening of exons 7 and 11 of MEF2A gene to assess the significance of MEF2A polymorphism/mutations in Northern Chinese CHD population in the case-control study. PATIENTS AND METHODS1. Patients726 individuals who underwent standardized coronary angiography in Department of Cardiology of Northern Hospital were enrolled in the study from November 2003 to May 2006(male 492, female 234, mean age 57.2±10.5 years old). The study was entitled by the Hospital Ethics Committee and informed written consent was obtained from all participants.2. PCR amplificationGenomic DNA was extracted using phenol/chloroform and PCR amplification was performed as usual. Primers were designed using Primer 5.0 software. The PCR products must overlap the special regions where the conflicting results were raised among different researches.3. PCR-SSCP for detection of mutation in exon 7Preliminary experiments were necessary to determine the specificity of PCR products and sensitivity of the denatured gel electrophoresis. PCR product was denatured at 99 oC for 30 minutes, chilled in ice for 5 minutes and then loaded for electrophoresis at 4 oC, 250 V for 16 hours. Next, the gel would be silver stained for coloration. Samples with skeptical mutations would repeat twice by different technicians. And then the PCR product would be purified and prepared for sequencing to determine whether mutations exist indeed.4. Genotyping for exon 11According to the preliminary experiments and sequencing results, PCR product with (CAG)n=11 would be kept as internal standard and accompany with other PCR product for electrophoresis at room temperature, 300 v for 5 hours. And then the gel would be silver stained for coloration. Distinguished patterns would repeat twice by two technicians. Skeptical PCR products would be purified for sequencing.5. STATISTICAL ANALYSISAllele frequencies in patients and controls were compared through a chi-square test. The x 2 test was also used to determine if the observed genotype frequencies in patients and controls differed from those expected under the Hardy-Weinberg equilibrium. Logistic regression analysis was performed for the determination of the independent predictors for CHD. The odds ratios as estimators of relative risk together with their 95 % approximate confidence intervals were calculated to assess the association with and without CHD. Jonckheere-Terpstra trend test was for the analysis of the correlation between allelic polymorphism and the extent of CHD. All statistical analyses were performed with the SPSS 13.0 statistical software package.RESULTS1. Of the 726 individuals, 378 had CHD. The CHD patients had a higher prevalence of traditional atherosclerotic risk factors at baseline than did the healthy control, such as smoking, hypertension, diabetes mellitus and so on.2. Mutation screening in MEF2A gene exon 7 No mutations were detected in exon 7 of MEF2A gene in patients and controls by PCR-SSCP analysis.3. Genotyping for (CAG)n allele in MEF2A gene exon 11The (CAG)n genotype frequencies [(CAG)n=12 (0.3 %), (CAG)_n=11 (46.7 %), (CAG)_n=10 (20.9 %), (CAG)_n=9 (29.9 %), (CAG)_n=8 (1.0 %), (CAG)_n=7 (0.8 %), (CAG)_n=5 (0.4 %)] were in Hardy-Weinberg equilibrium. The distribution of the CAG repeat polymorphism of MEF2A was different between two groups (p<0.001) as a whole. Compared to the healthy control, the frequency of (CAG)n=11 allele was significantly lower (p=0.001) whereas (CAG)_n=9 was higher in patients group (p<0.001, OR=1.958, 95 %CI=1.412-2.714), which implied that (CAG)_n=9 might be associated with CHD. The Logistic regression was applied to determine the predictive value of (CAG)_n=9 for CHD. After the adjustment of baseline characteristics, we concluded that (CAG)_n=9 could be an independent risk factor for CHD.Among the individuals, the proportion of patients carrying (CAG)_n=9 allele was highest in those with triple vessel disease (39.7 %) while lowest among those without CHD (23.7 %). A highly significant association toward a harmful effect of the polymorphism was demonstrated (p trend=0.000). CONCLUSIONSIn summary, the CAG repeat polymorphism of MEF2A gene is associated with coronary heart disease in studied Northern Chinese population. And the (CAG)_n=9 allele may be an independent predictive factor for CHD.