Abstract
Background: In this study we aimed to determine the possible risks for the development of coronary artery disease (CAD) in diabetic (DM+) and non-diabetic (DM−) patients according to the −374T/A polymorphism of the receptor for advanced glycation end products (RAGE) gene which affects the function of RAGE itself. Materials and Methods: This study was carried out in 52 non-diabetic and 62 diabetic patients with CAD, and 55 CAD-free, healthy volunteers as controls. The A-T transversion polymorphism at position −374 in the promotor region of the RAGE gene was analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) techniques. Results: The −374T/A AA genotype frequency was statistically higher in the whole patient group when compared with the control group (p=0.034), and statistically higher in the DM+ group when compared with the control group (p=0.003). Homozygosity for the −374A allele was found to be higher, but not statistically meaningful, in DM− patients (17.3%) when compared with the control group (13.2%). In this study, in contrast with other studies, we found possesion of the A allele to be an independent risk factor in CAD in patients with diabetes mellitus. Conclusion: Possesion of the −374A allele may contribute to the CAD in diabetic patients with triggering macrophages by increased levels of AGEs.
Atherosclerotic coronary artery disease, by the interaction of multiple genetic and environmental risk factors, is a complex disease and a major cause of morbidity and mortality in the Western population (1-4). Diabetes mellitus is the most prevalent metabolic syndrome which is characterized by hyperglycaemia, resulting in various short-term metabolic changes in lipid and protein metabolism and long-term irreversible vascular changes (5). Such chronic hyperglycemia results in the formation and accumulation of irreversible advanced glycation end products (AGEs) that have a plethora of adverse effects, resulting in dysfunction of the vasculature and effects shown to be mediated by specific AGE binding and degrading receptors (receptor for AGEs, RAGE) which are present in the non-diabetic population, but are two- to five-fold higher in diabetics (5-12).
Various lines of evidence suggest that genetic factors contribute significantly to the risk of CAD (1, 2, 13). The RAGE gene has been proposed as having a role in diabetic atherosclerosis (14). Its gene product, 35 kDa polypeptide RAGE, is a multiligand receptor of the immunoglobulin superfamily expressed at low levels in adult tissues in homeostasis, but highly overexpressed at sites of vascular pathology (12, 15-17). The RAGE gene is located on chromosome 6p21.3 in the major histocompatibility complex and has 11 exons (15, 16).
The hyperglycaemic state seen in diabetes mellitus is associated with the development of diabetes-specific microvascular complications (retinopathy, nephropathy and neuropathy) and accelerated macrovascular disease (atherosclerosis leading to heart disease, stroke and peripheral vascular disease). Evidence implicates the formation and subsequent effects of AGEs as a contributing cause. AGEs have a number of deleterious effects, including cross-linking of proteins, modification of matrix components, platelet aggregation, defective vascular relaxation, abnormal lipoprotein metabolism, and induction of cytotoxic pathways that may affect the pathophysiology of the vascular wall. AGE-specific cellular receptor (RAGE) complexes identified on different cell types, including endothelial cells, smooth muscle cells, lymphocytes, and monocytes, where they mediate AGE removal as well as multiple biological responses, including the triggering of proinflammatory and procoagulatory genes and cellular migration and proliferation. Accumulation of AGE/RAGE can be seen at sites of vascular disease in both animal models of diabetes and human diabetics (5-11, 18, 19).
The concept RAGE contributes to the development of coronary atherosclerosis is gaining favour. Several polymorphic sites have so far been found within the RAGE gene (13, 15). A functional −374T/A polymorphism in the promoter of the RAGE gene which has been associated with vascular complications has been shown to exert significant effects on transcriptional activity. Investigation of this common variant revealed that the introduction of the −374A allele abolished a nuclear protein binding site, supporting the role of this polymorphism in affecting RAGE transcriptional repression. The −374T/A variant has thus become a topic of great interest (13, 14, 20).
Accordingly, this study was to determine the role of this polymorphism in CAD in diabetic and non-diabetic Turkish patients.
Materials and Methods
Study participants and clinical investigation. The RAGE −374T/A gene polymorphism was studied in 52 non-diabetic (DM−) [11 (21.2%) women, 41 (78.8%) men] and 62 diabetic (DM+) [26 (41.9%) women, 36 (58.1%) men] cases with CAD.
Patients with severe coronary vascular disease were documented by angiography. Angiographic inclusion criteria were: ≥50% stenosis of at least one major coronary vessel because of atherosclerosis, and a vascular event, defined as myocardial infarction, percutaneous transluminal coronary angioplasty, or coronary artery bypass grafting. Patients were included irrespective of concomitant risk factors for atherosclerosis such as smoking, arterial hypertension and diabetes mellitus. Participants were of both sexes and the height and weight of each were also recorded, thus permitting calculation of the body mass index (BMI): [weight (kg)/height2 (m)]×100.
Healthy persons [27 (49.1%) women, 28 (50.9%) men] without any symptoms of diabetes mellitus and CAD were selected for the control group. Coronary angiography was not performed on these individuals, and therefore the presence of atherosclerotic coronary arteries could not be excluded. However, none of these individuals had any history of any vascular event.
Polymerase chain reaction (PCR)-based detection of RAGE mutation. Blood specimens were collected in tubes containing EDTA, and DNA samples were extracted from whole blood with a salting-out procedure (21).
RAGE −374T/A genotype was analysed by PCR-restriction fragment length polymorphism. The −374T/A mutation detected by cutting the PCR product with the restriction endonuclease Tsp509 I (New England Biolabs, Beverly, MA, USA). The digested DNAs were separated on 3% agarose gel in 1XTris borate EDTA buffer followed by staining with ethidium bromide solution (13, 20, 22-24). The RAGE genotypes were typed by visualization under ultraviolet light photographed using a Polaroid camera.
Statistical methods. Statatistical analysis was performed by using SPSS software package (revision 11.5 SPSS Inc., Chicago, IL, USA). Clinical laboratory data are expressed as mean±SD. Mean values were compared between patients and controls by unpaired Student's t-test. Differences in the distribution of RAGE −374 T/A genotypes and alleles between cases and controls were tested using the Chi-square statistic. Allele frequencies were estimated by gene counting methods. Values of p<0.05 were considered statistically significant.
Results
Demographic characteristics are summarised in Table I. DM− CAD, DM+ CAD patients and the control group were compared with advanced chi-square analysis. In DM-patients, there was a higher ratio of male gender (p=0.008), higher levels of total cholesterol (p=0.053), smoking (p=0.001) and less frequent family history of CAD (p=0.024). In DM+ patients, higher systolic (p=0.064) and diastolic pressures (p=0.019), and a higher prevalance of hypertriglyceridemia (p=0.014) were found. When the disease-specific complications, such as left ventricular hypertrophy (LVH), left atrium dilatation (LAD) and hypertension, were compared, there was no significance for LVH and LAD but hypertension was significantly more frequent in the DM+ group (p=0.001).
We found a higher ratio of male gender (p=0.037), higher systolic (p=0.058) and diastolic pressures (p=0.013), and higher prevalance of hypertriglyceridemia (p=0.044) in the patient group (DM+ and DM− CAD) when compared with the control group (Table I).
The frequency of the wild-type T allele in the control group was statistically higher (p=0.034) when compared with the whole patient group, while the AA genotype frequency was statistically higher in the whole patient group versus controls (28.3% vs. 13.2%). Moreover, when compared with DM+ CAD patients, the T allele frequency was statistically higher (p=0.003) in the control group, and the AA genotype frequency was significantly lower (13.2% vs. 38.9%) (Table II).
There was no statistically assosiation between RAGE −374T/A polymorphism and cardiovascular risk factors in the whole patient group (p>0.05).
In DM+ CAD patients, serum triglyceride (p=0.007) and VLDL-cholesterol (p=0.01) levels were statistically higher in individuals with the TT genotype than in those with the AA or AT genotype.
DM− patients with AA or AT genotype had a significantly higher ratio of male gender (p=0.028) (Table III) and a trend for a smoking habit (p=0.054) (Table IV) when compared with those with the TT genotype. When the individual and common effects of environmental factors and smoking habit in the development of CAD as a genetic factor was analyzed, in DM− CAD patients having either A or T allele and smoking habit, the risk was lower. Therefore, our findings shows no relationship between smoking habit and −374T/A polymorphism in the development of CAD (Table IV). Like smoking habit, the possible risks in the development of CAD, the individual and common effects of being male in gender and having either A or T allele is shown in Table III. In DM+ patients, RAGE −374T/A polymorphism and both being male in gender and having a smoking habit had no effect on the odds ratio for the development of CAD.
Demographic and biochemical characteristics of the study population.
In the controls together with AA and AT genotype, the systolic (130.46±29.79 vs. 121.66±20.07) and diastolic (81.40±15.30 vs. 76.94±11.89) blood pressures were higher in those with the TT genotype but the difference was not statistically meaningful.
Discussion
Coronary artery disease is a multifactorial disease in which genetic and environmental factors play an important role. These factors may be different in each race and ethnic group (3, 4, 25, 26).
Protein glycation is the most important process after synthesis (27) and the biological activity of sugar, such as differentiation, development and growth, covers a wide range (28-32). Non-enzymatic glycolysation, which leads to the formation of AGEs has many adverse effects and depends on the concentration of glucose; therefore, in diabetes mellitus increased glycolysation levels are detected which are responsible for the chronic complications of diabetes (6, 19). AGEs show their effects in extracellular matrix, in vasculature and by binding to their receptors (RAGE). Because of their prothrombotic and proinflammatory effects, the single nucleotide polymorphisms of the RAGE gene are gaining importance as being a candidate gene for atherosclerosis and other associated diseases.
Genotype and allele frequencies of RAGE −374T/A gene in study groups.
The individual and common effects of male gender and −374 T or −374 A allele on the odds ratio of the possible risks for the development of CAD in non-diabetic patients.
The individual and common effects of smoking habit and −374 T or −374 A allele on the odds ratio of the possible risk for the development of CAD in non-diabetic patients.
Comparison of previous studies of the RAGE −374T/A genotypes and cardiovascular diseases.
We investigated associations of RAGE polymorphism with the risk of CAD with or without diabetes mellitus in a case-control based study in a Turkish population. Our study is the first study to examine these associations. The causal role of RAGE in the pathogenesis of CAD has been examined in several studies (Table V). In this study, we observed an association between −374T/A polymorphism and diabetic CAD as a independent risk factor for the development of the disease and no association between −374T/A polymorphism and non-diabetic CAD or the whole patient group. These findings were similar to those of population studies but not all (Table V).
Our findings of an association of RAGE −374T/A polymorphism with diabetic and non-diabetic CAD patients differed from some of the studies shown in Table V. Pettersson-Fernholm et al. (33) and Falcone et al. (20, 22, 24) found that because the conversion of T →A caused the repression of receptor expression, resulting in inefficiency in cellular signalling, the A allele had a protective effect for the development of CAD. However Hudson et al. (34), Kirbis et al. (35) and Jixiong et al. (36) suggested no association between A allele and vascular diseases (Table V). The difference between those studies and our study could be because of the size of the study groups and ethnic origins of the population and/or differences in the smoking habit and the other risk factors for CAD.
In this study, we found the possession of the A allele to be an independent risk factor for CAD. The repression of RAGE expression due to the A allele results in higher blood levels of deleterious AGEs, HbA1c, glycated-LDL and glycated collagen. The contribution of the cross-linking of AGEs with vessel proteins that causes the thickening and leakeage of the wall in vascular diseases are well defined (37).
In conclusion, our results demonstrated that in non-diabetics, being male in gender and having a smoking habit were the most important risk factors for CAD, and in diabetics, the AA genotype had more significance than the other variants for the development of CAD. As such, larger numbers of study groups are required to establish whether −374T/A polymorphism has a causative role in the pathogenesis of CAD.
Acknowledgements
This present work was supported by the Research Fund of Istanbul University, project number: T-967/06102006.
- Received June 17, 2009.
- Revision received August 10, 2009.
- Accepted September 21, 2009.
- Copyright © 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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