Abstract
Background/Aim: Genetic polymorphism is a strong risk factor for coronary artery disease (CAD). In the present study, our aim was to evaluate angiotensin-converting enzyme (ACE) gene I/D polymorphism and interleukin-4 (IL-4) gene Intron 3 variable number of tandem repeat (VNTR) polymorphism in CAD. Materials and Methods: One hundred and twenty-four CAD patients and one hundred and twenty-three controls were enrolled. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR) analyses. Results: The risk associated with inheriting the combined genotypes for the two polymorphisms were evaluated and it was found that the individuals who were P2P2-homozygous at IL-4 gene intron 3 VNTR and DD-homozygous at ACE gene I/D have a higher risk of developing CAD. Conclusion: Although, there is no correlation between IL4 VNTR polymorphism and ACE gene polymorphism and CAD, there is a strong association between CAD and co-existence of IL-4 VNTR and ACE gene polymorphisms in the Turkish population.
Coronary artery disease (CAD) is a public health problem that has high morbidity and mortality rates accounting for up to 40% of all deaths in industrialized countries (1). Genetic polymorphism is a risk factor for CAD as well, as it may predispose to risk factors like hyperlipidaemia, obesity, hypertension, left ventricular hypertrophy, diabetes mellitus (DM), etc. (2). Mainly, it is known that CAD is a multi-factorial disease and includes gene–gene and gene–environment interactions (3). Nonetheless, the detection of possible genetic risk factors on the development of CAD is important for early diagnosis due to evaluating the individual cardiovascular risk profile.
In the pathogenesis of CAD, the main problem is atherosclerosis due to inflammation (4). Interleukin-4 (IL-4) is a potent cytokine secreted by T-helper 2 cells, eosinophils and mast cells. It plays a role in the formation of endothelial cell adhesion molecules, chemotaxis of immune cells and anti-inflammation. The IL-4 gene has been mapped to the q arm of chromosome 5 in a cluster of cytokine genes. Variable number of tandem repeat (VNTR) polymorphism with a unit size of 70-bp sequence is a frequent polymorphism of IL-4. It is located in the third intron of the gene and it contains two alleles, P1 (183 bp) and P2 (253 bp) (5). In literature, IL-4 VNTR polymorphism has been found to be related with many diseases, as rheumatoid arthritis (RA), bladder cancer, ischemic stroke, multiple sclerosis (MS), alopesi areata (AA) and diabetic neuropathy (6-9). In the present study, we hypothesized that the IL-4 gene VNTR polymorphism may play a primary or secondary role in the pathogenesis of CAD due to its role on anti-inflammation.
The angiotensin-converting enzyme (ACE), which is one of the key components of the renin–angiotensin system (RAS), is a zinc metallopeptidase that participates in the formation of the vasoconstrictor angiotensin II (Ang II) and reduces the vasodilator bradykinin-2 (10). Besides, ACE has effects on glucose metabolism (11). The human ACE gene polymorphism that affects ACE activity is localized in the long arm of chromosome 17 and it includes 26 exons and 25 introns. The insertion or deletion (I/D) polymorphism includes a 287-bp fragment in intron 16. ACE D/D and I/I homozygotes and I/D heterozygotes are genotypes of I/D polymorphism (10). ACE gene I/D polymorphism may be a risk factor for CAD due to RAS activation.
To the best of our knowledge, there is no report to have investigated both ACE gene I/D polymorphism and IL-4 gene VNTR polymorphism in CAD patients. In the present study, we aimed to experimentally explore these two polymorphisms for CAD patients in a Turkish population. We hypothesized that these polymorphisms may have an effects on CAD due to atherosclerosis or other risk factors like diabetes mellitus and hypertension.
Baseline clinical and demographic characteristics of the patients with CAD and healthy controls.
Materials and Methods
Subjects. The study group consisted of 124 unrelated patients with CAD (81 male and 43 female; mean age: 59.88±8.185; years±standard deviation [SD]) and 123 (67 male and 56 female; mean age: 58.75±10.617 years) unrelated healthy controls. CAD patients were recruited consecutively and prospectively including those whom were treated and followed-up in the Cardiology Department of Gaziosmanpasa University Research Hospital, Tokat, Turkey. The diagnosis of CAD was confirmed by coronary angiography performed by experienced cardiologists and it was defined as the presence of a stenosis of greater than 50% in one or more of the main coronary arteries. All control subjects were confirmed free from coronary artery disease by either angiography or clinical symptom together with electrocardiogram (ECG) examinations. Exclusion criteria for both groups included the presence of congenital heart disease, cardiomyopathy, valvular diseases or autoimmune diseases. All participants, patients and healthy controls, were of Turkish origin from the inner Central Black Sea region of Turkey. The healthy controls were matched for age and gender with RAS patients (Table I). The study protocol was approved by the Local Ethics Committee of Gaziosmanpasa University, Faculty of Medicine and written informed consent was obtained from the study participants.
Genotyping. Genomic DNA was extracted from whole venous blood samples using a commercial DNA isolation kit (Sigma-Aldrich, Taufkirchen, Germany). The ACE gene I/D (rs5186) and the IL-4 gene 70 bp VNTR (rs8179190) polymorphisms were analyzed by polymerase chain reaction (PCR). The PCR amplifications were carried out in a total volume of 25 μl reaction containing 100 ng of genomic DNA, 2.5 μl of 10X PCR buffer, 200 μM dNTP, 10 pM of each primer and one unit of Taq DNA polymerase. The ACE gene I/D polymorphism was analyzed by using forward (F) 5’-CTG GAG ACC ACT CCC ATC CTT TCT-3’ and reverse (R) 5’-GAT GTG GCC ATC ACA TTC GTC AGA T-3’ primers. The amplification conditions consisted of an initial melting step of 5 minutes at 94°C; followed by 30 cycles of 1 min at 94°C, 1 min 45 sec at 60°C and 1 min 30 sec at 72°C; and a final elongation step of 5 min at 72°C. In the absence of the 287 bp in intron 16 of the ACE gene, this PCR method resulted in a 190 bp product (D allele) and in the presence of the 287 bp, resulted a 477 bp product (I allele). In heterozygote samples, two bands (477 and 190 bp) were detected. For IL-4 gene 70 bp VNTR polymorphism, amplification was carried out using F 5’- AGG CTG AAA GGG GGA AAG C-3’, R 5’-CTG TTC ACC TCA ACT GCT CC-3’ primers with initial denaturation at 95°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 45 s, extension at 72°C for 1 min and final extension at 72°C for 10 min. The PCR products were visualized on 3% agarose gel stained with ethidium bromide. The PCR product was of 183 bp for P1 allele and 253 bp for P2 allele. A second PCR was performed to confirm samples with no conclusive results.
Statistical analysis. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS Statistics, version 20; IBM, Address) and OpenEpi Info software package version 3.01 (www.openepi.com). The Chi-square (χ2) test was used to evaluate the Hardy-Weinberg equilibrium (HWE) for the distribution of the genotypes of the patients and the controls. The relationships between ACE gene I/D and IL-4 gene 70 bp VNTR polymorphisms and the clinical as well as demographic characteristics of patients were analyzed by using the χ2 test, Fisher's exact or analysis of variance (ANOVA) statistics. The χ2 test and Fisher's exact test were used to compare categorical variables appropriately; odds ratio (OR) and 95% confidence interval (CI) were used for the assessment of risk factors. All p-values were 2-tailed and p-values less than 0.05 were considered significant.
Results
The baseline clinical and demographic characteristics of CAD patients and controls are presented in Table I. Gender, age, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, smoking, history of hypertension and history of diabetes mellitus of RAS patients and controls were compared. Among these characteristics, LDL cholesterol and history of hypertension showed statistically significant differences between patients and controls (p=0.002 and p=0.002, respectively). Clinical and demographic characteristics of CAD patients (gender, age, disease duration, HDL cholesterol, LDL cholesterol, triglycerides, history of hypertension, history of diabetes mellitus and smoking) stratified according to ACE gene I/D and IL-4 gene intron 3 VNTR polymorphisms are shown in Table II. Any association was not found between clinical and demographic characteristics of CAD patients and the ACE gene I/D and the IL-4 gene 70 bp VNTR polymorphisms (p>0.05) (Table II).
Clinical and demographic characteristics of CAD patients stratified according to ACE gene I/D and IL-4 gene intron 3 VNTR polymorphisms.
Allelic and genotypic distributions of the ACE gene I/D and the IL-4 gene intron 3 VNTR polymorphisms are shown in Table III. Genotype and allele frequencies did not show any significant differences between patients and controls according to ACE gene I/D and the IL-4 gene VNTR polymorphisms (p>0.05) (Table III).
We also examined the risk associated with inheriting the combined genotypes for the two polymorphisms (Table IV). Only the homozygosity for P2P2 at IL-4 gene intron 3 VNTR and homozygosity for DD at ACE gene I/D encoded a significant p-value of 0.031. Thus, individuals who were P2P2-homozygous and DD-homozygous have a higher risk of developing CAD. The observed and expected frequencies of IL-4 gene intron 3 VNTR polymorphism were in Hardy-Weinberg equilibrium in the control group but not in the patient group. However, the observed and expected frequencies of ACE gene I/D polymorphism were in Hardy-Weinberg equilibrium in both groups.
Discussion
In the current study, it was found that there was no correlation between CAD and IL-4 gene VNTR polymorphisms in the Turkish population from the Central Black Sea region. It is known that anti-inflammatory cytokines play a key role due to prevention of atherosclerosis in the development of CAD. IL-4 is one of the anti-inflammatory cytokines also known for its effects on haematopoiesis, inhibition of nitric oxide synthase and superoxide, and anti-tumour activities (12). There are several published studies that have investigated the association between different genetic polymorphisms and CAD in the Turkish population, such as APOE, NF-κB1A promoter and LOX-1 gene polymorphisms (13, 14, 15). To the best of our knowledge, there is no study on IL-4 VNTR polymorphism and CAD in Turkish population. Sobti et al. has studied the association of IL-4 gene VNTR polymorphism with CAD in Indian population and, similar to our results, they reported that there was no correlation between this polymorphism and CAD. They suggested that IL-4 VNTR polymorphism acted minimally in atherogenesis. Differently from our results, they suggested that this polymorphism was associated with other risk factors like diabetes mellitus (DM), hypertension, family history and mental stress in CAD, indicating probably that this polymorphism was not a primary risk factor of CAD but could play a role as a secondary risk factor due to CAD (16). CAD occurs from accumulation of lipids, calcium and inflammatory cells throughout the wall of coronary arteries. This accumulation causes narrowing in lumen and reduction in blood flow (17). Inflammation plays a key role on pathogenesis of CAD. IL-4 is an anti-inflammatory cytokine. In a study by Jha et al., IL-4 serum levels were found higher in a CAD group compared to the normal group and they suggested that serum levels of vascular inflammation markers might help determine patients with high risk for CAD (18). Unfortunately, the relation with genetic polymorphism of IL-4 VNTR and serum levels of IL-4 is not as yet completely clear. Although it is known that IL-4 has a role in the pathogenesis of CAD, the present study does not support the effects of IL-4 VNTR polymorphism in CAD patients in the studied Turkish population.
Genotype and allele frequencies of IL-4 and ACE gene polymorphisms in patient and control groups.
In the present study, no correlation was found between CAD and ACE gene I/D polymorphism in the Turkish population from the Central Black Sea region. Ozturk et al. has reported that the D allele may affect pulse pressure (PP) in patients with first acute anterior myocardial infarction in ACE gene I/D polymorphism. Also, in this study, they found no correlation between ACE gene I/D polymorphism and risk factors like hypercholesterolemia, DM, hypertension and obesity (19). Similar to our results, Agirbasli et al. suggested that there is no significant association between ACE gene I/D polymorphism and CAD in Turkish population (3). Inversely, Guney et al. reported that the DD genotype and the D allele may be related with the severity of CAD in Turkish population. They suggested that the ACE I/D polymorphism may be used as a risk factor for CAD (20).
Comparative analysis of combined genotypes of CAD patients and controls.
It was found that patients with the P2P2 genotype at IL-4 gene intron 3 VNTR and DD genotype at ACE gene I/D were susceptible for developing CAD in the current study. This result indicates that the co-existence of both genetic polymorphisms increase the probability of CAD in Turkish population. The combination of these gene polymorphisms is important. CAD is a multi-factorial disease; thus, the interactions of several genetic polymorphisms may be susceptible to developing CAD via different pathways that cannot be presently evaluated due to the lack of similar studies.
In conclusion, there is no correlation between IL4 VNTR polymorphism and ACE gene polymorphism and CAD. Additionally, there is a strong association between CAD and co-existence of IL-4 VNTR and ACE gene polymorphisms in the Turkish population studied. Further studies that include serum levels of IL4 and ACE are required to explain the exact mechanism of this cooperation in the pathogenesis of the disease. Finally, genetic factors are related to many complex diseases such as CAD, however it is not true to charge only one genetic polymorphism. It is better to find interaction of different genetic factors with each other in the pathogenesis of complex diseases.
Footnotes
-
Conflicts of Interest
The Authors have no conflicts of interest regarding the current study.
- Received June 3, 2014.
- Revision received July 15, 2014.
- Accepted July 16, 2014.
- Copyright © 2014 The Author(s). Published by the International Institute of Anticancer Research.
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.