Abstract
Background/Aim: Coronary artery disease (CAD) is a chronic inflammatory disease seen as formation of atherosclerotic plaques (atheroma) in coronary arteries. Recent published papers show that DNA damage and repair mechanisms play a crucial role on the development and severity of atheromas. In this study, we investigated nucleotide excision repair (NER) pathway-related gene polymorphisms in atherosclerosis. XPD, encoded by ERCC2 gene, is an ATP-depended helicase enzyme involved in the NER pathway. Ribonucleotide reductase (RR) is a tetra meric enzyme, synthesizing deoxyribonucleotides from ribonucleotides for DNA synthesis. RR is encoded by the RRM1 and RRM2 genes, which are two subunits of RR enzyme. Materials and Methods: DNA samples isolated from peripheral blood were genotyped with real-time polymerase chain reaction (RT-PCR) for RRM1 (rs12806698), RRM2(rs6859180) and ERCC2 (rs13181) genes. Results: The frequency of the RRM1 AC heterozygote genotype was found to be significantly lower (odds ratio (OR)=0.369, 95% confidence interval (CI)=0.179-0.760; p=0.006), whereas the CC homozygote genotype was found to be significantly higher in patients compared to controls (OR=7.636, 95% CI=2.747-21.229; p=0.000). In addition, the RRM1 A allele was higher in control group (p=0.000, OR=0.131 95%CI=0.047-0.364). For the ERCC2 gene, GG genotype was significantly higher in control group (p=0.017, OR=0.387, 95%CI=0.175-0.152) and TT genotype (p=0.021) was higher in CAD group. TT genotype had a ~3-fold increased risk (OR=3.615, 95%CI=1.148-11.380) for CAD. Carrying T allele appears to be a risk factor for CAD (p=0.017, OR=2.586, 95%CI=1.173-5.699), while the G allele might be a risk-reducing factor (p=0.021, OR=0.277, 95%CI=0.088-0.871) for CAD. Conclusion: RRM1 and ERCC gene polymorphisms, having homozygous mutant genotype, might be a risk factor for CAD. RRM1 and ERCC wild type alleles are risk-reducing factor for CAD. Also, carrying RRM1 A allele might have a protective effect for smokers.
Coronary artery disease (CAD) is known to have the highest mortality rate among other atherosclerotic diseases. Atherosclerotic plaque formation occurs in coronary arteries after prolonged inflammatory processes and fatty depositions (1, 2). CAD is etiologically related to several different factors, including lipid abnormalities, oxidative stress and DNA damage (3, 4). Recent publications report that low-density lipoprotein (LDL) oxidation and lipid peroxidations might cause DNA modifications, such as DNA strand breaks and bulky DNA lesions (5). Nucleotide excision repair (NER) pathway is mainly responsible for DNA repair of the disrupted parts on helical structure caused by physical or chemical agents (6). NER can be divided into two sub-pathways as global-genomic NER (GG-NER) and transcription coupled-NER (TC-NER). Those two are only differing in how they recognize DNA damage, while excision and synthesis mechanisms are same for GG-NER and TC-NER (7). The ERCC2 gene encodes XPD protein that has an ATP-dependent helicase activity in NER pathway (8). Studies about ERCC2 polymorphisms have shown that ERCC2 genetic variations might have roles on the development of some cancer types and atherosclerosis (9-14). RRM1 (ribonucleoside-diphosphate reductase large subunit) and RRM2 (ribonucleotide reductase small subunit) genes are encoding large and small subunits of the ribonucleotide reductase (RR) enzyme, respectively. RR is a tetrameric enzyme that synthesizes deoxyribonucleotides for DNA and plays an important role in cell-cycle regulation and DNA damage response (15, 16). RR also has a restraining effect on NER by inhibition (17). The aim of this study was to investigate the possible effects of ERCC2, RRM1 and RRM2 polymorphisms on CAD.
Demographic characteristics of patient and control groups.
Materials and Methods
Patient and control groups were selected after detailed clinical examinations in Marmara University Cardiovascular Surgery Department. After obtaining informed consent of each subject, blood samples were collected in EDTA containing tubes. DNA isolations were performed by Invitrogen iPrep Purification Instrument and Invitrogen iPrep PureLink gDNA Blood Kits (Invitrogen, Life Technologies, Carlsbad, CA, USA) from 350 μl whole peripheral blood samples. With the isolation procedure, performed by iPrep Purification Instrument, 100 μl DNA was collected. Sample DNA concentrations and optical density ratios were measured by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Genotyping was performed by Applied Biosystems Fast Real-Time polymerase chain reaction (RT-PCR) instrument and TaqMan Reagents primer-probe sets (Applied Biosystems, Foster City, CA, USA), specifically designed for RRM1 gene rs12806698, RRM2 gene rs6859180 and ERCC2 gene rs13181 polymorphisms. PCR reaction mixture was containing 10 μl X Genotyping Master Mix, 0.5 μl 40X TaqMan Genotyping Assay (TaqMan Reagents; Applied Biosystems), 8.5 μl PCR grade water and 1 μl of sample DNA. PCR conditions were 10 min of hold stage at 95°C and 40 cycles of 15 sec denaturation at 92°C and 60 sec of annealing/extension at 60°C as recommended by supplier. Allelic discrimination was done by software of 7500 Fast Real-Time PCR instrument by interpreting the fluorescent data of hybridizing probes. Statistical analyses were performed using SPSS Ver. 23 software (SPSS Inc, Chicago, IL, USA). Significant differences between groups were determined by Student's t-test, while demographics were compared by χ2 and Fisher's exact tests. Risk estimations were examined with Binary Logistic Regression analysis as odds ratio (OR) at 95% confidence interval (CI). p<0.05 was denoted as statistically significant.
Genotypic and allelic frequencies of patient and control groups.
Results
The demographic characteristics of CAD and control groups were given in Table I. The CAD group had, as expected, significantly higher levels of LDL-cholesterol (p=0.049) and lower levels of HDL-cholesterol (p=0.00).
RRM1, RRM2 and ERCC2 genotypic and allelic frequencies of CAD and control groups are given in Table II. There were significant differences for RRM1 and ERCC2 gene regions between CAD and control groups. AC genotype (p=0.006 and OR=0.369, 95%CI=0.179-0.760) and CC genotype (p= 0.00) of RRM1 gene had significant differences between groups. CC genotype was ~7.63-fold increased (OR=7.636, 95%CI=2.747-21.229) in CAD and A allele was found to be higher in control group (p=0.00, OR=0.131 95%CI=0.047-0.364) compared to CAD. For ERCC2 gene, GG genotype was significantly higher in control group (p=0.017, OR=0.387, 95%CI=0.175-0.152) and TT genotype (p=0.021) was higher in CAD group. TT genotype had a ~3-fold increased risk (OR=3.615, 95%CI=1.148-11.380) for CAD. Carrying T allele seemed to be a risk factor for CAD (p=0.017, OR=2.586, 95%CI=1.173-5.699), whereas G allele might be a risk-reducing factor (p=0.021, OR=0.277, 95%CI=0.088-0.871) for CAD. There were no significant differences between CAD and control groups neither for RRM2 genotypes nor alleles.
Genotypic and allelic frequencies between smokers and non-smokers of patient and control groups.
Considering the smoking habit, RRM1 and ERCC2 polymorphisms have a significant association with cigarette-smoking in CAD and control patients (p=0.001) than non-smoking patients (Table III). For cigarette-smoking patients, RRM1 gene AA genotype (p=0.009, OR=0.143, 95% CI=0.029-0.700) and carrying A allele (p=0.002, OR=0.068, 95% CI=0.008-0.564) appeared to decrease the risk for CAD. CC genotype (p=0.002, OR=14.636 95% CI=1.772-120.878) and carrying C allele (p=0.009, OR=7.000, 95% CI=1.429-34.286) had increased risk for CAD. Also, homozygous CC genotype (mutant genotype) frequency was increased in CAD group than AA genotype (wild type genotype) (p=0.026) in smoking CAD patients. ERCC2 gene GG genotype (p=0.003, OR=0.143, 95% CI=0.036-0.566) had decreased risk, TT genotype (p=0.078, OR=5.68895% CI=0.677-47.798) and carrying T allele (p=0.003, OR=7.00 CI=0.021-1.478) had increased risk for CAD. However, for non-smoking patients, RRM1 gene CC genotype has increased risk (p=0.012, OR=4.636, 95% CI=1.325-16.219), whereas carrying A allele seems to decrease the risk (p=0.012, OR=0.216, 95% CI=0.620-0.755) for CAD. According to comparison of genotype and smoking status within the CAD group (Table IV) for both RRM1 and ERCC2 genes, carrying homozygous wild type genotype has a protective effect, while carrying mutant allele appears to be a risk factor for CAD for cigarette smokers. There were no significant differences between CAD and control groups for RRM2 gene polymorphisms considering cigarette smoking.
Effect of nucleotide excision repair (NER)-related polymorphisms on CAD considering cigarette smoking.
Discussion
Recent findings on the relationship between DNA repair and atheroma formation and its severity have changed the general outlook on CAD and atherosclerosis. As oxidative stress is a main risk factor for atherosclerotic diseases and oxidative stress-related DNA damages are considered to be responsible for the main consequences of the disease, polymorphisms of DNA repair-related genes might be risk factors for atherosclerotic diseases. Several publications have shown that DNA repair and anti-oxidant gene polymorphisms have significant relationships with atherosclerotic diseases (18-20). According to those publications, our study aimed to investigate the relationships of RRM1, RRM2 and ERCC2 polymorphisms in CAD risk.
Our results showed the association between NER-related gene polymorphisms in CAD. NER-related gene polymorphisms are found to have direct or synergistic effects on risk of atherosclerotic diseases (14, 20, 21). Concerning ERCC2 polymorphisms, a study on large arterial atherosclerosis had parallel findings as ours, as there was a significant correlation between ERCC2 polymorphism and large arterial atherosclerosis and, in addition, carrying ERCC2 mutant allele was increasing the risk for smokers (14). Currently, it is known that elevated levels of cholesterol and smoking are risk factors for CAD and, beside the atherogenesis theory, both smoking and elevated cholesterol levels are inducing DNA damage. Lipid peroxidation products are DNA damaging agents, such as hydroxynoneal and its derivatives. Nair et al. have shown that increased levels of etheno-DNA adducts in atheromas are related to smoking (5). In a 14-year follow-up study, Izotti et al. showed that survival of patients was affected by their cholesterol levels and smoking habitus due to identified DNA modification levels in atheromas and genetic polymorphisms of oxidative stress and oxidative DNA damage-related genes (18). There have been few studies about RRM1 polymorphisms, all about chemotherapeutic drug response in cancer patients, but no studies about CAD (22-25).
The present work reveals the importance of DNA repair-related gene polymorphisms. The results demonstrate that RRM1 and ERCC gene polymorphisms, in particular, might have effects on CAD development. For both gene polymorphisms, having homozygous mutant genotype might be a risk factor for CAD and carrying RRM1 wild type allele might have protective effect for smokers. To the best of our knowledge, this is the first study that investigated the relationship of RRM1 and RRM2 gene polymorphisms and CAD. Further studies with larger populations will be more informative for understanding the effect of RR enzyme and its genetic variations on CAD.
- Received June 7, 2016.
- Revision received July 1, 2016.
- Accepted July 4, 2016.
- Copyright © 2016 The Author(s). Published by the International Institute of Anticancer Research.
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