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Preliminary Study: DNA Repair Gene Polymorphisms (RRM1, RRM2, ERCC2) In Left Ventricular Hypertrophy

ÖZ ORIGINAL ARTICLE ORİJİNAL ARAŞTIRMA DOI: 10.26650/experimed.2019.19024 Preliminary Study: DNA Repair Gene Polymorphisms (RRM1, RRM2, ERCC2) In Left Ventricular Hypertrophy Sol Ventrikül Hipertrofisinde (RRM1, RRM2, ERCC2) DNA Tamir Gen Polimorfizmleri Fatma Tuba Akdeniz1 , Seda Güleç Yılmaz1 , Cemil Selim İsbir2 , Yaşar Birkan2 , David Sinan Esensoy3 , Gözde Özcan1 , Atike Tekeli Kunt4 , Turgay İsbir3 1Department of Molecular Medicine, Yeditepe University Institute of Health Sciences, İstanbul, Turkey 2Department of Cardiovascular Surgery, Marmara University Pendik Training and Research Hospital, İstanbul, Turkey 3Department of Medical Biology, Yeditepe University School of Medicine, İstanbul, Turkey 4Department of Cardiovascular Surgery, Ankara Numune Training and Research Hospital, Ankara, Turkey Cite this article as: Akdeniz FT, Güleç Yılmaz S, İsbir CS, Birkan Y, Esensoy DS, Özcan G, et al. Preliminary Study: DNA Repair Gene Polymorphisms (RRM1, RRM2, ERCC2) In Left Ventricular Hypertrophy. Experimed 2019; 9(3): 99-104. Objective: Left ventricular hypertrophy (LVH) accounts for one of the most important independent risk factors for cardiac diseases. In the present study, we investigate the relationship of DNA repair gene polymorphisms and LVH. Material and Method: 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: Although there were no significant differences for RRM2 (p=0.365) and ERCC2 (p=0.740) genes. RRM1 (A>C) CC genotype was significantly higher in the LVH than control groups (p=0.018). RRM1gene wild type A allele carriers were significantly higher in the healthy controls than the LVH group (p=0.029). Conclusion: RRM1 gene CC genotype could be a risk factor, whereas the RRM1 gene AC genotype and the A allele might play a protective role against LVH.


INTRODUCTION
During mammalian organogenesis heart is the first functionally formed organ. Each year approximately one million newborns are known to be born with heart defects, and one in every three people die because of heart diseases, thus cardiovascular diseases are becoming the leading cause of global mortality (1).
Left ventricular hypertrophy (LVH) is responsible for a compensatory mechanism to keep normal wall tension and to maintain cardiac output. When the left ventricle contracts, oxygenated blood is distributed to the body through the aortic valve. If hypertension exists making it difficult for blood to spread throughout the body, the left ventricle will strain to send the blood. This leads to an abnormal increase in the left ventricular (LV) mass, which is called LVH (2). LVH can occur because of hypertension, aortic valvular stenosis, aortic regurgitation, mitral regurgitation, and hypertrophic cardiomyopathy (3). Patients with LVH may experience car-diac failure, coronary artery disease, peripheral arterial disease and sudden death. Stroke risk is increased six-fold in LVH cases (4).
Ribonucleotide reductase (RR) is an enzyme catalyzing the conversion of Adenosine diphosphate (ADP) into Deoxyadenosine diphosphate (dADP) by removing the hydroxyl group from the second position of the ribose ring. Then, dADP is converted to Deoxyadenosine triphosphate (dATP) with the help of creatine kinase (5). The RR enzyme is required for the synthesis of deoxyribonucleotides, it is the rate-limiting step in DNA synthesis and it has two protein subunits important for its activity (6). Ribonucleotide reductase protein M1 (RRM1), is a catalytic activator, and ribonucleotide reductase protein M2 (RRM2), is a free radical-containing subunit (7,8). Cardiac muscles prefer dATP as a more effective substrate than ATP for contraction, and with an increase in dATP levels, force generation, cross bridge cycling, and calcium sensitivity in the myocardium are known to be increased (9,10). Excision Repair Cross-Complementing 2 (ERCC2) gene is a nucleotide excision repair mechanism (11).
There are only few studies examining the association between DNA repair genes and cardiovascular diseases. In this study, we aimed to investigate some DNA repair gene polymorphisms (RRM1, RRM2, ERCC2) and discuss their possible roles in the molecular mechanism of LVH.

Study participants
All participants were selected from Marmara University Cardiovascular Surgery Department after detailed clinical examinations. All individuals signed an informed consent which was conducted in accordance with the ethical principles stated in the "Declaration of Helsinki". The study consisted of 15 patients with LVH and 24 healthy individuals. Echocardiographic parameters were measured using standard methods. Left ventricular systolic functions were estimated by measuring the Ejection Fraction (LVEF, %).

Genotyping
Blood samples from all participants were collected in ED-TA-tubes and DNA isolations were performed using Invitrogen iPrep Purification Instrument and Invitrogen iPrep Pure Linkg DNA Blood Kits (Invitrogen, Life Technologies, Carlsbad, CA, USA). DNA concentrations and optical density ratios were measured using NanoDrop 2000 (Thermoscientific, Waltham, MA, USA). Genotyping was performed using Applied Biosystems Fast Real-Time polymerase chain reaction (RT-PCR) instrument and TaqMan reagents primer-probe sets designed for RRM1 gene (rs12806698), RRM2 gene (rs6859180) and ERCC2 gene (rs13181) polymorphisms (Applied Biosystems, Foster City, CA, USA).
LDL-HDL subfraction analysis: Serum samples were analyzed for LDL and HDL subfractions with the LIPOPRINT SYSTEM (Quantimetrix, CA, USA). This system is performed using high-resolution, polyacrylamide gel electrophoresis to separate lipo-protein particles into various fractions on the basis of size and density. LDL was analyzed with 7 subfractions including Large LDL (1 to 2) and Small LDL (3 to 7), while HDL was separated into 10 subfractions with Large HDL (1 to 3), Intermediate HDL (4 to 7) and Small HDL (8 to 10).

Statistical Analysis
Statistical analyses were performed using the Statistical Package for Social Sciences version 23 software (IBM Corp.; Armonk, NY, USA). Significant differences between groups were determined using the Student's t-test and demographic information was compared using Chi-square and Fisher's exact tests. Risk estimations were examined with Odds Ratio (OR) at 95% Confidence Interval (CI). p<0.05 was denoted as statistically significant.

RESULTS
The demographic characteristics of the study population are given in Table 1. This study was conducted 39 samples; 4 female, 11 male in LVH group and 10 female 14 male in healthy control group. There were no statistically significant difference between healthy group and LVH group as demographic characterization (p=0.359). The mean ages of the patients with LVH and the healthy control groups were 66.87±10.63. and 62.46±5.97 (p=0.105). Systolic Blood Pressures (BP) were significantly higher in patients with LVH than the healthy control group (p=0.017). However there were no significantly difference between the groups, LDL-HDL Subfractions values are given as demographic information and were not associated with polymorphism.
Echocardiographic parameters of LVH patients and healthy control group are given in Table 2. Genotype and allele frequencies between LVH patients and the healthy controls are listed in Table 3. Although there were significant differences for neither RRM2 (A>G) gene (p=0.365) nor ERCC2 (G>T) (p=0.740) genes, RRM1 (A>C) gene was significantly different in the LVH and control groups (p=0.018). RRM1 heterozygote AC genotype was significantly higher in the healthy control group (p=0.008), but homozygote mutant CC genotype was higher in the LVH group (p=0.029). Regarding RRM1 (A>C) polymorphism, in the patient group the percentage of wildtype A allele was 30% while mutant C allele was 70%. RRM1 A allele carriers were significantly higher in the healthy control group (p=0.029), moreover RRM1 A allele carriers had a ~4.5fold decreased risk for LVH (OR=0.22, 95%CI=0.056-8.889). Our results indicated that RRM1gene CC genotype carrying might increase the risk of LVH, whereas the RRM1 gene AC genotype may decrease the risk, and the A allele might have a protective role against LVH.
Although there was no statistically significant difference between the echocardiographic parameters and the RRM1 allele group, the echocardiographic parameters in the RRM1 A allele carrier group were lower than the non-A allele carrier group and the RRM1 C allele carrier group was higher than the non-C allele carier group (Table 4).

DISCUSSION
Despite advances in ventricular dysfunction epidemiology, the mechanism of genotypic and phenotypic variations remains unclear. In this study, the relationship of RRM1 (rs12806698), RRM2 (rs6859180) and ERCC2 (rs13181) polymorphisms on LVH were investigated. The AC genotype of RRM1 gene was higher in the control group, indicating that the AC genotype decreases the risk of LVH; The CC genotype was in a high proportion in LVH patients, so carrying the A allele could lower the risk for LVH as its frequency was higher in the healthy control group than in the LVH patients.   The clinical and laboratory studies showed that dATP acts as a substrate for de-membranated cardiac muscle contraction and is a more effective substrate than ATP. It increases left ventricular contractions both in normal and infarcted heart regions.
dATP can be synthesized through a reductive reaction catalyzed by the RRM1 enzyme. Furthermore it has been asserted that RR inhibition could be a therapeutic target of atheroproliferative disorders (12).   Unrepaired DNA base damage triggers apoptotic pathways (13) and apoptosis accelerates myocyte loss and causes myocardial dysfunctions (14). Apoptosis-induced myocyte damage causes abnormal loading and increased wall stress which results in heart failure. In spite of compensatory mechanisms, insufficient DNA repair could lead to myocyte apoptosis that begins at the onset of hypertrophy (14).
The RRM1 gene, is a molecular target of gemcitabine, and has very important roles. Increased RRM1 expression levels were correlated with longer survival rates, while it is a disadvantage to have high levels of RRM1 expression because of a decreased efficacy of chemotherapy. Two single nucleotide polymorphisms were discovered upstream of the first exon of the RRM1 gene. They are an adenine/cytosine change at (-) 37 nucleotide position and a cytosine/thymidine change at (-) 524 nucleotide position. These polymorphisms affect promoter activity highly associated with overall survival (15).
Ribonucleotide reductase activity has a role not only in cell cycle but also in the proper functioning of myocytes. RR overexpression increases dATP, affecting the contractility of cardiomyocytes. Elevated cardiomyocyte dATP levels via RR protein increases muscle contractility, subsequently basal cardiac function increases by actin-myosin binding and cycling. Thus, even in cardiac overload, myocytes could maintain normal myocardial energetics (9). Furthermore, it has been claimed that cardiac-specific RR gene therapy may reverse cardiac dysfunction. In the animal model, heart failure treated with the RR gene was inducing myosin activation (10).
According to analysis of sex differences, genders may account for the risk of cardiovascular diseases. In terms of cardiac adaptation, the left ventricular dimensions are smaller and left ventricular performance is higher in women compared to men. Sex differences can play a role in LVH however, in this study, there was no statistical significance related to gender and LVH (16).
One of the most important factors in the pathogenesis of LVH is systolic BP. It has been shown that there is a direct relationship between the incidence of LVH and the level of the systolic BP. Our study supports this finding due to the fact that the difference in the systolic BP between the groups is statistically significant and the mean is higher in patients with LVH than the healthy control group (17).