Asian Cardiovasc Thorac Ann 2005;13:153-156
© 2005 Asia Publishing EXchange Ltd
Renin-Angiotensin System Polymorphisms and Coronary Artery Surgery Patients
Kanat Ozisik, MD,
Muge Misirlioglu, PhD1,
Tulga A Ulus, MD2,
Serdar Tuncer, MD1,
Mustafa Emir, MD2,
Fehmi Katircioglu, MD2
Department of Cardiovascular Surgery, Ankara Numune Education and Research Hospital
1 Metis Biotechnology Ltd.
2 Department of Cardiovascular Surgery, TYIH, Ankara, Turkey
For reprint information contact: Kanat Ozisik, MD Tel: 90 505 290 1885 Fax: 90 312 310 3460 Email: sozisik2002{at}yahoo.com, 23.cad Kirkkonaklar mah. Simkent Sitesi 2.Blok No:6/13, 06610 Ankara, Turkey.
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ABSTRACT
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The frequencies of angiotensin-converting enzyme gene insertion/deletion, angiotensinogen-M253T, and angiotensin II type 1 receptor-A1166C polymorphisms were analyzed in 105 patients undergoing coronary artery bypass grafting (group 1) and a control group of 105 non-cardiac patients (group 2). Blood samples were obtained for biochemical analyses and DNA extraction. Genotyping was performed by polymerase-chain-reaction-based restriction analysis. According to the angiotensin-converting enzyme gene insertion/deletion polymorphism, 36.3% of patients in group 1 and 30.7% in group 2 were homozygous for the DD allele. This difference was not statistically significant. Angiotensin II type 1 receptor-A1166C genotype polymorphism was also not significantly different between the groups. The results showed the angiotensinogen-M235T polymorphism to be heterogenous. The MM homozygote frequency was significantly higher in controls (72.3%), whereas 80% of the TT homozygote frequency was in the surgical group ( p = 0.001). These results show that although there were no significant differences in angiotensin-converting enzyme gene insertion/deletion and angiotensin II type 1 receptor-A1166C genotype polymorphisms between the groups, angiotensinogen-M235T polymorphism of TT homozygote frequency was significantly associated with patients undergoing coronary artery bypass surgery.
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INTRODUCTION
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Coronary artery disease (CAD) is a multifactorial disease influenced by environmental and genetic factors. The genes conferring susceptibility to CAD are largely unknown. The renin-angiotensin system (RAS) plays a pivotal role in the pathogenesis of cardiovascular disease.1 The reported association of insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene with myocardial infarction (MI) and CAD has thus generated continuing interest.2,3 However, these relationships could not be confirmed in other studies, and evaluation of ACE I/D polymorphism and the risk of CAD in the large prospective American Physicians Study also failed to confirm this association.4–6 Furthermore, there was no evidence in a case-controlled study or in a retrospective cohort study, based on the Copenhagen City Heart Study, that ACE I/D polymorphism played a role in the development of MI or CAD.7 A recent study in the Australian population showed a strong association of the ACE DD genotype with the presence of CAD or a history of MI, but not with the severity of CAD, in comparison to a cohort of healthy school children.8 In some studies, the presence or absence of CAD was not determined by angiography and, as a consequence, false-negative control populations might have been used. It is to be expected that comparative genetic studies may offer a new and informative approach to polygenic disease such as CAD; so the ACE gene is postulated to be a candidate gene affecting the important clinical problem of CAD. The purpose of this study was to determine the frequencies of ACE I/D, angiotensinogen (AGT)-M253T, and angiotensin II type 1 receptor (AT1R)-A1166C polymorphisms in Turkish patients undergoing coronary artery bypass grafting (CABG).
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PATIENTS AND METHODS
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This study was approved by the Medical Ethics Committee of the institution. Informed consent was obtained from each patient included in the study. From April 2001 until July 2001, 105 patients (80 male and 25 female; mean age, 65 ± 7 years) undergoing elective CABG were included (group 1). The control group consisted of 105 non-cardiac patients (78 male and 27 female; mean age, 63.9 ± 7 years) who had a cardiological check-up because of their medical history and their complaints, however subsequent angiography indicated normal coronary vessels. Patients with diabetes mellitus, history of coagulopathy or ongoing anticoagulation therapy, those receiving ACE inhibitor medication for more than 3 months (because therapy with ACE inhibitors could influence the progression of CAD and the development of MI), those with chronic obstructive pulmonary disease, renal insufficiency, liver disease, and any who smoked or used steroids, nonsteroidal anti-inflammatory drugs or aspirin within 5 days preoperatively were excluded from the study. Standard preoperative evaluation using electrocardiography, radiography, blood and urine biochemistry, hematologic tests, Doppler echocardiography, and coronary angiography was performed for each patient. Among the group 1 patients, 21 had single-vessel disease (20%) and the others had multivessel disease.
Genomic DNA was extracted from peripheral blood lymphocytes according to standard procedures. Polymerase chain reaction (PCR) amplifications of the related regions were carried out in 50 µL volumes of reaction mixture containing 75 mM Tris-HCl (pH 8.8), 200 mM (NH4)2SO4, 0.1% Tween-20, 2.0 mM MgCl2, 50 mM of each dNTP, 50 pmol of each set of specific primers, 1.25 unit of Taq DNA polymerase (DNAmp Ltd., UK), and 0.2–0.5 µg DNA sample. Thirty cycles of 94°C – 30 sec, 60°C – 45 sec (67°C – 35 sec for ACE insertion sequence specific region), and 68°C – 1 min were performed in an automated thermal cycler (MJR PTC-200; MJ Research, Watertown, MA, USA). The amplified products were analyzed by electrophoresis on 2% agarose gel.6 The ACE D and I alleles resulted in ~190 bp and ~490 bp amplicons, respectively. The DNA samples of DD genotypes were subjected to a second PCR amplification with an insertion-specific primer set.9 In the presence of I allele 335-bp, product was obtained, but there was no product in samples homozygous for DD. The T
C base substitution causes replacement by methionine of threonine in the protein. To determine the angiotensinogen M235T genotype, 104 bp PCR products were digested with MspI restriction enzyme. The homozygote C-type variant digestion products 73 and 31 bp were analyzed on 4% High Melt-Small Fragment AgarGel (CLP Inc., USA) electrophoresis. The heterozygotes show 3 bands, whereas homozygote T-type variant is not digested and shows only the 104 bp band. AT1R-A1166C genotypes were analyzed by DdeI restriction enzyme digestion, which cuts 850 bp PCR products into 600 and 250 bp long. An additional DdeI recognition site is created in the C-type variant, at nucleotide 1166, which is found in the 250 bp fragment. Homozygote CC, homozygote AA, and heterozygotes produce 3, 2, and 4 bands (600, 250, 140, and 110 bp long), respectively.
Data were expressed as mean ± standard error of mean. Differences between groups were determined using the Mann-Whitney U test. Genotypes and allele frequencies were evaluated by the chi-squared test. Allele frequencies were estimated by gene counting methods. A p-value < 0.05 was considered to be statistically significant.
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RESULTS
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Statistically significant differences were observed between the 2 groups in some of the demographic and hemodynamic parameters. Hypertension, hypercholesterolemia, angina, previous MI, ejection fraction, and left ventricular end-diastolic pressure ratios were different in the CABG group (Table 1
). There were 3 (2.9%) operative deaths in the CABG group. According to ACE I/D polymorphism, 36.3% of the patients in group 1 and 30.7% in group 2 were homozygous for the DD allele. The ID heterozygous frequency was 40.7% in group 1 and 47.2% in group 2. The II allele homozygous was 24.5% in group 1 and 20.8% in group 2. These differences were not statistically significant (Figure 1). AT1R-A1166C genotype polymorphism did not differ significantly between the two groups (Figure 2). The AA homozygote frequency was 59.8% in group 1 and 67.3% in group 2. The CC homozygote frequency was 5.9% in group 1 and 5.0% in group 2. The AC heterozygous frequency was 34.3% in group 1 and 27.7% in group 2. The results showed the M235T polymorphism to be heterogenous. The MM homozygote frequency was significantly higher in controls (72.3%), whereas 80% of the TT homozygote frequency (Figure 3) was in the surgical group ( p = 0.001).
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Figure 2. Distribution of angiotensin II type 1 receptor A1166C genotypes and allele frequencies among the groups ( p > 0.05).
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Figure 3. Distribution of angiotensinogen M235T genotypes and allele frequencies among the groups ( p = 0.001).
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DISCUSSION
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Human gene cloning for the ACE, AGT, and AT1R has led to several polymorphisms which may play important roles in the progression of cardiovascular diseases. The level of plasma ACE is strongly genetically determined.2 ACE is a ubiquitous carboxypeptidase. Among other actions, it catalyzes the conversion of angiotensin I to angiotensin II and the breakdown of bradykinin to kinin degradation products. Both angiotensin II and bradykinin are powerful vasoactive molecules with acute and chronic effects on the cardiovascular system. Therefore, with its pivotal role in two important cardiovascular hormonal regulatory systems, the RAS and the kallikrein-kinin system, ACE has an important impact on cardiovascular structure and function.10 Angiotensin II causes vasoconstriction, increases the proliferation of vascular smooth muscle cells, and causes hypertrophic changes of the vessel wall.11 In 1990, Rigat and colleagues12 described an I/D polymorphism of the ACE gene that accounted for 40% of the inter-individual variation in serum and cardiac ACE activity. ACE levels are highest in individuals who are homozygous for the D allele, lowest in those homozygous for the I allele, and intermediate in I/D heterozygous individuals.13
In a retrospective study in France and Ulster, men with the DD genotype were found to have 1.34 times the risk of MI compared to those with the DI or II genotype; moreover, in a low-risk subgroup of these patients, those with the DD genotype had an even higher risk of MI (odds ratio, 3.2).14 This association has been attributed to increased formation of angiotensin II in individuals who carry the ACE D allele. Most of the population studies have attributed increased ACE plasma level associated with the D allele to cardiovascular risk. However, there is a small increase in the risk of MI, but not the severity of atherosclerosis as in individuals with homozygous DD allele.6,11,15 In this study, the frequency of the ACE I/D allele was not found to be different in the CABG and control subjects. The significant difference in the D allele was not considered to be very important unless it is confirmed by other tests, adding to the results of previous studies.
Several studies demonstrated a significant association between CAD and M235T polymorphism.16,17 However, in 12 combined studies, the T allele was not associated with atherosclerotic cardiovascular complications.18 The pooled excess risk was 17% in TT. The MM homozygotes did not exceed the threshold of statistical significance in 9 reports on CAD, including MI. In our study, MM homozygote frequency was significantly higher in the controls, and TT homozygote frequency was higher in the CABG group. This study in Turkish CABG patients indicates that the M235T polymorphism results were very important for the TT homozygous genotype.
At least two studies observed synergistic effects of ACE I/D and AT1R-A1166C genotype polymorphisms on the risk of MI or CAD.19,20 In a case-controlled study of 613 MI cases and 723 age-matched controls, the odds ratio associated with ACE DD genotype was 1.05 in subjects without the C allele of the AT1R gene, 1.52 in AC heterozygous, and 3.95 in CC homozygous. The results of our study indicate that AT1R-A1166C genotype polymorphism is not significantly different in CABG patients. Our results reveal that although there are no significant differences in ACE I/D and AT1R-A1166C genotype polymorphisms among the groups, the M235T polymorphism of TT homozygote frequency is significantly associated with Turkish patients who require CABG surgery. It is believed that further studies with close perioperative evaluation of patients with CAD undergoing CABG are required, not only to detect whether these polymorphisms have any effect on the progression of the CAD, but also the influence of ACE inhibitor treatment with regard to the individual ACE polymorphism.
Presented at the 12th Annual Meeting of the Asian Society for Cardiovascular Surgery , Istanbul, Turkey, April 18–22, 2004.
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