Association between the eNOS (Glu298Asp) and the RAS genes polymorphisms and premature coronary artery disease in a Turkish population

Clinica Chimica Acta 351 (2005) 87 – 94 www.elsevier.com/locate/clinchim

Association between the eNOS (Glu298Asp) and the RAS genes polymorphisms and premature coronary artery disease in a Turkish population Afig Berdelia, Cevad Sekurib,*, F. Sirri Camc, Ertugrul Ercand, Abdi Sagcane, Istemihan Tengizd, Erhan Eserf, Mustafa AkVng a Ege University, Faculty of Medicine, Department of Pediatri, Izmir, Turkey Celal Bayar University, Faculty of Medicine, Department of Cardiology, Manisa, Turkey c Celal Bayar University, Faculty of Medicine, Department of Medical Biology and Genetics, Manisa, Turkey d Central Hospital, Izmir, Turkey e Atakalp Hospital, Izmir, Turkey f Celal Bayar University, Faculty Of Medicine, Department of Public Health, Manisa, Turkey g Ege University, Faculty of Medicine, Department of Cardiology, Izmir, Turkey b

Received 1 March 2004; received in revised form 23 July 2004; accepted 4 August 2004

Abstract Background: The renin–angiotensin system (RAS) and endothelial nitric oxide (NO) affect the pathogenesis of atherosclerosis and prognosis of coronary artery disease (CAD). Previous epidemiologic data suggested that genetic factors are more likely to affect young rather than old people. Our objective was to investigate the association between the polymorphisms of eNOS (Glu298Asp) and the RAS genes and premature CAD in a Turkish population. Methods: A total of 115 Turkish patients with premature CAD and 83 controls were included in the study. ACE I/D, AT1R A/C, AGT T/M and eNOS Glu298Asp gene polymorphisms were analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Results: It was found that increased premature CAD risk is associated with higher frequencies of the ACE DD [OR: 2.600 (CI 95% 1.395–4.847, p=0.002)], AGT MM [OR=2.407 (CI 95% 1.267–4.573, p=0.007)] and eNOS 894TT [OR=17.000 (CI 95% 3.952–73.125, pb0.001)] genotypes. Carriers of ACE DD+eNOS 894TT ( p=0.002), AGT MM+eNOS 894TT ( p=0.001), AT1R AA+eNOS 894TT and AT1R non-AA+eNOS 894TT ( p=0.002) genotypes were significantly associated with the risk of premature CAD.

* Corresponding author. Erzene Mah. 113 sok. No: 31, Camyuva, 9 Bornova-Izmir, Turkey. Tel.: +90 232 375 66 48; fax: +90 232 386 70 71. E-mail address: [email protected] (C. Sekuri). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.08.015

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Conclusions: This study indicates a synergistic contribution of RAS genes (ACE I/D, AGT T/M, AT1R T/C) and eNOS Glu298Asp polymorphisms to the development of the premature CAD. D 2004 Elsevier B.V. All rights reserved. Keywords: Renin–angiotensin system; Endothelial nitric oxide synthase; Gene polymorphism; Premature coronary artery disease

1. Introduction It is clear that endothelial cells play critical roles in the progression and clinical manifestation of the atherosclerotic process [1]. The endothelium is thought to link the fibrinolytic system and the renin–angiotensin system (RAS) [2]. RAS is an endocrine system that is important in controlling the circulatory homeostasis. The angiotensin-converting enzyme (ACE), the key enzyme in the renin– angiotensin system, is released from the cell membrane. It converts the angiotensin I to the angiotensin II and inactivates the bradykinin and tachykinins; thereby regulating its effects on fibrinolysis, platelet activation and aggregation [3]. The human ACE gene is found on chromosome 17, and a polymorphism consisting of the presence or the absence of a 287-base pair (bp) Alu repeat has been identified [4]. There are three genotypes in this polymorphism: DD and II homozygotes, and ID heterozygote. The II genotype is associated with lower tissue and plasma levels of ACE, whereas the DD genotype is associated with higher ACE levels [5]. Other major components of the RAS are angiotensinogen which is the precursor of angiotensin I and the angiotensin II type-1 receptor (AT1R) which is the cell surface receptor for angiotensin II [6]. Previous studies have shown that the DD genotype of the ACE gene is associated with development of left ventricular hypertrophy, myocardial infarction (MI) and remodeling. The TT genotype of the AGT gene is an independent risk factor for coronary heart disease [7], and individuals with AT1R-CC genotype would be at increased risk for MI [8]. One of the most important products of endothelial cells is the nitric oxide (NO), a major mediator of endothelium dependent vasodilation made in endothelial cells from l-arginine through the action of the homodimeric enzyme endothelial nitric oxide synthase (eNOS). Also, NO modulates the action of angiotensin II in cardiovascular tissue, and angioten-

sin II modulates NO synthesis [9,10]. In addition to vasodilation, NO inhibits platelet aggregation, proliferation of vascular smooth muscle cells, leukocyte adhesion to endothelial cells [11,12], vascular smooth muscle cell migration and growth [13,14], and the oxidation of atherogenic low-density lipoproteins (LDLs) [15]. Therefore, eNOS may have an important atheroprotective role through these mechanisms [16]. The gene that encodes eNOS is located on chromosome 7q35–36 and consists of 26 exons with a total size of 21 kb [17]. The eNOS gene is expressionally and functionally regulated through multiple regulatory steps [18,19]. It depicts several polymorphisms [20], some of which bear functional consequences. Endothelial NOS gene Glu298Asp polymorphism (G894T) in exon 7 was reported to be associated with essential hypertension [21], acute MI [22,23] coronary artery spasm [24] and coronary artery disease (CAD) [16]. Because the Glu298Asp mutation occurs in exon and results in an amino acid change, it could have functional effects. Tesauro et al. reported that eNOS isoforms are processed differentially depending on the presence of aspartate or glutamate at position 298. Thus, the 894 G/T polymorphism is not silent, and may possibly be perceived by an endogenous protease as a target for cleavage [25]. Also, this mutation may result in a decrease in eNOS activity by inducing a conformational change from helix to tight turn in the eNOS [26]. The aim of the present study was to investigate the association between the eNOS (Glu298Asp) and the RAS genes polymorphisms and premature CAD in a Turkish population.

2. Materials and methods The eNOS and the RAS genes polymorphisms were analysed in 115 unrelated Turkish patients with a diagnosis of premature CAD who were admitted to the Cardiology Departments of three centers in the

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Eagean region, West of Turkey. Eigthy-three consecutive healthy control subjects without a history of CAD were recruited among those who presented to the check-up clinics in the three hospitals during November and December 2002. The study was approved by the Ethics Committee of the Celal Bayar University Hospital, and all subjects gave written informed consents. The inclusion criteria for the patients were: (1) age at the time of CAD diagnosis was 55 years or less in men and 65 years or less in women; (2) stenosis of at least 50% in a major coronary artery, or one of their branches, as determined by angiography. The extent of the disease was defined as the number of arteries with at least 50% stenosis in a single or multiple vessels. The coronary angiography was performed by Judkin’s method at the Catheterization Laboratories. Diagnosis of MI was confirmed using patients’ medical records using the WHO criteria [27] based on symptoms, elevation in cardiac enzymes or electrocardiographic changes. All patients were informed about coronary risk factors such as diabetes mellitus, hypertension, hypercholesterolemia and cigarette smoking. Triglycerides, total cholesterol, high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) levels were measured by the conventional methods of clinical chemistry. Arterial hypertension was defined as systolic blood pressure equal to or greater than 140 mm Hg and/or diastolic blood pressure equal to or greater than 90 mm Hg in more than one instance. Patients with a history of diabetes or basal glycemia greater than 120 mg/dl were defined as diabetic. Smoking habit from all the subjects was defined as a daily intake of more than five cigarettes. Body mass index was defined as increased when greater than 25 kg/m2. A familial history of CAD was determined by interviewing patients and controls. 2.1. Genetic analysis Genomic DNA was extracted from 200 Al of EDTA-anticoagulated peripheral blood leucocytes using the QUIAmp Blood Kit (Quiagen, Ontaria Canada, Cat. no. 51106). Amplification of DNA for genotyping, the ACE I/D polymorphism was carried out by polymerase chain reaction (PCR) in a final volume of 15 Al containing 200 AM dNTP

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mix, 1.5 mM MgCl2, 1 Buffer, 1 unit of AmpliTaq polymerase (PE Applied Biosystems) and 10 pmol of each primer. The primers used to encompass the polymorphic region of the ACE were 5V-CTGGAGACCACTCCCATCCTTTCT-3V and 5V-ATGTGGCCATCACATTCGTCAGAT-3V [4]. DNA is amplified for 35 cycles, each cycle comprising denaturation at 94 8C for 30 s, annealing at 50 8C for 30 s, extension at 72 8C for 1 min with final extension time of 7 min. The initial denaturation stage was carried out at 95 8C for 5 min. The PCR products are separated on 2.5% agarose gel and identified by ethidium-bromide staining. Each DD genotype was confirmed through a second PCR with primers specific for the insertion sequence [28]. To analyse AGT M235T polymorphism, genomic DNA was PCR-amplified with primers 5V-GATGCGCACAAGGTCCTG-3Vand 5V-CAGGGTGCTGTCCACACTGGCTCGC-3V, respectively [29], and the AT1R A1166C polymorphism was analysed with primers 5V-GCAGCACTTCACTACCAAATGAT-3V and 5V-TGTTCTTCGAGCAGCCGT-3V as previously described [30]. Endothelial NOS genotyping for the Glu298Asp mutation was performed as described by Hingorani et al. [16]. The primers used were 5V-CATGAGGCTCAGCCCCAGAAC-3V (forward) and 5V-AGTCAATCCCTTTGGTGCTCAC-3V(reverse). DNA is amplified for 30 cycles, each cycle comprising denaturation at 95 8C for 1 min, annealing at 60 8C for 1 min, extension at 70 8C for 1 min with final extension time of 5 min at 70 8C. The initial denaturation stage was carried out at 95 8C for 5 min. PCR products were digested with the restriction enzyme MboI at 37 8C for 16 h. In the presence of T at nucleotide 894 which corresponds to Asp 298, the 206-bp PCR product is cleaved into two fragments of 119 and 87 bp. The PCR products are separated on 2.5% agarose gel and identified by ethidium-bromide staining. 2.2. Statistical analysis Statistical analysis was carried out using the SPSS v10.0 for Windows statistical package (SPSS, Chicago, IL, USA). Variables are presented as meanFS.D. A p value of 0.05 or less was considered significant. Univariate analysis was performed by chi-

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square, odds ratios (ORs) and Mann–Whitney U test. The Hardy–Weinberg’s equilibrium for genotype distribution was estimated by the chi-square test. The researchers also calculated the genotypic OR for premature CAD and their 95% confidence interval (CI) with two-tailed p values, using multiple logistic regression analysis adjusted for family history of CAD, diabetes, hypertension, total cholesterol, triglycerides, HDL-C, LDL-C, BMI and smoking habit. The Backward Wald method was used for logistic regression analysis.

3. Results Table 1 shows the clinical characteristics of the 115 patients with the premature CAD and 83 subjects of the control group. The patient group had a higher prevalence of hypertension, diabetes, smoking and the family history of premature CAD, compared to the control group. The patients also had higher BMI, total cholesterol, LDL-C and triglycerides levels ( pb0.05). According to the achieved results, family history, hypertension, diabetes, smoking, obesity, high total cholesterol, LDL-C and triglycerides levels, low HDLC, ACE DD, AGT MM and eNOS TT genotypes were increased risk factors of premature CAD (Table 2). The distribution of the ACE I/D, AGT T/M, AT1R A/C and eNOS T/G genotypes and allele frequencies in patients and controls were compatible with Hardy– Weinberg’s equilibrium as presented in Table 3 (ACE

Table 1 The demographic characteristic and distribution of risk factors in patients and control subjects Age (years) Male/female Family history of CAD Hypertension Smoking habit (z5 per day) BMI (kg/m2) Diabetes Triglycerides (mg/dl) Total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl)

Patients (n=115)

Controls (n=83)

48.1F7.9 89/26 49 (43%) 44 (38%) 77 (67%) 26.3F2.5 24 (21%) 179.5F76.5 202.3F34.6 41.7F5.3 131.3F27.1

44.6F1.4 65/18 12 (15%) 14 (17%) 33 (40%) 24.6F2.8 3 (4%) 156.0F62.9 186.1F29.8 43.2F5.0 122.8F25.3

BMI: body mass index; CAD: coronary heart disease; HDL: highdensity lipoprotein; LDL: low-density lipoprotein.

Table 2 Odds ratios of traditional cardiovascular risk factors and, ACE DD, AGT MM, AT1R AA and eNOS TT genotypes in premature CAD Gender Hypertension Diabetes Smoking BMI (kg/m2) Family history of premature CAD Total cholesterol LDL cholesterol HDL cholesterol Triglycerides AGT MM AT1R AA eNOS TT ACE DD

OR (CI 95%)

p

0.903 (0.459–1.776) 3.054 (1.537–6.069) 7.033(2.041–24.236) 2.953 (1.645–5.300) 3.311 (1.815–6.042) 4.393 (2.149–8.977) 3.359 (1.598–7.059) 2.088 (0.996–4.376) 2.066 (1.042–4.094) 2.621 (1.462–4.697) 2.407 (1.267–4.573) 0.636 (0.360–1.125) 17.00(3.952–73.125) 2.600 (1.395–4.847)

0.76 0.001 b0.001 b0.001 b0.001 b0.001 0.001 0.048 0.036 0.001 0.007 0.119 b0.001 0.002

BMI: body mass index; CAD: coronary heart disease; HDL: highdensity lipoprotein; LDL: low-density lipoprotein.

I/D: p=0.002, v 2=9.30; AT1R A/C p=0.119, v 2=2.42; AGT T/M: p=0.007, v 2=7.39; eNOS T/G: p=0.001, v 2=23.89). The ACE D, AGT M, eNOS 894T alleles

Table 3 Genotype distribution according to the presence of premature CAD Patients (n=115)

Controls (n=83)

ACE genotypes DD ID II Allele frequencies I/D

52 (45.2%) 46 (40.0%) 17 (14.8%) 0.348/0.652

20 (24.1%) 40 (48.2%) 23 (27.7%) 0.518/0.482

v 2=9.30 p=0.002

AT1R genotypes AA AC CC Allele frequencies A/C

55 (47.8%) 50 (43.5%) 10 (8.7%) 0.695/0.305

49 (59.0%) 30 (36.2%) 4 (4.8%) 0.771/0.229

v 2=2.42 p=0.119

AGT genotypes MM MT TT Allele frequencies M/T

46 (40.0%) 42 (36.5%) 27 (23.5%) 0.583/0.417

18 (21.7%) 47 (56.7%) 18 (21.6%) 0.500/0.500

v 2=7.39 p=0.007

eNOS Glu298Asp genotypes TT 34 (29.5%) TG 37 (32.2%) GG 44 (38.3%) Allele frequencies T/G 0.456/0.544

2 (2.4%) 24 (28.9%) 57 (68.7%) 0.168/0.832

v 2=23.89 pb0.001

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were significantly more common in the patient group compared to the control group (Table 3). There was a significant association between the DD genotype and the premature CAD [ACE DD vs. ID and II; OR=2.600 (CI 95% 1.395–4.847, p=0.002)]. Also, it was observed that increased premature CAD risk was associated with higher frequencies of the AGT MM genotype in patients compared to the controls [AGT MM vs. TT and MT, OR=2.407 (CI 95% 1.267–4.573, p=0.007)]. We did not find a significant association between the AT1R AA genotype and the premature CAD [AT1R AA vs. AC and CC, OR=0.636 (CI 95% 0.360–1.125, p=0.119)]. There was a significant association between the TT genotype and the premature CAD [ eNOS TT vs. TG and GG; OR=17.000 (CI 95% 3.952–73.125, p=0.001)] (Table 2). 3.1. Combined analysis of RAS and eNOS genes The frequencies of ACE DD+eNOS TT; AGT MM+eNOS TT; AT1R AA+eNOS TT; AT1R nonAA+eNOS TT individuals were significantly increased ( p=0.002; p=0.001; p=0.001; p=0.002) in patients (13/ 115; 17/115; 17/115; 18/115, respectively) compared to the controls (0/83; 1/83; 0/83; 2/83, respectively). In the logistic regression analysis, the carriers of ACE DD+eNOS TT ( p=0.002), AGT MM+eNOS TT ( p=0.001), AT1R AA+eNOS TT ( p=0.001) and AT1R non-AA+eNOS TT ( p=0.002) genotypes were significantly associated with the risk of premature CAD (Table 4). In addition, there was not any association between the subjects with ACE-DD+eNOS non-TT and ACE non-DD+eNOS TT genotypes and premaTable 4 Combined analysis of RAS and eNOS genotypes a

ACE DD–eNOS 894TT AT1R AA–eNOS 894TTa AGT MM–eNOS 894TT ACE non-DD–eNOS 894TT AT1R non-AA–eNOS 894TT AGT non-MM–eNOS 894TT ACE DD–eNOS non-TT AT1R AA–eNOS non-TT ACE non-DD–eNOS non-TT AGT non-MM–eNOS non-TT a

OR (CI 95%)

p

0.551 0.541 14.22 0.258 7.515 12.30 1.555 0.360 0.208 0.245

0.002 b0.001 0.001 b0.001 0.002 0.003 0.172 0.001 b0.001 b0.001

No case in the control group.

(0.484–0.628) (0.474–0.619) (1.85–109.17) (0.139–0.479) (1.693–33.359) (1.591–95.087) (0.823–2.935) (0.201–0.645) (0.112–0.385) (0.130–0.460)

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Table 5 Adjusteda odds ratios for cardiovascular risk factors in premature coronary artery disease Risk factors

OR (95% CI)

p

Family history of premature CAD Diabetes mellitus Smoking BMI (kg/m2) Total cholesterol HDL cholesterol eNOS 894TT ACE–DD

4.132 (1.66–10.29) 7.996 (1.747–36.60) 3.005 (1.381–6.540) 3.478 (1.586–7.626) 4.875 (1.826–13.02) 3.357 (1.325–8.502) 20.08(4.083–98.752) 5.058 (2.156–11.86)

0.002 0.007 0.006 0.002 0.002 0.011 b0.001 b0.001

BMI: body mass index, CAD: coronary artery disease, HDL: highdensity lipoprotein. a Logistic regression.

ture CAD. On the other hand, we did not find any interaction among RAS genotypes when we performed stratified analysis among both cases and controls ( pN0.05). By the multiple regression analysis, it became clear that family history of premature CAD, smoking, diabetes, obesity, total and HDL-cholesterol, ACE DD [OR=5.058 (CI 95% 2.156–11.864, p=0.001)] and eNOS 894TT [OR=20.080 (CI 95% 4.083– 98.752, p=0.001)] genotypes were independent risk factors of CAD (Table 5).

4. Discussion Cardiovascular disease is the leading cause of mortality in Turkey as in the world. The most important recent epidemiological investigation on Turkish population, the TEKHARF study (Turkish Adults Risk Factors Study), has shown that the incidence of CAD in young individuals is common in this population [31]. Coronary artery disease has multifactorial etiologies resulting from the interaction of genetic predisposition and environmental risk factors. In such a manner, cardiovascular risk factors, such as diabetes mellitus, dyslipidemia, hypertension and obesity may have both genetic and environmental components [32]. The premature CAD is known to have a particularly strong genetic component. Previous epidemiologic data have suggested that genetic factors are more likely to affect young rather than old people [33]. The renin–angiotensin system may play an important role in the progression of atherosclerosis and its

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clinical manifestations. In cases with atherosclerosis, endothelial dysfunction contributes to the abnormal vasomotor activities. In addition, endothelial dysfunction suggests that reduced bioavailability of NO may even have prognostic implications and contribute to the progression of coronary atherosclerosis due to impaired endothelium-mediated vasomotion [34]. In the present study, we demonstrated an increased premature CAD risk associated with higher frequencies of the ACE DD, AGT MM and eNOS 894TT genotypes. In addition, such results have suggested that family history of the premature CAD, smoking, diabetes, total and HDL-cholesterol, obesity, ACE DD and eNOS 894TT genotpyes were independent risk factors of the development of premature CAD. Fatini et al. [35] reported that the ACE DD and AT1R CC genotypes were independent predictors of the development of CAD in Italian population. In contrast to this study, our results did not support the interaction of AT1R CC genotypes and the premature CAD. In another study, Petrovic et al. [36] showed that ACE DD genotype is an independent risk factor for premature MI and they found evidence of an interactive effect on MI risk among genotypes of RAS. On the other hand, Ferna´ndez-Arca´s et al. [37] reported that the AGT M allele is an independent risk factor for MI. In agreement with these findings, these two studies revealed that ACE DD and AGT MM polymorphisms are associated with CAD. In the CORGENE study, a cross-sectional study involving 463 Caucasians who underwent coronary angiography for established or suspected CAD (156 patients with previous MI, 307 patients without MI), Jeunemaitre et al. [38] demonstrated no significant association between these ACE, AGT, and AGTR1 polymorphisms and the clinical characteristics of MI and non-MI patients. Ichihara et al.’s [39] study on 327 Japanese patients with CAD and 352 matched controls did not find an association between AGT genotype and CAD. Several studies have shown that the polymorphisms of the eNOS gene are associated with hemodynamics. Shimasaki et al. [22] reported that the frequency of the Asp298 allele was significantly higher in MI patients compared to the controls in Japan. Similarly, Hingorani et al. [16] found that the Glu298Asp polymorphism is associated with angiographically demonstrated CAD and MI in patients in

the United Kingdom. In contrast to the results of the present and the aforementioned studies, Aras et al. [40] indicated that eNOS gene Glu298Asp polymorphism is not an independent risk factor for CAD and MI in Turkish population. Karvonen et al. [41] reported that the Glu298Asp variant of the eNOS gene does not seem to be a major risk factor for cardiovascular alterations in the general population. Nassar et al. [42] found that the frequencies of the Glu298Glu, Glu298Asp and Asp298Asp genotypes were similar in patients and controls (34.9%, 46.3% and 18.8% for G1 and 29.3%, 56% and 14.7% for G2, respectively, p=0.29). Their study does not support that the eNOS Asp298 allele contributes to the development of premature CAD. In the current study, we have investigated the association between the eNOS Glu298Asp and the RAS genes polymorphisms, and premature CAD in a Turkish population. In a combined analysis of RAS and eNOS genes, carriers of ACE DD+eNOS 894TT, AGT MM+eNOS 894TT, AT1R AA+eNOS 894TT and AT1R non-AA+eNOS 894TT genotypes were significantly associated with higher risk of premature CAD. The synergistic effect between the ACE DD, AGT MM, AT1R non-AA and eNOS 894TT genotypes might be a result of the fact that RAS and eNOS interact in the cardiovascular system. Especially, the carriers who are ACE DD and the eNOS 894TT could display the highest endothelial dysfunction and a higher risk of development of premature CAD. The interaction between RAS genes and other gene variants were studied by Batalla et al. [43], and they suggested that a synergistic effect between the APOE and AGT polymorphisms and early MI. Nakagami et al. investigated the ACE and eNOS 4a/b gene polymorphism and combined analysis of these genes in CAD patients. In this study, the ACE genotype was associated with CAD, while the eNOS 4a/b genotype was not. A combined analysis of these genes did not enhance the predictability of CAD [44]. In a study conducted in Spain, Alvarez et al. [45] reported that there is a synergistic relation between the eNOS 786CC and the ACE DD genotypes in the risk of developing early CAD. To the best of our knowledge, no previous reports have investigated the association between the eNOS Glu298Asp and the RAS genes polymorphisms and premature CAD.

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In multiple regression analysis, family history of premature CAD, smoking, diabetes, obesity, total and HDL-cholesterol, ACE DD and eNOS 894TT genotypes were independent risk factors of CAD. This finding suggested that these gene polymorphisms might be important predictors for the premature CAD. In conclusion, this study indicates a synergistic contribution of RAS genes (ACE I/D, AGT T/M and AT1R A/C) and eNOS Glu298Asp polymorphisms to the development of the premature CAD. This finding is potentially important, but it requires further confirmation in other populations.

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