Endothelial nitric oxide synthase gene polymorphisms -786T >C and 894G >Tin coronary artery bypass graft surgery patients

Atherosclerotic coronary artery disease (CAD) is the most common form of cardiovascular disease. CAD is a multifactorial disease of complex aetiology, influenced by both genetic and environmental determinants [1]. Several risk factors for CAD have been established, such as hypertension, diabetes mellitus, lipid disorders and smoking. To comprehend fully the aetiology of CAD, special attention has been given to genetic factors. Although many candidate gene studies have been published in the past decade, the genetic background of CAD remains poorly defined [2].

Numerous studies suggest that endothelial dysfunction plays a crucial role in the initiation and progression of atherosclerosis, the fundamental pathology of CAD. A key element in endothelial dysfunction is the loss of endothelium-derived nitric oxide (NO). NO has multiple roles in cardiovascular physiology and pathophysiology, including the regulation of vasomotor tone, cell adhesion to the endothelium, inhibition of platelet aggregation and vascular smooth muscle cell proliferation and limitation of atherogenic low-density lipoprotein oxidation [3].

Endothelial nitric oxide synthase (eNOS) catalyses the biosynthesis of NO. Two functional polymorphisms have been identified in the eNOS gene: a single nucleotide polymorphism (SNP) in position 786 of the 5' flanking region of the eNOS gene (-786T >C, rs2070744) that reduces eNOS gene promoter activity by approximately 50 per cent [4] and a 894G >T polymorphism leading to amino acid substitution at position 298 (Glu298Asp, rs1799983)[5], which results in enhanced proteolytic cleavage of the mature enzyme. These two polymorphisms are not in linkage disequilibrium, according to data derived from the HapMap database (http://www.hapmap.org). The dysregulation of eNOS caused by these gene polymorphisms leads to diminished NO production and is thought to contribute to the pathogenesis of several cardiovascular diseases, including myocardial infarction (MI) and CAD.

Candidates for coronary artery bypass grafting (CABG) represent a group of patients with well-documented and severe CAD. Selection for CABG surgery verifies the critical endpoint of severe atherosclerotic disease and constitutes a good model for its study. To the best of our knowledge, there are no data on the presence of eNOS gene polymorphisms exclusively in cohorts of CABG patients. The aim of the present study was to evaluate the association of two eNOS gene polymorphisms with CAD in CABG patients compared with non-atherosclerotic individuals. We further analysed the association of the examined polymorphisms in CABG patients with the presence of previous MI, hypertension and diabetes or smoking status.

Approximately 3 ml blood was collected in tubes containing ethylene diamine tetraacetic acid (EDTA). Genomic DNA was extracted from peripheral blood leucocytes by the Puregene^® DNA Purification System (Gentra, Minneapolis, MN, USA), according to the manufacturer's instructions.

Candidates for CABG represent a group of patients with well-documented and severe CAD. Selection for CABG surgery verifies the critical endpoint of severe atherosclerotic disease and constitutes a good model for its study. Few genetic association studies have examined CABG patients as a separate, clinically defined model of severe atherosclerosis. In these patients, we investigated the association of -786T >C and 894G >T polymorphisms of the eNOS gene with CAD. Our results show that eNOS -786C and 894T variants are not independent predisposing factors for severe CAD. We found, however, an interaction between the 894T allele and smoking in CABG patients. This gene-smoking interaction supports the idea that smoking enhanced endothelial damage in the presence of specific gene variants, such as 894T, of the eNOS gene in our study.

Several studies have investigated the possible association of eNOS gene polymorphisms with CAD, with conflicting results. Similar to our results, some studies have reported a lack of association of eNOS -786T >C and 894G >T polymorphisms with CAD in other populations of Caucasian or Asian origin [11–16]. There have, however, been a few studies showing a positive association of eNOS gene polymorphisms with CAD [17, 18]. Two meta-analyses conducted by Casas et al. showed that the eNOS -786C allele is associated with slightly increased risk for CAD [19] and that homozygosity for the 894T allele is associated with a moderately increased risk for ischaemic heart disease [20]. Both studies, however, noted the substantial heterogeneity of included studies, and in the last updated meta-analysis they highlighted that they observed statistical evidence of small-study bias in studies of the G894T and -786T >C polymorphisms [19]. Our approach was to use a patient population with well-documented, severe athero-sclerotic disease, assessed by experienced clinicians.

It has been suggested that interaction of eNOS gene polymorphisms with environmental factors such as smoking may influence the late stages of CAD and also alter the early pathogenesis of atherosclerosis by modulation of endothelial function [21]. In the present study, we found that the eNOS 894T allele had a higher prevalence in current/past smoker than never smoker CABG patients. Smoking induces oxidative stress, thereby suppressing eNOS activity, and also increases peroxynitrate formation, causing a further suppression of eNOS activity, because peroxynitrate inhibits eNOS production [22]. It also changes the function of eNOS, from catalysing the synthesis of NO to catalysing the synthesis of oxygen radicals, finally it decreases pteridine-tetrahydrobiopterin levels, causing the uncoupling of eNOS [3]. Since both polymorphisms in the eNOS gene cause loss of function and reduction in eNOS enzymatic activity, NO levels in subjects carrying these polymorphisms are even lower, resulting in further endothelial damage. The interaction of smoking with the G894T polymorphism was present solely in CABG patients. A possible explanation for this finding derives from recent demonstration that there is a relationship between endothelial dysfunction and increased Rho kinase (ROCK) activity in smokers and that the activation of ROCK contributes to the reduced NO bioavailability in humans with atherosclerosis via eNOS suppression [23, 24]. Thus, the interaction we found between the eNOS 894T allele and smoking in CABG patients might also be mediated through ROCK activation. Despite the number of studies examining the association between eNOS gene polymorphisms and CAD, there are only a few reports on their interaction with pro-atherogenic risk factors such as smoking. For the 894T allele, it has been shown that carriage of this allele increases the likelihood of smoking-associated endothelial dysfunction [21] and of angiographically proven CAD, [25] a result which is in agreement with our finding of a higher frequency of the 894T allele in current/past smoker than never smoker CABG patients.

Our study has certain limitations. CABG patients and non-CAD controls were matched for sex but significantly differed in age, lipid profile and incidence of hypertension and diabetes. Firstly, CABG patients were younger than non-CAD controls. This is a welcome difference, however, since it indicates that control subjects have been disease-free for a longer time and, presumably, the possibility of them developing CAD complications in the future is reduced. Differences in lipid profile could possibly be attributed to the fact that most CABG patients were receiving medications (mostly statins) which affect lipid profile, while most control subjects did not receive any such drugs. Finally, the incidence of hypertension and diabetes was lower in non-CAD controls, as it was not possible to find enough subjects with this condition among our control population, CAD being the main complication of these pathological conditions. We found no association between any of the studied polymorphisms and diabetes or hypertension in the analysis performed in all subjects, however (data not shown).

In conclusion, we studied two polymorphisms in the eNOS gene in Greek CABG patients, which have both been subject to controversy concerning their relevance to CAD. Our results show that in this patient population, the -786C and 894T alleles of the eNOS gene are not associated with CAD. The eNOS 894G >T polymorphism is associated with CAD in current/past smoker patients, however; a finding which suggests that this polymorphism may be associated with differences in the response of the endothelium to smoking status.