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J Am Coll Cardiol, 2003; 41:930-937, doi:10.1016/S0735-1097(02)03012-7 © 2003 by the American College of Cardiology Foundation |
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* Servizio di Emodinamica and Divisione di Cardiologia Ospedale di Cittadella, University of Padova, Padova, Italy
Department of Clinical and Experimental Medicine, Clinica Medica 4, University of Padova, Padova, Italy
Manuscript received March 13, 2002; revised manuscript received October 7, 2002, accepted November 22, 2002.
* Reprint requests and correspondence: Prof. Gian Paolo Rossi, Department of Clinical and Experimental Medicine, Clinica Medica 4 University Hospital, via Giustiniani, 2, 35126 Padova, Italy.
gianpaolo.rossi{at}unipd.it
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Abstract |
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BACKGROUND: Endothelial dysfunction foretells cardiovascular events and can be genetically determined.
METHODS: We genotyped for the promoter (T-786C) and exon 7 (Glu298Asp, G894T) polymorphisms in 1,225 subjects; 1,106 were consecutive patients undergoing coronary angiography and 119 control subjects without any cardiovascular risk factors. Genotyping was performed with melting curve analysis of polymerase chain reaction products from allele-specific acceptor and donor probes that were 5'- and 3'-end labeled with LCRed640 and fluorescein, respectively; CAD was assessed by quantitative coronary angiography. We performed multiple logistic regression analysis for the effect of the T-786C, the missense Glu298Asp variant, and other coronary risk factors on two- and three-vessel CAD.
RESULTS: The overall genotype distribution of T-786C (CC = 17.7%, CT = 40.4%, and TT = 41.9%) and Glu298Asp (GG = 43.3%, GT = 37.0%, and TT = 19.7%) was consistent with the Hardy-Weinberg equilibrium. The regression analysis showed that the T-786C, but not the missense Glu298Asp variant, significantly predicted CAD, independent of other risk factors. Compared with TT homozygous, subjects carrying the C allele had a significant (p = 0.002) increase in the odds ratio of harboring two- or three-vessel CAD of 1.672 (95% confidence interval, 1.062 to 2.527). A subgroup analysis confirmed this effect of the T-786C polymorphism in men (p = 0.007), cigarette smokers (p = 0.001), subjects older than 60 years of age (p = 0.007), with hypercholesterolemia (p = 0.011), low high-density lipoprotein cholesterol (p = 0.006), and overweight or with obesity (p = 0.041).
CONCLUSIONS: The C allele at the T-786C endothelial nitric oxide synthase polymorphism is associated with a higher risk of multivessel CAD in Caucasians.
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An impaired endothelium-dependent vasodilation (4) has been consistently reported in conditions that are associated with accelerated atherosclerosis, such as arterial hypertension, cigarette smoking, diabetes mellitus, hypercholesterolemia, hyperhomocyst(e)inemia, and aging (5,6). Furthermore, a blunted endothelium-dependent vasodilation was found to predict CV events independently of the common risk factors (7) and, therefore, might be a precursor of CAD.
Nitric oxide (NO) is 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). In addition to its key role in the regulation of vascular tone and blood pressure (8,9), NO is thought to be involved in atherogenesis (8,10,11), development of heart failure, and congenital septal defects and vascular remodeling, as shown by data in mice lacking the eNOS gene (10,12). In fact, NO blunts the activity of the nuclear factor-kappaB family of transcription factors (13,14) and, thereby, can prevent the endothelial expression of adhesion molecules and inflammatory cytokines, which are instrumental for the triggering of atherogenesis (15). Accordingly, a genetically determined impaired availability of NO might be a major risk factor for development of CAD.
The eNOS gene (16) is expressionally and functionally regulated through multiple regulatory steps (14,17) and entails several polymorphisms (18), some of which bear functional consequences. Therefore, this gene is a likely candidate for impaired endothelium-dependent vasodilation and CAD. Some eNOS gene polymorphisms have indeed been linked to CV phenotypes and events (19–28), and two of them to coronary spasm (23,29). Of the latter, the T-786C is located in the promoter region, which is the most important for the regulation of the transcription rate of the eNOS gene (29). The mutant C allele implies a blunted transcription rate and was found to be far more common in Japanese patients with vasospastic angina than in controls (29); furthermore, we recently reported that this allele was associated with a blunted forearm vasodilation in response to acetylcholine in hypertensive patients (30).
Another eNOS polymorphism, the GAG to GAT substitution in exon 7, results in the replacement of glutamate by aspartate (Glu298Asp, G894T), which might impair the function of eNOS by causing a tight turn of the alpha-helix (23), as suggested by some studies (31–34).
As both polymorphisms were found to be associated with an altered coronary vasomotor reactivity and with an impaired endothelium-dependent vasodilation (29,30), we hypothesized that they might play a role for the triggering of atherogenesis and CAD occurrence. However, only limited and conflicting results were available so far to support this hypothesis. No association of the T-786C polymorphism with CV events (35) and of the Glu298Asp polymorphism with MI was seen in Caucasians (36). However, both these studies relied on CV events and not on coronary angiography for the assessment of CAD.
Thus, within the Genetic and ENvironmental factors In Coronary Atherosclerosis (GENICA) study, a large prospective investigation of the genetic determinants of CAD, which was performed in consecutive Caucasian patients undergoing coronary angiography, we sought to determine if the T-786C and the Glu298Asp polymorphisms predicted multivessel CAD.
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Methods |
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50%) CAD was eventually ruled out by coronary angiography; group 2 comprised 119 consecutive healthy normotensive blood donors enrolled at the local blood bank during the same period. In these latter subjects, it was unethical to perform coronary angiography to rule out the presence of asymptomatic CAD. Therefore, the following inclusion criteria were used: negative family history of CAD, MI, and stroke; nonsmoking status; absence of hypercholesterolemia, hypertriglyceridemia, diabetes mellitus, all defined as specified in the following text. Based on available data from epidemiologic and family studies, a cohort fulfilling these criteria is expected to have a very low prevalence of asymptomatic CAD.
Demographic and clinical measurements.
A standard questionnaire was used to carefully ascertain medical history in all participants (transient ischemic attack, stroke, angina, MI, coronary artery bypass, percutaneous transluminal coronary angioplasty, renal failure, peripheral artery disease, and history of vascular surgical interventions), smoking habits, presence/absence of hypertension, diabetes, hypercholesterolemia, hypertriglyceridemia, and current medications. Body mass index was calculated as weight/height2 (kg/m2). Patients were classified into three groups: current smokers, nonsmokers, and ex-smokers (who had stopped smoking for at least one year). Diabetes mellitus (type I or II) was defined as a previous diagnosis of the disease, history of antidiabetic medications, or plasma fasting levels of glucose 126 mg/dl (7.0 mmol/l) on at least two occasions. Impaired glucose tolerance was defined as plasma fasting levels of glucose ranging between 110 to 126 mg/dl (6.1 to 6.9 mmol/l) (38). Hypercholesterolemia was defined as a low-density lipoprotein (LDL) cholesterol 100 mg/dl according to the National Cholesterol Education Program guidelines for patients with CAD (39); hypertriglyceridemia was defined as plasma fasting levels 134 mg/dl, that is, higher than the 95th percentile value of our group 1 control subjects. Blood pressure was measured by mercury sphygmomanometer using Korotkoff phase V for diastolic, according to the World Health Organization guidelines. Hypertension was defined as systolic pressure 140 mm Hg, and/or diastolic pressure 90 mm Hg (40), or use of any antihypertensive agents.
Coronary angiography.
Angiography was carried out and evaluated by experienced cardiologists who were blinded to patients’ genotype. The severity of CAD was determined by the number of significantly stenosed coronary arteries (41). Patients were classified as follows: code 1 = normal vessels; code 2 = <50% stenosis; code 3, 4, and 5 = stenosis 50% in one, two, or three major coronary arteries, respectively.
Laboratory measurements. Each patient was studied between 8:30 and 12:00 A.M. Before coronary angiography, blood samples were taken from the femoral artery and were immediately put on ice and centrifuged at 3,000x g (at 4°C for 10 min). Total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, glycemia, sodium, potassium, blood urea nitrogen, and creatinine levels were measured with conventional methods.
Extraction of deoxyribonucleic acid and eNOS genotyping. The blood was stored at –20°C until deoxyribonucleic acid was extracted using a commercially available kit (DNA Blood Extraction Fast KIT, Analitica Srl., Padova, Italy). Details of the methodology used for genotyping at each polymorphism, including oligonucleotides serving as amplification primers and fluorescence resonance energy transfer probes, were recently reported (30).
Statistical analysis.
One-way analysis of variance followed by Bonferroni’s post-hoc test was used to compare quantitative variables between CAD patients and control subjects. Chi-squared analysis was used to compare the frequencies of the categorical coronary risk factors and of eNOS gene variants between the CAD and the control groups. Consistency of the genotype frequencies with the Hardy-Weinberg equilibrium was also tested by chi-squared analysis. To identify the independent risk factors for CAD, we performed multiple logistic regression analysis for the effect of the T-786C, the missense Glu298Asp variant, and the other coronary risk factors. To this end, independent variables were coded as dummy variables as follows: age, 0 for <60 years, 1 for 60 years; gender, 0 for female, 1 for male; body mass index, 0 for <26 kg/m2, 1 for 26 kg/m2; hypertension, diabetes mellitus, and LDL cholesterol, 100 mg/dl, 0 for absence, 1 for presence; cigarette smoking, 0 for nonsmoker, 1 for current or ex-smoker; the T-786C, 0 for CC, 1 for CT, and 2 for TT; missense Glu298Asp variant, 0 for GG, 1 for TG, and 2 for TT (42). Because the aim of this study was to identify predictors of multivessel CAD, the dependent variable was coded as follows: 0 for normal coronary artery and for stenosis <50%; 1 for stenosis 50% in two or three major epicardial vessels. The forward stepwise selection (Wald criterion) was used. Odds ratios (approximating to relative risks) were calculated as a measure of the association between the eNOS genotype and the CAD phenotype, with the effect of the mutant allele C for the T-786C polymorphism assumed to be dominant (TT vs. CT and CC) or additive (TT and CT combined vs. CC). Similarly, for the Glu298Asp polymorphism, the effect of the mutant allele was investigated according to a dominant (TT and TG combined vs. GG) or additive (TT vs. GT and GG) model of inheritance. For each odds ratio, we calculated two-tailed p values and 95% confidence intervals. Statistical significance was defined as p < 0.05. All analyses were performed using SPSS 10.0 for Windows (SPSS Italy Inc., Bologna, Italy).
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50%) stenosis in one, two, or three major epicardial vessels, respectively. The eNOS polymorphism genotypes were available in 1,106 (87%) of all patients. A preliminary regression analysis on all subjects revealed a borderline significant (p = 0.034) association between the T-786C eNOS genotype and one-, two-, or three-vessel CAD. Therefore, to maximize contrast at the phenotype level, it was decided to combine two- and three-vessel CAD patients, as CAD group, and normal vessel and <50% stenosis as control group (group 1); thus, the CAD group and group 1 comprised 749 and 330 patients, respectively. Of the latter, 21% were healthy individuals, 39% had valvular heart disease, 15% had cardiomyopathy (13.7% dilated, 1.3% hypertrophic), 7% had hypertensive heart disease, and 18% had ischemic cardiomyopathy. The latter diagnosis was based on resting and stress electrocardiogram and on results of stress myocardial scintigraphy that showed inducible ischemia. The demographic and clinical characteristics of the CAD patients and of group 1 and 2 (119 subjects) controls are shown in Table 1. The CAD patients were older, had higher plasma glucose and triglycerides, and lower HDL cholesterol than control subjects. There were more males in CAD patients than in group 1 (p < 0.001), but not group 2 control subjects. Compared with both control groups, more CAD patients had diabetes mellitus, hypercholesterolemia (both p < 0.001), hypertriglyceridemia (p < 0.001), and were smokers or ex-smokers (p < 0.0001). Compared with controls, the CAD patients more commonly reported a history of myocardial infarction (p < 0.0001), coronary artery bypass surgery (p < 0.0001), percutaneous transluminal coronary angioplasty (p < 0.0001), peripheral artery disease (p < 0.001), vascular surgery (p < 0.001), but not of transient ischemic attack or stroke.
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We investigated whether there was a linkage between the T-786C and the Glu298Asp polymorphism, by Kendall’s tau b and Spearman’s correlation analysis, and found a highly significant (p < 0.001) relationship between the two polymorphisms (Kendall’s tau b = 0.303 ± 0.025; Spearman’s correlation = 0.330 ± 0.027).
Association between T-786C, Glu298Asp, eNOS variants, and CAD. We found with the logistic regression analysis that the T-786C polymorphism significantly predicted CAD. In Table 2 we ranked the variables that entered into the model according to the value of the Wald coefficient, which estimates the usefulness of the parameter to the model. Besides confirming that male gender, age, hypertriglyceridemia, and cigarette smoking were significant predictors of CAD, we found that the C allele also significantly predicted CAD, being as useful as cigarette smoking to this end. At variance, other "classical" CV risk factors, such as arterial hypertension, diabetes mellitus, obesity, and elevated LDL cholesterol, did not remain in the model and, therefore, were less useful than the C allele as predictors of CAD. Subjects carrying the C allele, either homozygous or heterozygous, had a 69.2% increased risk of having significant multivessel CAD. We observed similar results when the C allele was examined according to an additive model, that is, CC homozygous versus CT and TT (Table 2). In contrast, no effect of the Glu298Asp missense variant on CAD was detected. The association of the C allele with multivessel CAD was even stronger (p < 0.001) when the 18% of group 1 control patients with ischemic cardiomyopathy were excluded from the analysis.
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Discussion |
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Reporter gene studies showed that the T-786C substitution in the promoter region of eNOS reduced by 50% the rate of transcription of eNOS, both under baseline conditions and in response to hypoxia (29), and was associated with decreased serum levels of nitrite/nitrate (43). These effects might depend upon the fact that a mutant allele can bind the replication protein A1, which acts as a gene repressor protein (43). The mutant C allele was found to be quite rare in healthy Japanese subjects (3%) and more common (15%) in patients with coronary spasm, thus indicating a highly significant (p < 0.0001) association with an altered coronary vasomotor responses in this ethnic group (44). We found the C allele in 38% of our cohort, which is consistent with the 37.8% recently reported in British middle-age men from primary care practices (35). Thus, this allele seems to be much more common in Caucasian than in Japanese individuals, albeit the underlying reasons remain hypothetical.
To our knowledge there was no data supporting the relevance of the T-786C polymorphism in non-Japanese populations, because no association with CAD was found in one study (35). Our results filled this gap by showing that the C allele was associated with CAD in Caucasian subjects. The logistic regression (Table 2) showed that subjects who carried the C allele exhibited a 73% increase of the risk of harboring significant multivessel coronary artery narrowing. This significant association does not imply, per se, a causal relationship; neither does it clarify the basic mechanisms. However, it is worth pointing out that, in a large series of mild-to-moderate uncomplicated essential hypertensive patients who underwent an infusion of the endothelium-dependent vasodilator acetylcholine into the brachial artery, the TT homozygous subjects exhibited a significantly enhanced vasodilation, as compared with CT and CC individuals (30). Thus, it would appear that, in a condition that implies enhanced oxidative stress, such as arterial hypertension, the genetic predisposition to generate less NO can become apparent and, in the long run, might be detrimental and result in susceptibility to atherogenesis.
This contention is further supported by the present subgroup analysis. We found that the concomitance of at least one major CV risk factor further enhanced the additional risk associated with the C allele and that the concurrence of multiple risk factors markedly enhanced the nefarious effects of the C allele (Fig. 1). Thus, under most conditions that imply oxidative stress, such as aging, smoking, hypercholesterolemia, male gender (lack of estrogens), and overweight or obesity, the genetic predisposition to generate less NO associated with the C allele could contribute to decreasing the bioavailability of NO and, thus, to the triggering of coronary atherogenesis.
Recent studies showed an association of the T (mutant) allele at the Glu298Asp polymorphism with human phenotypes that can be relevant for CV disease, including vascular adaptation to pregnancy (34), endothelial response to smoking and n-3 fatty acids (32), high-altitude pulmonary edema (33), and a lower mean age at end-stage renal disease in patients with autosomal dominant polycystic kidney disease (31). We found that frequency of the T (mutant) allele was 38%, that is, much higher than in Japanese (6.7% to 10.7% in a study [42], 9.8% to 25.6% in another study [33]), but similar to that observed in Caucasians from continental Europe (31% to 34%) (31,45), albeit lower than in U.K. patients (68.8% and 52.2% in control subjects and CAD patients, respectively) (36). We found no evidence for an association of this polymorphism with severity of CAD in agreement with earlier and more recent studies (26,35,42,45,46) but in contrast to reports of an association of the Glu298 with MI (36,42). This might not be surprising because: 1) the replacement of aspartate for glutamate is deemed to be a conservative one, and 2) recombinant eNOS Asp298 and Glu298 enzymes showed no discernible difference in the Michaelis constant (Km) or the maximum velocity (Vmax) (18). However, at variance with this latter finding, it was found that the enzymatic activity of eNOS was systematically decreased in renal artery specimens of patients harboring the T allele (31). Thus, either the functional impact of this polymorphism can differ between the renal and the coronary artery bed or this polymorphism could only act as a marker for a functional locus elsewhere in the gene, as suggested by the observation of a linkage disequilibrium with other eNOS polymorphisms (35). Of much interest, we found evidence of a significant linkage between T-786C and the Glu298Asp polymorphism, at variance with a previous report (47). Thus, available conflicting results on the association of the Glu298Asp polymorphism with CAD or CV events might be explained on the basis of a variable association of this variant with the T-786C polymorphism in different populations. However, individuals carrying the T allele (TT and TG) were found to have significantly higher pressor responses to phenylephrine than GG homozygous, thus suggesting that the T allele might increase vascular reactivity (48), possibly because the Asp298, but not the Glu298, variant of eNOS is susceptible to cleavage (49). Whatever the functional relevance of the missense Glu298Asp variant might be, our results in individuals from southern Europe, consistent with earlier studies (26,35,42), do not support the contention that Glu298Asp polymorphism is, per se, strongly associated with CAD in Caucasian patients.
This cross-sectional study comprised exclusively Caucasians, and, therefore, our findings might not apply to other ethnic groups or indicate a prognostic value of the T-786C polymorphism, which might be revealed by an ongoing follow-up study. Furthermore, only a minority of our study population was women, while estrogens are known to enhance NO availability and to account for protection of fertile women from CV events. We could confirm the usefulness of the T-786C polymorphism as a predictor of CAD in men but not in women; however, we cannot exclude the possibility that this study was underpowered to detect an association of the T-786C eNOS polymorphism with CAD in both genders.
In conclusion, we have found that a functional polymorphism of the eNOS gene, the T-786C, which is located in the promoter region, was significantly associated with severe CAD, independent of the other common CV risk factors in Caucasian patients.
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Footnotes |
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References |
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C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999;99:2864–2870Asp) is a major risk factor for coronary artery disease in the UK. Circulation. 1999;100:1515–1520C mutation associated with coronary spastic angina. Hum Mol Genet. 2000;9:2629–2637C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with myocardial infarction, especially without coronary organic stenosis. Am J Cardiol. 2000;86:628–634[CrossRef][Medline]
C and missense Glu298Asp variants. J Investig Med. 2000;48:367–374[Medline]
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