METHODS

Study population

We studied 201 patients consecutively admitted to our institution with angiographically proven coronary artery disease (more than 50% stenosis affecting at least one vessel) and 114 control subjects recruited from patients admitted for valve replacement, in whom angiographic examination excluded the presence of coronary artery disease. All the patients and controls were interviewed and data on smoking habits, hypertension, diabetes, dyslipidaemia, and family history of coronary artery disease were recorded. Informed consent was obtained from all patients and controls, as required by our ethics committee.

For coronary risk factors, the following definitions were used: subjects were defined as hypertensive if their blood pressure was > 140/90 mm Hg or if they were receiving any antihypertensive treatment; those with a history of diabetes or who were receiving any antidiabetic drugs were considered to be diabetic; those with a total plasma cholesterol concentration of ≥ 5.70 mmol/l or a triglyceride concentration of ≥ 2.26 mmol/l, or who were receiving lipid lowering drugs, were considered dyslipidaemic. Smoking history was coded as never, ex (for least six months), and current. A positive family history was the presence of a first degree relative with coronary artery disease at the age of ≤ 55 years for men and ≤ 65 years for women.

Angiographic study

All patients and controls underwent coronary angiography. Coronary stenosis was considered significant in the presence of a luminal diameter narrowing of ≥ 50% of at least one epicardial coronary artery. The severity of coronary artery disease was expressed by the number of affected vessels (one, two, or three vessel disease) and also by means of the Duke scoring system¹³—a prognostic index that includes the number of diseased major vessels, the presence of left main coronary artery disease, the percentage narrowing of the major vessels, and involvement of the left anterior descending coronary artery, particularly when the proximal segment shows severe stenosis (≥ 95%). The Duke score ranges from 0–100 (0 = no disease, 100 = the most severe disease).

Analysis of Glu²⁹⁸→ Asp polymorphism on exon 7 of eNOS gene

Genomic DNA was extracted from samples of whole blood by standard methods.¹⁴ The coding sequence variant was a G→T substitution at position 894 in exon 7 which determines the Glu to Asp amino acid substitution (in codon 298) in the mature eNOS protein. According to previously described procedure, genotyping of all subjects was performed by polymerase chain reaction amplification of exon 7 with the primers 5`-CATGAGGCTCAGCCCCAGAAC-3` (sense) and 5`-AGTCAATCCCTTTGGTGCTCAC-3` (antisense) followed by MboI restriction enzyme digestion for 16 hours at 37°C.¹² In the presence of a T at nucleotide 894 which corresponds to Asp 298, the 206 base pair (bp) polymerase chain reaction product is cleaved into two fragments of 119 and 87 bp. The products of the digestion process were highlighted by electrophoresis on a 1.5% agarose gel.

Statistical analysis

All statistical analyses were conducted with the Statview statistical package, version 5.0.1 (SAS Institute). Data are expressed as mean (SEM). Differences between the means of the two continuous variables were evaluated by Student's t test. Differences in non-continuous variables, genotype distribution, and the Hardy–Weinberg equilibrium were tested by χ² analysis. One way analysis of variance was used to analyse the relations between genotypes and the general characteristics and severity of coronary artery disease, in terms of the number of diseased vessels and the Duke score. Logistic regression analysis was used to assess the independent effect of each risk factor on the occurrence of coronary artery disease. A probability value of p < 0.05 was considered to be significant.

RESULTS

Comparison of the two study groups

Demographic and clinical characteristics of patients and controls were given in table 1. The prevalence of atherogenic risk factors (including age, sex, hypertension, diabetes, cigarette smoking, dyslipidaemia, and a family history of coronary artery disease) was significantly higher in the patient group.

Distribution of the Glu²⁹⁸→Asp polymorphism of the eNOS gene

Although the distribution of genotypes in both coronary artery disease cases and controls satisfied the Hardy–Weinberg equilibrium, the Glu²⁹⁸→Asp polymorphism in exon 7 of the eNOS gene was significantly associated with the presence of coronary artery disease in our patients (table 2). The proportion of Asp²⁹⁸ homozygotes was 15.9% in the coronary artery disease cases and 6.1% in control subjects (χ² = 8.589, p = 0.0136).

In comparison with Glu²⁹⁸ homozygotes, the odds ratio (OR) for coronary artery disease associated with the Asp/Asp genotype was 2.4 (table 3). Because Glu/Asp carriers were not at increased risk of coronary artery disease (OR 0.66, 95% confidence interval 0.4 to 1.1), the odds ratios associated with the Asp/Asp genotype were therefore computed with Glu/Glu + Glu/Asp considered as the reference group. As shown in table 3, in comparison with individuals homozygous and heterozygous for Glu²⁹⁸, the odds ratio for coronary artery disease among Asp/Asp carries was 2.9. Multivariate analysis showed that the Asp/Asp genotype was an independent risk factor for coronary artery disease (table 4).

Glu²⁹⁸→Asp polymorphism of the eNOS gene and severity of coronary artery disease

The relations between several variables and Glu²⁹⁸→Asp genotype were studied in coronary artery disease patients (table 5). We did not find any association between the genotype and hypertension, dyslipidaemia, diabetes, smoking status, or family history of coronary artery disease.

We found a significant association between the Glu²⁹⁸→Asp variant of the eNOS gene and the severity of the disease in terms of the number of stenosed vessels. eNOS Glu²⁹⁸→Asp polymorphism was also associated with the extent and the severity of coronary artery disease evaluated by the Duke scoring system (mean (SEM): 56.1 (3.1) for Asp/Asp, 46.7 (2.0) for Glu/Asp, and 46.1 (1.9) for Glu/Glu; p = 0.02) (fig 1).

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Figure 1

Glu²⁹⁸→Asp polymorphism of the eNOS gene and the severity of coronary artery disease assessed by the Duke scoring system.

DISCUSSION

We report the association between the common Glu²⁹⁸→Asp polymorphism of the eNOS gene and the occurrence of coronary artery disease in the Italian population. We found an excess of homozygosity for the Asp²⁹⁸ variant among coronary artery disease cases compared with controls, and the risk of developing coronary artery disease was about threefold higher for Asp²⁹⁸ homozygotes than in persons with a Glu²⁹⁸ allele in the eNOS gene. Multivariate analysis showed that this association was independent of other factors related to coronary artery disease risk. Our study provides the first evidence for an association between Glu²⁹⁸→Asp polymorphism and the extent and severity of coronary artery disease.

There was the expected clustering of coronary artery disease risk factors among cases. However, we did not detect an association between the Glu²⁹⁸→Asp polymorphism and any of these possibly confounding variables.

Glu²⁹⁸→Asp polymorphism of the eNOS gene and risk of atherosclerosis related disease

Up to now, Glu²⁹⁸→Asp polymorphism of the eNOS gene has been linked to an increased risk of stroke, coronary atherosclerosis, and acute myocardial infarction.^15,16 Previous studies from Japan and the UK have already suggested a role for Glu²⁹⁸→Asp polymorphism in the development of coronary atherosclerosis, with the excess risk being confined to Asp²⁹⁸ homozygosity,^12,17 as in our study. These studies, however, also showed that the genotype frequency of Glu²⁹⁸→Asp polymorphism can vary substantially among different populations. For example, while in the Japanese population this polymorphism could only explain a small part of the genetic susceptibility to acute myocardial infarction, as the Asp/Asp genotype was present in only five of 226 patients (2.2%),¹⁷ in the UK the Asp/Asp genotype was found in 107 of 298 patients (35%) and in 45 of 249 patients (18.1%) with coronary artery disease and acute myocardial infarction, respectively.¹² Our genotype frequencies in both cases and controls were in agreement with those recently reported by Lembo and colleagues among Italian subjects who had atherosclerotic plaques on their carotid arteries and control subjects without carotid plaques.¹⁵ In that study, Asp²⁹⁸ homozygosity was an independent risk factor for the development of carotid plaques, but no association was found between Glu²⁹⁸→Asp polymorphism of the eNOS gene and the degree of involvement of the various segments of the carotid arteries. Nevertheless, it is important to emphasise that some groups have failed to find any relation between the Asp²⁹⁸ variant and the risk of atherosclerosis.^18–21 Elbaz and colleagues even found a significant association between the Glu/Glu genotype and the risk of brain infarction.²¹

Functional significance of the Glu²⁹⁸→Asp polymorphism of the eNOS gene

The fact that in our study the risk for coronary artery disease was confined to Asp²⁹⁸ homozygotes suggested that homozygosity for aspartic acid in position 298 could produce a reduction in the amount or enzymatic activity of eNOS. If the Asp²⁹⁸ variant of eNOS leads to altered NO synthesis, this could provide a mechanism for both its increased prevalence among patients with coronary artery disease and its association with the extent and severity of the disease. Several experimental studies have in fact shown that a reduction in the endothelial production of NO appears to be critical for the evolution, progression, and clinical manifestations of the atherosclerotic process.^22,23 It is noteworthy that in our study an association was observed between the Glu²⁹⁸→Asp polymorphism and both the number of diseased vessels and the Duke scoring system—a prognostic index that also includes the percentage narrowing of the major vessels and the anatomical localisation of the stenosis. Thus our data suggested that the Asp²⁹⁸ variant of eNOS could contribute to the generalised architecture of the vessels. This hypothesis is supported by in vivo evidence that eNOS mutant mice display a paradoxical increase in wall thickness accompanied by a hyperplastic response of the arterial wall after carotid artery ligation.²⁴ This suggests that a primary defect in the NOS/NO pathway may promote abnormal remodelling and pathological changes in vessel wall morphology associated with atherosclerosis. Thus it is possible that in the process of atherosclerotic remodelling of adult human vessels, alterations in NO production resulting from the substitution of Glu²⁹⁸ with Asp²⁹⁸ could have a major impact on smooth muscle cell migration and proliferation. Indeed, despite the apparently conservative nature of the Glu→Asp amino acid substitution, there is evidence that a similar substitution in other enzymes can alter protein function.²⁵ Recently, Philip and colleagues showed that enhanced vascular responsiveness to phenylephrine was associated with the Asp²⁹⁸ allele,²⁶ and a significant reduction in endothelium dependent dilatation has been correlated with Glu²⁹⁸→Asp polymorphism in early pregnancy.²⁷ On the other hand, a study by Schneider and associates appeared to exclude an effect of eNOS Glu²⁹⁸→Asp polymorphism on endothelium dependent vasodilatation.²⁸ Furthermore, Sofowora and colleagues reported that Asp²⁹⁸ homozygotes excreted significantly less nitrate/nitrite than Glu²⁹⁸ homozygotes without affecting nitric oxide mediated vascular responses.²⁹ Thus the impact of Glu²⁹⁸→Asp polymorphism on endothelial NO function remains to be clarified.

Finally, genetic contributions of eNOS to plasma NO metabolite concentrations have been recently reported. The mutant allele of the T⁷⁸⁶→C polymorphism in the promoter region of the eNOS gene has been associated with a reduced promoter activity and endothelial synthesis of NO, both of which predispose to coronary spasm in the Japanese population.³⁰ Moreover, Yoshimura and colleagues found that the T⁷⁸⁶→C variant is in linkage disequilibrium with the eNOS gene intron 4b/a polymorphism,³¹ which is also reported to be involved in smoking dependent coronary artery disease,³² suggesting that the T⁷⁸⁶→C mutation underlies the functional characteristics of the intron 4a allele. It is not known whether the associations that we reported in this study reflect a hypofunctional enzyme or linkage disequilibrium between the Glu²⁹⁸→Asp polymorphism and another functional variant within the eNOS gene or another gene.

Conclusions

We observed that the Glu²⁹⁸→Asp polymorphism of the eNOS gene is associated with the presence, extent, and severity of angiographically assessed coronary artery disease in the Italian population. Because this polymorphism has also recently been associated with carotid atheroma in the same population, more studies are needed to investigate whether the Glu²⁹⁸→Asp polymorphism of the eNOS could represent an useful genetic marker to identify individuals prone to the development of atherosclerotic diseases.