Cardiovascular diseases, particularly coronary artery diseases, represent a major cause of mortality worldwide, with an increasing burden in low- and middle-income countries such as Benin [1][2]. Coronary artery diseases result from a complex atherosclerotic process influenced by both environmental and genetic factors [3]. Among the molecular factors involved in the pathophysiology, nitric oxide (NO) plays a crucial role as a vasodilator, anti-inflammatory, and antithrombotic mediator [4]. The synthesis of nitric oxide in the endothelium is catalyzed by the enzyme endothelial nitric oxide synthase (eNOS), encoded by the NOS3 gene [5]. The eNOS gene, located on chromosome 7 (7q35-36), spans 21 kb and comprises 26 exons and 25 introns [6]. Approximately 10 polymorphic loci are distributed throughout the promoter, exonic, and intronic regions of the eNOS gene [7]. A common mutation resulting in amino acid substitutions in mature proteins is the G894T or Glu298Asp (rs1799983) variant, where a guanine-to-thymine substitution leads to the replacement of glutamic acid (Glu) with aspartic acid (Asp) at position 298 in exon 7 [7]. This genetic variant has been associated with impaired NO production and endothelial dysfunction, potentially contributing to the development of atherosclerosis and coronary artery diseases [7][8]. Studies conducted across diverse populations have demonstrated significant associations between the G894T polymorphism and coronary artery disease risk, although findings sometimes vary across ethnic and geographic contexts [9][10]. In sub-Saharan Africa, data regarding this association remain scarce, particularly in Benin, where cardiovascular diseases are increasingly prevalent yet remain understudied from a molecular perspective. This study aims to investigate the contribution of the eNOS G894T polymorphism to coronary artery disease in the Beninese population of Cotonou, with the objective of enhancing understanding of underlying genetic factors.

This study investigated cases with coronary artery disease and control subjects with a mean age of 53.09 ± 11.03 years. Our findings are consistent with research by Arafa et al. [13] in 2018, who reported a mean age of 55 ± 9.9 years in an Egyptian case-control population, though their study examined both eNOS and Apo E genes as potential risk factors for coronary artery disease. Similarly, work by Younan et al. in 2014 identified a mean age of 51.71 ± 8.97 years in an Egyptian population with coronary and carotid atherosclerotic disease [14].

The results of our study revealed a high frequency of metabolic disorders among patients compared to controls, particularly regarding glycemic parameters, lipid profile, and renal function. Fasting hyperglycemia was observed in 28.9% of cases versus 2.6% of controls, suggesting a higher prevalence of glucose regulation disorders among cases. Hyperglycemia is a well-established risk factor for renal and cardiovascular complications and is frequently associated with lipid abnormalities such as hypertriglyceridemia and reduced HDL cholesterol [15]. Regarding lipid profile, one case exhibited total hypercholesterolemia compared to two controls. HDL-C hypocholesterolemia was significantly more prevalent in cases (47.4%) than controls (21.1%), consistent with the typical dyslipidemic phenotype observed in patients with chronic kidney disease (CKD) [15][16]. This observation aligns with work by Shiqi et al. in 2021, which demonstrated that an elevated TG/HDL-C ratio serves as an independent predictor of declining glomerular filtration rate (GFR), particularly in elderly subjects [17]. Abnormal serum creatinine levels were present in 34.2% of cases compared to only 2.6% of controls, indicating moderate to severe renal impairment. This trend was confirmed by GFR assessment using the MDRD formula, which revealed reduced GFR in 52.6% of cases versus a single control (2.6%). These findings are consistent with existing literature. In a retrospective cohort study, Nagayama et al. observed that lipid disorders, particularly elevated triglycerides and reduced HDL-C, are significantly associated with progressive GFR decline [18]. Furthermore, work by James demonstrated that the TG/HDL-C ratio is a robust predictive marker for chronic kidney disease risk, independent of other metabolic factors [19]. This association may be explained by mechanisms of lipotoxicity, endothelial dysfunction, and chronic inflammation observed in contexts of lipid abnormalities and metabolic stress [20].

The human endothelial nitric oxide synthase (eNOS) gene is considered one of the genes associated with cardiovascular conditions. In the present study, homozygosity for the Asp298 allele of the eNOS gene was identified in 7.89% of cases versus 2.63% of controls. Although this difference was not statistically significant due to the relatively small sample size, it suggests a potential association between the Asp/Asp genotype and coronary artery disease occurrence. This finding aligns with several previous studies indicating that the Asp298 variant of the eNOS gene may impair endothelial nitric oxide synthase activity, thereby reducing nitric oxide (NO) production—a crucial vasodilator regulating vascular tone and endothelial function [7][8][21]. NO plays an important protective role in cardiovascular function by inhibiting platelet aggregation, vascular smooth muscle cell proliferation, and leukocyte adhesion to the vascular wall [22]. Decreased NO production may promote prothrombotic phenomena, vascular inflammation, or endothelial dysfunction, all of which are implicated in the pathophysiology of cardiovascular diseases [4][23].

Recent and classic studies show that the Glu298Asp (rs1799983) polymorphism of the eNOS (NOS3) gene plays an important role in the pathophysiology of coronary artery disease. In a study conducted in 2024 by Vecoli1 et al. involving 506 patients suspected of having stable coronary artery disease, the Asp298 allele was independently associated with induced myocardial ischemia, even after adjusting for obstructive coronary lesions and other traditional risk factors [24]. This is consistent with the results found in our study, except that the sample size in our study is not significant. In terms of coronary heart disease (CHD), a meta-analysis of 39 case-control studies (7489 cases, 7051 controls) showed that the Asp genotype (vs. Glu) is significantly associated with increased susceptibility to CHD [25]. Earlier, another meta-analysis of 26 studies (23,028 subjects) had already shown that Asp/Asp homozygosity moderately increases the risk of ischemic heart disease (OR ≈ 1.31) [26].

A major meta-analysis combining 39 studies involving over 10,000 coronary artery disease cases revealed a significant association between Asp/Asp homozygosity and increased coronary artery disease risk (OR = 1.31; 95% CI: 1.13 - 1.51) [24][25]. Similarly, a study conducted in Greece demonstrated that individuals carrying the Asp298 allele had an almost twofold increased risk of myocardial infarction [26][27]. However, the low frequency of this homozygosity in both groups, along with the modest difference between cases and controls, requires cautious interpretation. Moreover, studies conducted in different populations have reported variable frequencies of this mutation, suggesting potential influences of ethnic or environmental factors on the distribution of eNOS gene polymorphisms [28][29]. However, the low frequency of this homozygosity in both groups, as well as the modest difference between cases and controls, must be interpreted with caution.

It is important to note that multifactorial diseases such as coronary artery diseases sometimes exhibit variable phenotypes across populations [30]. It should be noted that Fathelbab’s 2025 study of 100 patients with acute coronary syndrome who underwent coronary angiography showed that the c.894G>T polymorphism of eNOS is associated with an increased risk of coronary artery dilation but does not appear to contribute significantly to atherosclerotic coronary artery disease [31]. Although several studies have reported an association between eNOS gene polymorphism and coronary artery diseases, other investigations, particularly those by Hibi et al. and Yoon et al., found no relationship between this polymorphism and coronary artery disease risk [32][33].

It is important to note that multifactorial diseases such as coronary artery disease sometimes present a variable phenotype from one population to another [30]. Although several studies have reported a link between eNOS gene polymorphism and coronary artery disease, other studies, notably those conducted by Hibi et al. and Yoon et al., have reported that there is no link between this polymorphism and the risk of developing coronary artery disease [28][34]. This perfectly illustrates the multifactorial nature of coronary artery disease, with interaction between genetic susceptibility factors and environmental factors.

Our study warrants further investigation on a larger sample size in order to analyze the combined effect of this mutation with other cardiovascular risk factors (hypertension, smoking, diabetes, dyslipidemia, etc.). In addition, a functional analysis could be considered in order to better understand the pathophysiological impact of the Asp/Asp genotype in the Beninese population.

This study has several limitations. The relatively small sample size limits statistical power and may restrict the detection of modest associations. Selection bias cannot be ruled out due to the participant recruitment methods. Finally, despite adjusting for relevant variables, residual confounding factors remain possible, particularly those that were not measured.

This study demonstrated a high prevalence of metabolic abnormalities among cases, including fasting hyperglycemia, low HDL cholesterol, moderate hypertriglyceridemia, and impaired renal function. The findings suggest a potential association between Asp298 allele homozygosity of the eNOS gene and cardiovascular disease development. Although the frequencies observed in our sample do not establish statistical significance, they align with existing literature that attributes a potential role to this polymorphism in reducing nitric oxide bioavailability and promoting endothelial dysfunction. However, ethnic variability and genotype-environment interactions may influence both the distribution and clinical expression of this variant. Future large-scale studies incorporating combined genetic approaches and precise clinical data are necessary to confirm these observations and further elucidate the contribution of the Asp298 polymorphism to cardiovascular risk in the Beninese population.