Endothelial Nitric Oxide Synthase Glu298Asp Research Articles

Polymorphisms of endothelial nitric oxide synthase and angiotensin-converting enzyme in systemic sclerosis

A previously published study in this journal reported significant associations of angiotensin-converting enzyme insertion/deletion (ACE I/D) and endothelial nitric oxide synthase (eNOS) Glu298Asp (G894T) polymorphisms with systemic sclerosis.1

Endothelial nitric oxide synthase Glu298Asp gene polymorphism, blood pressure and hypertension in a general population sample

The Glu298Asp (E/D) polymorphism of the endothelial nitric oxide synthase (eNOS) gene has been related to hypertension. Since several studies have produced contradictory results, this issue is still subject to ongoing debate. We investigated the association of the eNOS E298D polymorphism with hypertension and with blood pressure (BP) in a large population-based sample of Caucasian ethnicity. Cross-sectional study in a random sample of the general population. The eNOS E298D polymorphism was determined by 5'-exonuclease assay among 4219 participants aged 20-79 years of the Study of Health in Pomerania (SHIP). The percentages of the EE298, ED298 and DD298 genotypes were 49.2, 42.0 and 8.8%, respectively. The D allele frequencies did not differ between the groups of normotensive and hypertensive subjects (29.7 versus 29.9%, P = 0.812). Similarly, no association could be established between E298D genotype and prevalent hypertension, neither for D allele carriership (multivariate odds ratio 0.97, 95% confidence interval 0.83-1.12) nor for DD homozygosity (multivariate odds ratio 1.10, 95% confidence interval 0.84-1.43). Likewise, genotype groups did not differ as to the distribution of systolic (ANCOVA P = 0.917) or diastolic BP values (ANCOVA P = 0.657). Nearly identical results were obtained if the analyses were repeated sex-specifically or if subjects on antihypertensive medication were excluded. In a population-based cohort of Caucasians covering a broad age range, the eNOS E298D polymorphism is neither associated with prevalent hypertension nor with systolic or diastolic BP. These results do not support the hypothesis that the E298D polymorphism contributes to the genetic susceptibility to hypertension.

Association of the endothelial nitric oxide synthase (NOS3) Glu298Asp gene polymorphism with the risk of Alzheimer's disease

Sirs: Molecular epidemiological studies have presented contradictory results concerning a potential role of the NOS3 Glu298Asp gene polymorphism in Alzheimer’s disease (AD). Single studies may have been underpowered to detect interactions or even overall effects. Given the amount of accumulated data, it was deemed important to perform a quantitative synthesis of the evidence using a rigorous method. We therefore conducted a meta-analysis of all available studies relating the NOS3 polymorphism to the risk of AD. We considered all studies that examined the association of the NOS3 polymorphism with AD. Sources included MEDLINE, EMBASE, and the HuGENet database (last search update September, 2004). References of retrieved articles were also screened. The metaanalysis was designed on the same principles as a previous metaanalysis of the cathepsin D gene polymorphism and AD performed by our team [9]. Data were combined using both fixed effects (Mantel-Haenszel) and random effects (DerSimonian and Laird) models. Random effects incorporate an estimate of the betweenstudy variance and provide wider confidence intervals, when the results of the constituent studies differ among themselves. Thirteen studies probing the relationship between the NOS3 polymorphism and AD susceptibility were identified [1–4, 6–8, 10–15]. One eligible study [2] contained two different series of AD cases and controls, thus a total of 14 separate comparisons were considered. The eligible studies included a total of 2,688 cases with AD and 2,304 controls of which 2,686 and 2,290, respectively, had genotype data. The distribution of genotypes in control groups was consistent with Hardy-Weinberg equilibrium in all studies, except for three study comparisons [2, 6, 8], where there was a significant deficit of both homozygote genotypes. A sensitivity analysis was thus performed excluding these comparisons. There was no evidence that the G allele may confer increased susceptibility to AD (Fig. 1). The summary OR was 1.09 by both random (p = 0.14) and fixed effects (p = 0.065) without significant between-study heterogeneity (Table 1). In subgroup analyses, no differences were observed in allele LETTER TO THE EDITORS

Prevalence of eNOS Glu298Asp Polymorphism in Healthy Volunteers from a Region of Northern India

Objective: Endothelial nitric oxide synthase (eNOS) Glu298Asp polymorphisms are under extensive study worldwide due to their suggested role in cardiovascular disorders. This polymorphism had gained more attention since several reports suggest its association with hypertension and coronary artery disease (CAD). Asian Indians are highly susceptible to ischemic heart dis eases. We determined the prevalence of eNOS Glu298 Asp polymorphism in 139 healthy volunteers from Delhi and the surrounding areas. The subjects were recruited from those who willingly participated in this study in response to a publicized call and a standard questionnaire. Male to female ratio was 2.7:1 due to the larger number of male participants in this investigation. This, however, does not represent normal male to female distribution in the area. Despite the male bias, this investigation was justified. The prevalence of CAD in males is about 3 times higher in this region and no data had so far been available on the distribution of this polymorphism from India. Method: The eNOS Glu298Asp polymorphism was studied by PCR-RFLP. Results: Distribution of genotype GG, GT and TT in the study subjects was found to be 71.22, 28.06 and 0.72%, respectively, and allele frequency was G,0.853;T,0.147. Conclusion: T allele had been described as susceptibility allele for CAD in several population studies. The frequency of the T allele was found to be two times higher in our subjects than that reported for Japanese and Korean populations. This study does not provide any direct evidence for eNOS gene disease associations but is the first report on the prevalence of eNOS Glu298Asp gene polymorphism in Indian subjects. Whether the observed pattern of eNOS Glu298Asp polymorphism contributes to the greater susceptibility of Asian Indians to CAD as compared to the other population groups, needs to be investigated.

Functional comparison of the endothelial nitric oxide synthase Glu298Asp polymorphic variants in human endothelial cells

The G894T endothelial nitric oxide synthase (eNOS) polymorphism results in a Glu to Asp substitution at position 298. This position is located externally on the protein and as the regulation of eNOS is dependent on its subcellular localization and interaction with modulatory proteins, we aimed to address whether the substitution of Asp at 298 had any effect on these mechanisms. Initially, we developed a novel method to accurately determine molar quantities of each variant by expressing them as green fluorescent protein (GFP) fusion proteins and using recombinant adenoviruses to facilitate transient infection of human microvascular endothelial cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting of eNOSAsp revealed a 135-kDa proteolytic fragment which was not present with eNOSGlu. This proteolysis was prevented by using LDS buffer confirming that this differential cleavage is an artefact of sample preparation and unlikely to occur intracellularly. Nitric oxide was measured following stimulation with calcium ionophore or oestrogen in the presence of varying sepiapterin concentrations. GFP fluorescence was used to quantify the amount of fusion protein and calculate intracellular specific activity. There was no significant difference in intracellular specific activity between Glu and Asp eNOS in response to calcium ionophore or oestrogen. Tetrahydrobiopterin supplementation increased eNOS activity of both variants in an identical manner. The presence of the GFP also facilitated the visualization of the variants by confocal microscopy and demonstrated that both localized to the plasma membrane and the Golgi. These findings demonstrate that the Asp substitution at 298 does not have a major effect in modulating eNOS activity in vivo.

The Influence of G‐Protein β3‐Subunit Gene and Endothelial Nitric Oxide Synthase Gene in Exon 7 Polymorphisms on Progression of Autosomal Dominant Polycystic Kidney Disease

Background: A significant phenotypical variability is observed in autosomal dominant polycystic kidney disease (ADPKD). The variability cannot be fully explained by the genetic heterogeneity of the disease. We examined the influence of G‐protein β3‐subunit C825T polymorphism and endothelial nitric oxide synthase Glu298Asp polymorphism on the progression of ADPKD towards end stage renal failure (ESRF). Methods: 306 ADPKD patients (pts) were analyzed; 261 pts (136 males, 125 females) with ESRF, with subgroup of 73 pts (44 males, 29 females) with ESRF before 45 years (rapid progressors), 46 pts (20 males, 26 females) with ESRF later than in 63 years (slow progressors) and 45 ADPKD pts (17 males, 28 females) in mean age 51 years with serum creatinine under 110 µmol/L (slow progressors) and 100 genetically unrelated healthy Czech subjects. DNA samples from collected blood were genotyped for G‐protein β3‐subunit C825T genotype in exon 10 and for endothelial nitric oxide synthase Glu298Asp genotype in exon 7. Results: The G‐protein β3‐subunit C825T genotype exhibited no significant differences among the groups of slow progressors (6.6% (6/91) TT, 54.9% (50/91) CT, 38.8% (35/91) CC), rapid progressors (9.6% (7/73) TT, 46.6% (34/73) CT, 43.8% (32/73) CC), ADPKD group with ESRF between 40–63 years (9.2% (13/142) TT, 50% (71/142) CT, 40.8% (58/142) CC) and control group (12% TT, 44% CT, 44% CC). When comparing the ages of ESRF of all patients with ESRF, we did not find significant differences in the ages: males TT—51.7 ± 8.8 years, CT—51.9 ± 10.3 years, CC—49.7 ± 10.2 years and females TT—56 ± 9.9 years, CT—53.2 ± 8.5 years, CC—53.9 ± 8.7 years. The endothelial nitric oxide synthase Glu298Asp and Asp29Asp genotypes were significantly more frequent in rapid progressors (9.6% (7/73) Asp/Asp, 39.7% (29/73) Asp/Glu, 50.7% (37/73) Glu/Glu) and in ADPKD group with ESRF between 40–63 years (11.3% (16/142) Asp/Asp, 41.5% (59/142) Asp/Glu, 47.2% (67/142) Glu/Glu) in comparison with slow progressors (8.8% (8/91) Asp/Asp, 24.2% (22/91) Asp/Glu, 67.0% (61/91) Glu/Glu) and with control group (8% Asp/Asp, 32% Asp/Glu, 60% Glu/Glu) (Chi‐square test, p < 0.05). Comparing the ages of ESRF of all patients with ESRF, we did not find significant differences in the ages in males with Asp/Asp—54.9 ± 10.4 years, Asp/Glu—50.2 ± 9.4 years, Glu/Glu—51.0 ± 10.4 years. We found out in homozygous Asp/Asp females significantly earlier onset of ESRF (49.2 ± 5.6 years) in comparison with heterozygous females (53.3 ± 7.2 years) and with Glu/Glu homozygous females (54.8 ± 9.7 years) (t‐test, p < 0.05). Conclusion: We excluded the significance of G‐protein β3‐subunit C825T polymorphism on the progression of ADPKD. We established the negative prognostic value of the carriers of Asp variant of eNOS polymorphism. Finding of new modifiers could have in future clinical consequences for ADPKD patients.

Genetic and Environmental Contributions to Severe Preeclampsia: Lack of Association with the Endothelial Nitric Oxide Synthase Glu298Asp Variant in a Developing Country

Objective: We recently identified a missense variant (Glu298Asp) within exon 7 of the endothelial nitric oxide synthase (eNOS) gene that is associated with severe preeclampsia in the Japanese population. Our objective was to analyze the association between the Glu298Asp eNOS gene variant and severe preeclampsia in a developing country where the incidence of preeclampsia is more frequent. Methods: The study included 112 Bangladeshi women exhibiting severe preeclampsia and 119 control subjects. Screening for the Glu298Asp eNOS gene variant was carried out by analysis of the polymerase chain reaction-restriction fragment length polymorphism. Results: The analysis revealed that the frequencies of the Glu298Asp variant (Glu298Asp homozygotes and heterozygotes) in the preeclampsia (n = 40; 35%) and control (n = 42; 35%) groups were not significantly (p > 0.05) different. Conclusions: We conclude that the presence of the Glu298Asp eNOS gene is not a marker for the increased risk of preeclampsia in a population where the level of prenatal care is such that preeclampsia is still frequently seen.

The endothelial nitric oxide synthase (Glu298Asp and - 786T>C) gene polymorphisms are associated with coronary in-stent restenosis

Aims Coronary stent deployment is a major advance in percutaneous treatment of ischaemic heart disease, but 10–40% of patients still develop angiographic restenosis by 6 months due to neointimal hyperplasia. Patient-specific factors, including genetic factors, can contribute to this process. We have conducted a prospective study to examine the involvement of genetic risk factors (eNOS, ACE, MMP-3, IL-6, and PECAM-1) in restenosis following coronary stent deployment. Methods and Results A total of 226 patients who underwent elective and successful coronary artery stenting to de novo lesions in native coronary arteries were studied. Two hundred and five (90·7%) patients were restudied by coronary angiogram at 6 months and the stented lesions were assessed using an automated quantitative angiography system. Genotype was determined by polymerase chain reaction (PCR) and restriction enzyme digestion. Restenosis rate, defined as ≥50% diameter stenosis, was 29·3%. The overall genotype frequency distributions were in Hardy–Weinberg equilibrium for all variants. Carriers of the 298Asp allele of the eNOS Glu298Asp polymorphism showed a higher frequency of restenosis with an odds ratio of 1·88 (95%CI: 1·01–3·51, P =0·043) compared to 298Glu homozygotes. Carriers of the –786C allele of the eNOS –786T>C polymorphism also showed a higher frequency of restenosis with odds ratio of 2·06 (95%CI: 1·08–3·94, P =0·028). These effects were essentially additive and were independent of other classical risk factors. Other studied genes did not show significant association with coronary in-stent restenosis. Conclusion In patients with coronary artery disease, the possession of the 298Asp and –786C variants of the eNOS gene are a risk factor for coronary in-stent restenosis, demonstrating the importance of the nitric oxide system in restenosis. Copyright 2002 The European Society of Cardiology. Published by Elsevier Science Ltd. All rights reserved.

Endothelial nitric oxide synthase GLU298ASP polymorphism and blood pressure responses to salt

Clinical Pharmacology &amp; Therapeutics (2003) 73, P98–P98; doi:

The endothelial nitric oxide synthase (Glu298Asp and -786T>C) gene polymorphisms are associated with coronary in-stent restenosis.

Coronary stent deployment is a major advance in percutaneous treatment of ischaemic heart disease, but 10-40% of patients still develop angiographic restenosis by 6 months due to neointimal hyperplasia. Patient-specific factors, including genetic factors, can contribute to this process. We have conducted a prospective study to examine the involvement of genetic risk factors (eNOS, ACE, MMP-3, IL-6, and PECAM-1) in restenosis following coronary stent deployment. A total of 226 patients who underwent elective and successful coronary artery stenting to de novo lesions in native coronary arteries were studied. Two hundred and five (90.7%) patients were restudied by coronary angiogram at 6 months and the stented lesions were assessed using an automated quantitative angiography system. Genotype was determined by polymerase chain reaction (PCR) and restriction enzyme digestion. Restenosis rate, defined as >or=50% diameter stenosis, was 29.3%. The overall genotype frequency distributions were in Hardy-Weinberg equilibrium for all variants. Carriers of the 298Asp allele of the eNOS Glu298Asp polymorphism showed a higher frequency of restenosis with an odds ratio of 1.88 (95%CI: 1.01-3.51, P=0.043) compared to 298Glu homozygotes. Carriers of the -786C allele of the eNOS -786T>C polymorphism also showed a higher frequency of restenosis with odds ratio of 2.06 (95%CI: 1.08-3.94, P=0.028). These effects were essentially additive and were independent of other classical risk factors. Other studied genes did not show significant association with coronary in-stent restenosis. In patients with coronary artery disease, the possession of the 298Asp and -786C variants of the eNOS gene are a risk factor for coronary in-stent restenosis, demonstrating the importance of the nitric oxide system in restenosis.

Genetic evaluation of the common variant of the endothelial nitric oxide synthase (Glu298Asp) in patients with thrombangitis obliterans

Genetic evaluation of the common variant of the endothelial nitric oxide synthase (Glu298Asp) in patients with thrombangitis obliterans

High prevalence of polymorphisms of angiotensin-converting enzyme (I/D) and endothelial nitric oxide synthase (Glu298Asp) in patients with systemic sclerosis

Purpose Systemic sclerosis is characterized by progressive microvascular occlusion and fibrosis and by an imbalance in the fibrinolytic system. In vivo and in vitro studies suggest that the renin-angiotensin system partly regulates vascular fibrinolytic balance. Angiotensin II increases the production and secretion of plasminogen activator inhibitor-1, while angiotensin-converting enzyme (ACE) contributes to reduced production of tissue plasminogen activator and endothelial nitric oxide synthesis by bradykinin degradation. The aim of our study was to investigate the effects of ACE insertion/deletion (I/D) and endothelial nitric oxide synthase (eNOS) Glu298Asp (G894→T) and T–786→C polymorphisms in patients with systemic sclerosis. Subjects and methods We studied 73 consecutive patients (47 with limited and 26 with diffuse cutaneous systemic sclerosis) and 112 control subjects. ACE I/D and eNOS polymorphisms were genotyped by polymerase chain reaction–restriction fragment length polymorphism analysis. Results The ACE I/D and the eNOS G894→T polymorphisms were more common in patients than in controls (for the ACE D allele: odds ratio [OR] = 3.4; 95% confidence interval [CI]: 1.5 to 7.9; P = 0.003; for the eNOS T allele: OR = 1.9; 95% CI: 1.0 to 3.4; P = 0.04). There was no association between the eNOS T–786→C polymorphism and systemic sclerosis. Conclusion Our findings of an increased risk of systemic sclerosis in ACE D and eNOS 894T allele carriers suggest that these polymorphisms may contribute to the pathogenesis of the disease.

A Common Variant of Endothelial Nitric Oxide Synthase (Glu298Asp) Is an Independent Risk Factor for Carotid Atherosclerosis

Endothelium-derived NO is formed from L-arginine by endothelial NO synthase (eNOS) encoded by the NOS 3 gene on chromosome 7. Because several studies have indicated that NO plays a key role in the development of the atherosclerotic process, we investigated whether common variants in the eNOS gene are associated with an increased risk of plaque on carotid arteries. We studied 375 subjects attending the hypertension center of our institution to be screened for arterial hypertension. The examined subjects were classified according to the presence of carotid plaques (intima-media thickness >/=1.5 mm), and 2 intronic (CA and 27-bp repeats) polymorphisms and 1 exonic (Glu298Asp) polymorphism of the eNOS gene were explored. Only the Glu298Asp polymorphism of eNOS was associated with the presence of carotid plaques (P:<0.05). In particular, there was an excess of homozygotes for the Asp298 variant among subjects with carotid plaques, whereas the number of subjects who had the Glu298 allele in exon 7 of the eNOS gene was equally distributed in both study groups. Interestingly, the risk of having carotid plaques was increased approximately 3 times in subjects who were homozygotic for the Asp298 variant compared with subjects who were homozygotic for the Glu298 variant and was independent of the other common risk factors (age, blood pressure, and smoking). Homozygosity for Asp298, a common variant of the eNOS gene, is an independent risk factor for carotid atherosclerosis in this study population.