The missense Glu298Asp variant of the endothelial nitric oxide synthase gene is strongly associated with placental abruption

We recently identified a missense variant (Glu298Asp) that lies within exon 7 of the endothelial nitric oxide synthase (eNOS) gene, and that is associated with severe preeclampsia (proteinuric hypertension that develops as a consequence of pregnancy). Maternal hypertension is the most consistently identified factor predisposing to placental abruption. Our objective, therefore, was to analyze the association between the Glu298Asp eNOS gene variant and placental abruption. The study participants included 35 patients with histories of placental abruption and 170 control subjects. Screening for the Glu298Asp eNOS gene variant was carried out by analysis of polymerase chain reaction/restriction fragment length polymorphism. The analyses revealed that the frequency of the Glu298Asp variant (Glu298Asp homozygotes and heterozygotes) was significantly (P<0.001) higher in the placental abruption group (n=14; 40%) than in the control group (n=24; 14%). We conclude that the presence of the Glu298Asp eNOS gene variant could be a marker of increased risk of developing placental abruption.     

NO Level and Endothelial NO Synthase Gene Polymorphism （Glu298Asp） in the Patients with Coronary Artery Disease from the Turkish Population

A healthy endothelium plays a core role in cardiovascu-lar control [1]. In the endothelial cell, nitric oxide (NO) issynthesized by the endothelial nitric oxide synthase (eNOS)encoded by a 26-exon gene (NOS 3) located on chromo-some 7 [2]. Besides its regulatory functions on vasomotortone and blood flow, endothelial NO is known to inhibitthe platelet activation and modulate migration and growthof the vascular smooth muscle [3]. Indirect evidence sug-gests that alterations of the NO pathwa…     

Endothelial nitric oxide synthase gene polymorphism in the Indian and Mestizo populations of Mexico

Endothelium-derived nitric oxide (NO) is an important factor in vasodilation synthesized by endothelial nitric oxide synthase (eNOS). A polymorphism (894 G to T) in exon 7 of the eNOS gene causes the conversion of Glu to Asp in position 298. The Glu298Asp polymorphism has been extensively associated with cardiovascular disease. We determined the Glu298Asp polymorphism frequency in healthy Mexican Mestizo, Huastec, Mayo, and Mayan populations by the endonuclease restriction method. The four populations analyzed were in Hardy-Weinberg equilibrium. Allele frequencies were similar among Mexican populations but different when compared with Caucasians. However, when compared with allele frequencies in Asian populations, Mestizo and Huastec allele frequencies were significantly different. Genotypically, only the Mestizos presented Asp298 homozygosity. The absence of double mutants in Indian populations resembles that in Asians. With these data, we conclude that the low frequency of the eNOS Glu298Asp polymorphism in Indian and Mestizo populations of Mexico is related to the Asian origin of Amerindian groups.     

Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan

Vascular endothelial cells produce nitric oxide (NO), which contributes to the regulation of blood pressure and regional blood flow. Although Endothelial NO synthase (eNOS) gene polymorphisms have been shown to have a positive association with coronary artery disease, the linkage between eNOS gene polymorphisms and hypertension has been controversial. In the present study, therefore, we identified genotypes for Glu298Asp and variable number tandem repeats in intron 4 (4b/a) in 183 hypertensive and 193 normotensive populations. The Glu298Asp variant had a significant association with hypertension (odds ratio, 1.8; 95% confidence interval, 1.1-3.0). The allele frequencies of 298Asp for Glu298 in hypertensive patients were significantly higher than those in normotensive subjects (0.128 vs 0.080, p<0.05). Diastolic and mean arterial blood pressures were significantly higher in hypertensive subjects with the 298Asp allele than those without the variant allele (p<0.05). However, disequilibrium of 4b/a polymorphism was absent between these two groups. These results suggest that the Glu298Asp variant may be a genetic susceptibility factor for hypertension.     

Association of endothelial nitric oxide synthase gene polymorphisms with classical risk factors in development of premature coronary artery disease

Genetic polymorphism of the endothelial nitric oxide synthase (eNOS) affects the pathogenesis of atherosclerosis and associated with premature coronary artery disease (PCAD). We aimed to explore the association between Glu298Asp polymorphism of the eNOS gene and premature CAD in Egyptians, and the possible interaction between this polymorphism and other risk factors. The study population consisted of 116 patients with PCAD, and 119 controls. Glu298Asp polymorphism (rs1799983) of the eNOS gene was analyzed by polymerase chain reaction (PCR). We found that the TT genotype of the eNOS gene increased the risk of PCAD by 2.6. Hypertension, diabetes, smoking, total cholesterol, triglycerides, LDLc, HDLc and TT genotype of the eNOS gene were independent risk factors for the development of PCAD. We conclude that, the TT genotype of Glu298Asp polymorphism of eNOS gene is an independent risk factor of PCAD in Egyptians. The association of smoking, obesity, dyslipidemia and/or metabolic syndrome with the TT genotype increased the risk of the development of PCAD.     

Oxidative stress and eNOS (Glu298Asp) gene polymorphism in preeclampsia in Indian population

Oxidative stress causing widespread endothelial dysfunction has been proposed as a key factor involved in the development of preeclampsia (PE). With this background our objective was to study oxidative stress biomarkers like nitric oxide and malondialdehyde (MDA) and to correlate these markers with endothelial nitric oxide synthase (eNOS) (Glu298Asp) gene polymorphism. This cross-sectional study included 300 pregnant women diagnosed with PE and 200 women with normal pregnancy. Plasma NO and MDA levels were analyzed using student's t test and eNOS gene polymorphism was studied by performing polymerase chain reaction amplification and restriction length polymorphism and frequencies were distributed by using χ(2) analysis. The mean plasma levels of NO were significantly lower in study group while MDA levels were significantly higher in study group (P < 0.001). Genotypic and allelic frequency of eNOS gene in both groups was found to be significant (P < 0.05). The intergenotypic variation of NO and MDA levels was found to be significant (P < 0.001). We concluded that the plasma levels of NO are decreased while MDA levels are increased in subjects with PE and that might contribute to the pathophysiology of PE. As observed in this study Glu298Asp eNOS gene polymorphism showed significant association with PE.     

Glu298Asp polymorphism of the endothelial nitric oxide synthase gene in Turkish patients with ischemic stroke

The low plasma nitric oxide concentrations and reduced vascular reactivity are considered major proatherogenic mechanisms in cardiovascular diseases. The present study aimed to assess the allelic frequency and the genotypic distribution of the Glu298Asp gene polymorphism at exon 7 of endothelial nitric oxide synthase (eNOS) gene in Turkish ischemic stroke patients compared to appropriate healthy controls, and to correlate the genetic findings with stroke subtypes. The study population included 146 (75 males, 71 females) patients with ischemic stroke which were categorized according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) and 133 (34 males, 99 females) healthy subjects. The eNOS polymorphism was identified with a PCR followed by RFLP with the restriction enzyme BanII. Genotypes were defined as GG, GT, and TT according to the presence of the G and T alleles. In this case-control study, we did not find any significant difference in either the genotypic distribution or allelic frequency of Glu298Asp gene polymorphism between the patients and the controls. In addition, there was also no significant difference for the genotype distribution and the allelic frequency among the stroke subtypes. The results suggested the lack of the association between the Glu298Asp gene polymorphism and ischemic stroke or subtypes of ischemic stroke in the Turkish population.     

Genetic association studies of endothelial nitric oxide synthase gene polymorphisms in women with unexplained recurrent pregnancy loss: a systematic and meta-analysis

Recurrent pregnancy loss (RPL) is a multifactorial disorder, both genetic and environmental factors contribute to the development of RPL. Recently, the association between endothelial nitric oxide synthase (eNOS) gene polymorphisms and unexplained recurrent pregnancy loss (URPL) has been reported, and the results were inconsistent. Hence, we performed the meta-analysis to drive a more precise estimation of association between eNOS polymorphisms and URPL. Odds ratio (OR) and its 95 % confidence interval were calculated under co-dominant (AA vs. BB, TT vs. GG) and additive (A vs. B, T vs. G) genetic models. Studies of eNOS intron 4 VNTR and Glu298Asp were separated by ethnicities. 13 studies included 1,769 URPL cases and 1,376 healthy controls on eNOS intron 4 VNTR polymorphism, and 11 studies were involved in Glu298Asp polymorphism with 1,498 URPL cases and 1,123 healthy controls. The integrated results showed that eNOS Glu298Asp polymorphism was associated with URPL [ORs were 1.91 (1.42–2.56), P < 0.001; 1.67 (1.36–2.04), P < 0.001, respectively]. When analyses were separated by ethnic subgroups, the association between eNOS Glu298Asp polymorphism and URPL was only observed in East Asians [OR = 1.88 (1.52–2.33), P < 0.001 under additive model], and there was no association between eNOS intron 4 VNTR polymorphism and URPL in Caucasians and East Asians. The results indicated a significant association between eNOS Glu298Asp polymorphism and URPL in East Asians. No association was observed between eNOS intron 4 VNTR polymorphism and URPL in Caucasians and East Asians.     

Analysis of endothelial nitric oxide synthase gene polymorphisms with cardiovascular diseases in eastern Taiwan

A few studies have been carried out to address the correlation between the endothelial nitric oxide synthase (eNOS) gene polymorphisms and cardiovascular diseases (CVD) within the Taiwanese population. However, no report has documented the situations in eastern Taiwan, which has different ethnic groups from those in western Taiwan. In this study, we explored the relationship between polymorphic eNOS alleles and CVD in eastern Taiwan. DNA extraction and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis were employed for the detection polymorphism in exon 7 of the eNOS gene. A total of 198 subjects was included. The subjects were 120 patients with CVD such as hypertension, coronary artery disease (CAD), and stroke. Normal subjects (78) served as control. Analysis of the gene polymorphism revealed that the frequency of the eNOS gene variant containing a 27-bp repeat in intron 4 is similar between control subjects (aa:ab:bb = 0%:21.8%:78.2%), and patients with CVD (aa:ab:bb = 3.3%:21.7%:75.0%). The frequency of the Glu298Asp (894G --> T) polymorphism in exon 7 of the eNOS gene was significantly different between control subjects (TT:GT:-GG = 7.7%:29.5%:62.8%) and patients with CVD (TT:GT:GG = 5.0%:74.2%:20.8%). These results suggest that the Glu298Asp polymorphism in exon 7 of the eNOS gene is likely to be a risk factor for CVD in the eastern Taiwanese population.     

Association of polymorphisms of endothelial nitric oxide synthase (eNOS) gene with the risk of primary open angle glaucoma in a Brazilian population

The present study aimed to investigate the association of endothelial nitric oxide synthase (eNOS) gene polymorphisms with primary open angle glaucoma (POAG). We conducted a case-control study that included 90 patients with POAG and 127 healthy controls whose blood samples were genotyped for the functional polymorphisms T-786C and Glu298Asp of the eNOS gene by Taqman fluorescent allelic discrimination assay. The T-786C polymorphism was significantly associated as a risk factor for POAG among women (OR: 2.28; 95% CI: 1.11 to 4.70, p=0.024) and marginally associated to the risk of POAG in the patients ≥52 years of age at diagnosis (OR: 2.11; 95% CI: 0.98 to 4.55, p=0,055). However, these results was not confirmed after adjustments for gender, age, self-declared skin color, tobacco smoking and eNOS genotypes by multivariate logistic regression model (OR: 2.08; 95% CI: 0.87 to 5.01, p=0.101 and OR: 2.20; 95% CI: 0.95 to 5.12, p=0.067, respectively). The haplotype CG of T-786C and Glu298Asp showed a borderline association with risk of POAG in the overall analysis (OR: 1.76; 95% CI: 0.98 to 3.14, p=0.055) and among women (OR: 2.02; 95% CI: 0.98 to 4.16, p=0.052). Furthermore, the CG haplotype was significantly associated with the development of POAG for the age at diagnosis group ≥52 years (OR: 3.48; 95% CI: 1.54 to 7.84, p=0.002).We suggested that haplotypes of the polymorphisms T-786C and Glu298Asp of eNOS may interact with gender and age in modulating the risk of POAG.     

Glu298Asp polymorphism influences the beneficial effects of fish oil fatty acids on postprandial vascular function

Our objective was to determine whether the endothelial nitric oxide synthase (eNOS) Glu298Asp polymorphism influences vascular response to raised NEFA enriched with saturated fatty acids (SFA) or long-chain (LC) n-3 polyunsaturated fatty acids (PUFA). Subjects were prospectively recruited for genotype (Glu298, n = 30 and Asp298, n = 29; balanced for age and gender) consumed SFA on two occasions, with and without the substitution of 0.07 g fat/kg body weight with LC n-3 PUFA, and with heparin infusion to elevate NEFA. Endothelial function was measured before and after NEFA elevation (240 min), with blood samples taken every 30 min. Flow-mediated dilation (FMD) decreased following SFA alone and increased following SFA+LC n-3 PUFA. There were 2-fold differences in the change in FMD response to the different fat loads between the Asp298 and Glu298 genotypes (P = 0.002) and between genders (P < 0.02). Sodium nitroprusside-induced reactivity, measured by laser Doppler imaging with iontophoresis, was significantly greater with SFA+LC n-3 PUFA in all female subjects (P < 0.001) but not in males. Elevated NEFA influences both endothelial-dependent and endothelial-independent vasodilation during the postprandial phase. Effects of fat composition appear to be genotype and gender dependent, with the greatest difference in vasodilatory response to the two fat loads seen in the Asp298 females.     

The effects of NOS3 Glu298Asp variant on colorectal cancer risk and progression in Turkish population

Endothelial nitric oxide synthase (eNOS), coded by the gene NOS3, may play an important role in uncontrollable cellular growth in several cancer types. Our study was performed to test the association between Glu298Asp polymorphisms in the NOS3 gene and colorectal cancer risk and progression. In this study, NOS3 Glu298Asp polymorphism was genotyped in 84 patients with colorectal cancer and 99 healthy subjects using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis. There were significant differences in the distribution of NOS3 genotypes and frequencies of the alleles between colorectal cancer patients and controls (P = 0.016, P = 0.006, respectively). The increased frequency of NOS3 Glu298Asp homozygotes genotypes in patients who had advanced tumour stage was statistically significant (P = 0.042). Our findings have suggested that NOS3 Glu298Asp polymorphism might be associated with the risk and progression of colorectal cancer in Turkish population.     

Functional characterization of Glu298Asp mutant human endothelial nitric oxide synthase purified from a yeast expression system.

The Glu298Asp polymorphism of human endothelial nitric oxide synthase (eNOS) has been reported to be associated with several cardiovascular diseases, including hypertension and myocardial infarction. Therefore, we investigated the effect of the Glu298Asp (E298D) mutation on the function of purified recombinant eNOS expressed in the yeast Pichia pastoris. Wild type (WT) and mutant exhibited comparable affinities for L-arginine (K(m) values 4.4+/-0.6 and 5.2+/-0.8 microM, respectively) and V(max) values (142+/-36 and 159+/-29 nmol of L-citrulline/mg min, respectively). The E298D mutation affected neither electron transfer through the reductase domain (measured as cytochrome c reduction) nor reductive O(2) activation (measured either as NADPH oxidation or as H(2)O(2) formation in the absence of L-arginine and tetrahydrobiopterin (BH4)). The mutant was activated by BH4 with an EC(50) of 0.24+/-0.04 microM, a value comparable to that obtained with WT eNOS (0.22+/-0.02 microM). Activation of the enzyme by Ca(2+) was not affected (EC(50)=0.50+/-0.04 and 0.49+/-0.02 microM for WT and E298D eNOS, respectively). Calmodulin (CaM) affinity, studied by radioligand binding using 125I-labeled CaM, revealed virtually identical K(D) (3.2+/-0.5 and 4.0+/-0.3nM) and B(max) (1.4+/-0.2 and 1.2+/-0.3 pmol/pmol subunit) values for WT and E298D eNOS, respectively. Furthermore, E298D eNOS did not differ from the WT enzyme with respect to heme and flavin content or the ability to form SDS-resistant dimers. To summarize, we obtained no evidence for altered enzyme function of the eNOS mutant that could explain endothelial dysfunction associated with the E298D polymorphism.     

A missense Glu298Asp variant in the endothelial nitric oxide synthase gene is associated with coronary spasm in the Japanese

Coronary spasm plays an important role in the pathogenesis of not only variant angina but also ischemic heart disease in general. However, the precise mechanism(s) by which coronary spasm occurs remains to be elucidated. Coronary spasm may arise from interactions between environmental and genetic factors. Endothelial derived nitric oxide (NO) has been implicated in the control of vascular tone. We have recently shown that both basal and acetylcholine (ACh)-induced NO activities are impaired in the coronary arteries of patients with coronary spasm. The purpose of this study has been to elucidate the possible variants that occur in the coding region of the endothelial nitric oxide synthase (eNOS) gene and that may be associated with coronary spasm. After initial screening in the entire 26 coding regions of the eNOS gene, we found a missense Glu298Asp variant in exon 7 in patients with coronary spasm. We subsequently performed a larger scale study involving 113 patients with coronary spasm and 100 control subjects, who were all diagnosed by intracoronary injection of ACh. The analysis revealed a significant difference in the distribution of the variant between the coronary spasm group (21.2%) and control group (9.0%; P=0.014 for dominant effect). Thus, we have found the missense Glu298Asp variant in the eNOS gene by the analysis of its entire 26 coding regions. The variant is significantly associated with coronary spasm. Received: 2 February 1998 / Accepted: 9 April 1998     

Impact of angiotensin-converting enzyme, angiotensinogen, endothelial NO synthase, and bradykinin receptor B2 gene polymorphisms on myocardium in patients with hypertension and in athletes

We investigated the role of gene polymorphisms in angiotensin-converting enzyme (ACE), angiotensinogen, endothelial NO (eNO) synthase, and bradykinin receptor B2 in determining the cardiovascular system structure and function in hypertension and "athletic heart" syndrome. Using a PCR-based method, 114 hypertensive patients and 94 athletes were genotyped for I/D polymorphism of ACE, M235T angiotensinogen (ANG), Glu298 Asp endothelial synthase (eNOS), and type 2 receptor for bradykinin (BDKR2). Echocardiography and a 24 hour blood pressure monitoring being performed. The (+)-allel of BDKR2 gene was associated with the left ventricular hypertrophy and greater wall thickness in athletes and hypertensive subjects. The hypertensive patients, that were homozygous for Glu298 allele of eNOS, demonstrated a lower level of diastolic blood pressure than did those with Glu298 Asp and Asp298 Asp genotypes. At the same time, the ACE and AND gene polymorphisms displayed no association with the cardiac structure and function.     

Association between Glu298Asp/677C-T single nucleotide polymorphism in the eNOS/MTHRF gene and blood stasis syndrome of ischemic stroke

Blood stasis syndrome of ischemic stroke (BSS-IS) is a common clinical phenotype that may be affected by certain mutagenic environmental factors or chemotherapeutic drugs; however, the role of susceptibility genes remains unclear. Previous studies have shown that ischemic stroke (IS) was closely associated with the Glu298Asp polymorphism in the eNOS gene and the 677C-T (Ala→Val) polymorphism in methylenetetrahydrofolate reductase (MTHRF) gene. Therefore, these two single nucleotide polymorphisms (SNPs) were selected to detect their associations with BSS-IS in this study. A SNP chip was employed to screen the SNP variation between both groups, and the results were verified using denaturing high-performance liquid chromatography (DHPLC) and restriction fragment length polymorphism (RFLP). The results confirmed that the TT genotype of Glu298Asp in the eNOS gene may be one of the risk factors associated with BSS-IS, while the genotype of 677C-T (Ala→Val) in the MTHRF gene may not be relevant to BSS-IS.     

Right Ventricular Adaptation Is Associated with the Glu298Asp Variant of the NOS3 Gene in Elite Athletes

Nitric oxide (NO), an important endogenous pulmonary vasodilator is synthetized by the endothelial NO synthase (NOS3). Reduced NO bioavailability and thus the Glu298Asp polymorphism of NOS3 may enhance right ventricular (RV) afterload and hypertrophic remodeling and influence athletic performance. To test this hypothesis world class level athletes (water polo players, kayakers, canoeists, rowers, swimmers, n = 126) with a VO₂ maximum greater than 50ml/kg/min were compared with non-athletic volunteers (n = 155). Cardiopulmonary exercise tests and cardiac magnetic resonance imaging (cMRI) were performed to determine structural or functional changes. Genotype distribution of the NOS3 Glu298Asp polymorphism was not affected by gender or physical performance. Cardiac MRI showed increased stroke volume with eccentric hypertrophy in all athletes regardless of their genotype. However, the Asp allelic variant carriers had increased RV mass index (32±6g versus 27±6g, p<0.01) and larger RV stroke volume index (71±10ml versus 64±10ml, p<0.01) than athletes with a Glu/Glu genotype. Genotype was not significantly associated with athletic performance. In the non-athletic group no genotype related differences were detected. The association between the NOS3 Glu298Asp polymorphism and RV structure and dimension in elite athletes emphasizes the importance of NOS3 gene function and NO bioavailability in sport related cardiac adaptation.     

Consistent interethnic differences in the distribution of clinically relevant endothelial nitric oxide synthase genetic polymorphisms.

A maldistribution of endothelial nitric oxide synthase (eNOS) genetic variants may explain differences in NO-mediated effects and response to drugs among black and white subjects. While interethnic differences in the distribution of eNOS genetic variants exist in the American population, it is not known whether such interethnic differences exist in other populations. To test this possibility, we examined the distribution of genetic variants of three clinically relevant eNOS polymorphisms (T(-786)C in the promoter, the VNTR in intron 4, and the Glu298Asp variant in exon 7) in 136 black and 154 white subjects from a Brazilian population, which is very heterogeneous. We also estimated the haplotype frequency and evaluated associations between these variants. The Asp298 variant was more common in whites (32.8%) than in blacks (15.1%) (P < 0.004). Similarly, the C(-786) variant was more common in whites (41.9%) than in blacks (19.5%) (P < 0.0004). However, the 4a variant was more common in blacks (32.0%) than in whites (17.9%) (P < 0.003). The most common predicted haplotype in both ethnic groups combined only wild-type variants. While the second most common haplotype in blacks includes the variant 4a and the wild-type variants for the remaining polymorphisms, the second most common haplotype in whites includes the variants Asp298 and C(-786) and the wild-type variant for polymorphism in intron 4. The marked interethnic differences that we found in Brazilians are very similar to those previously reported in Americans. These findings strongly suggest a consistent difference in the distribution of eNOS genetic variants in blacks compared with whites and indicate that the interethnic differences do not vary with geographic origin.     

Endothelial Nitric Oxide Synthase Single Nucleotide Polymorphism and Left Ventricular Function in Early Chronic Kidney Disease

Background

Chronic kidney disease (CKD) is associated with accelerated cardiovascular disease and heart failure. Endothelial nitric oxide synthase (eNOS) Glu298Asp single nucleotide polymorphism (SNP) genotype has been associated with a worse phenotype amongst patients with established heart failure and in patients with progression of their renal disease. The association of a cardiac functional difference in non-dialysis CKD patients with no known previous heart failure, and eNOS gene variant is investigated.

Methods

140 non-dialysis CKD patients, who had cardiac magnetic resonance (CMR) imaging and tissue doppler echocardiography as part of two clinical trials, were genotyped for eNOS Glu298Asp SNP retrospectively.

Results

The median estimated glomerular filtration rate (eGFR) was 50mls/min and left ventricular ejection fraction (LVEF) was 74% with no overt diastolic dysfunction in this cohort. There were significant differences in LVEF across eNOS genotypes with GG genotype being associated with a worse LVEF compared to other genotypes (LVEF: GG 71%, TG 76%, TT 73%, p = 0.006). After multivariate analysis, (adjusting for age, eGFR, baseline mean arterial pressure, contemporary CMR heart rate, total cholesterol, high sensitive C-reactive protein, body mass index and gender) GG genotype was associated with a worse LVEF, and increased LV end-diastolic and systolic index (p = 0.004, 0.049 and 0.009 respectively).

Conclusions

eNOS Glu298Asp rs1799983 polymorphism in CKD patients is associated with relevant sub-clinical cardiac remodelling as detected by CMR. This gene variant may therefore represent an important genetic biomarker, and possibly highlight pathways for intervention, in these patients who are at particular risk of worsening cardiac disease as their renal dysfunction progresses.