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1.  Endothelial nitric oxide synthase gene polymorphisms -786T >C and 894G >T in coronary artery bypass graft surgery patients 
Human Genomics  2010;4(6):375-383.
Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene (-786T >C and 894G >T) enhance endo-thelial dysfunction and have been studied in relation to coronary artery disease (CAD). In the present study, we examined the association of the above polymorphisms with CAD, as well as with myocardial infarction (MI), hypertension, diabetes and smoking in CAD patients. Study subjects consisted of 154 consecutive coronary artery bypass graft (CABG) patients and 155 non-CAD controls. eNOS -786T >C and 894G >T polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism. The estimated frequencies of the -786C and 894T alleles did not differ between the two groups (p = 0.46 and p = 0.84, respectively). The prevalence of eNOS polymorphisms was not associated with MI, hypertension or diabetes in CABG patients; however, we found that the 894TT genotype and 894T allele were significantly more frequent in current/past smoker CABG patients (16.7 per cent and 39.6 per cent, respectively) compared with never smoker CABG patients (6.1 per cent and 24.4 per cent, respectively) (p = 0.01 and p < 0.01, respectively). We found no association of eNOS -786C and 894T variant alleles with CAD; however, within CABG patients, a gene-environment interaction was found between the eNOS 894T allele and smoking.
PMCID: PMC3525225  PMID: 20846926
coronary artery disease; coronary artery bypass graft surgery; eNOS -786T > C polymorphism, eNOS 894G > T polymorphism; smoking
2.  Association of endothelial nitric oxide synthase polymorphisms with coronary artery disease in Korean individuals with or without diabetes mellitus 
Polymorphisms of the endothelial nitric oxide synthase (eNOS) gene have been implicated in various diseases, but their roles as risk factors in type 2 diabetes mellitus (T2DM) with regard to coronary artery disease (CAD) are largely unknown. Therefore, we investigated the association of the genotypes and haplotypes of eNOS polymorphisms in CAD with T2DM. A case-control study was performed to evaluate the genotypes and haplotypes of the eNOS polymorphisms (-786T>C, 4a4b and 894G>T) in 192 CAD patients and 196 controls. The same population was also re-organized upon the status of T2DM. The genotypes of eNOS -786T>C, 4a4b and 894G>T polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism. We found that eNOS -786TC+CC and 4a4b+4a4a genotypes were significantly prevalent in the diabetic controls and the diabetic CAD patients compared to the non-diabetic controls or non-diabetic CAD patients, respectively. The frequency of the -786C-4a-894G haplotype was significantly greater in the diabetic CAD patients (p=0.001) and diabetic controls (p=0.023) compared to the non-diabetic controls, whereas the haplotype of -786T-4b-894G was less prevalent in the diabetic CAD patients compared to the non-diabetic controls (p=0.018). Significant associations of the genotypes and the haplotypes were consistently observed in the T2DM group compared to non-DM group, regardless of CAD status. Our finding suggests that the eNOS -786T>C and 4a4b polymorphisms and the -786C-4a-894G haplotype are risk factors for T2DM, whereas the haplotype of -786T-4b-894G has a protective effect against the development of T2DM.
PMCID: PMC3445891  PMID: 22993595
coronary artery disease; endothelial nitric oxide synthase; haplotype; polymorphism; type 2 diabetes mellitus
3.  Endothelial Nitric Oxide Synthase Gene T-786C Polymorphism in Renal Transplant Recipients 
Background: Nitric oxide (NO) is a major mediator in vascular biology, regulating regional blood flow. NO and the enzymes required for its production contribute to ischemia-reperfusion injury. The T-786C functional polymorphism in the promoter region substantially reduces promoter activity of the endothelial nitric oxide synthase (eNOS) gene and compromises endothelial NO synthesis.
Objective: To examine the association between T-786C (rs 2070744) single nucleotide polymorphism (SNP) in eNOS gene and the development of acute rejection in renal transplant patients.
Methods: 60 renal transplant recipients (30 with episodes of acute rejection (ARs) and 30 without rejection (non-ARs)), between June 2008 and March 2010, were included in this study. The polymorphism was determined by PCR-restriction fragment-length polymorphism analysis.
Results: The distribution of the genotypes were TT/TC/CC 60%, 33.4%, 6.6%, and 43%, 46.7%, 13.3% in ARs and non-ARs, respectively (p=0.28). The frequency of T-allele was 76.7% and 66.3%; and for C-allele was 66.6% and 33.3% in ARs and non-ARs, respectively (p=0.09). There were no significant associations between these polymorphisms and acute and chronic kidney allograft rejection.
Conclusion: We could not detect any significant association between polymorphism in T-786C of eNOS gene and the development of acute rejection.
PMCID: PMC4089251
Nitric oxide; Endothelial nitric oxide synthase; Polymorphism; Transplant; Acute rejection
4.  Is there a relationship between endothelial nitric oxide synthase gene polymorphisms and ankylosing spondylitis? 
Clinics  2013;68(3):305-309.
Nitric oxide is produced by endothelial nitric oxide synthase, and its production can be influenced by polymorphisms of the endothelial nitric oxide synthase gene. Because candidate genes responsible for susceptibility to ankylosing spondylitis are mostly unknown and available data suggest that there may be problems related to the nitric oxide pathway, such as endothelial dysfunction and increased asymmetric dimethylarginine, this study aimed to assess the association of common endothelial nitric oxide synthase gene polymorphisms with ankylosing spondylitis.
One hundred ninety-four unrelated Turkish ankylosing spondylitis patients and 113 healthy without apparent cardiovascular disease, hypertension or diabetes mellitus were included. All individuals were genotyped by PCR-RFLP for two single-nucleotide polymorphisms, namely 786T>C (rs2070744, promoter region) and 786 Glu298Asp (rs1799983, exon 7). Variable numbers of tandem repeat polymorphisms in intron 4 were also studied and investigated by direct electrophoresis on agarose gel following polymerase chain reaction analysis. The Bath ankylosing spondylitis metrology index of the patients was calculated, and human leukocyte antigen B27 was studied.
All studied polymorphisms satisfied Hardy-Weinberg equilibrium. Sex distributions were similar between the patient and control groups. No significant differences were found in the distributions of allele and genotype frequencies of the studied endothelial nitric oxide synthase polymorphisms between patients and controls. There were no correlations between endothelial nitric oxide synthase polymorphisms, disease duration, Bath ankylosing spondylitis metrology index or human leukocyte antigen B27.
The results presented in this study do not support a major role of common endothelial nitric oxide synthase polymorphisms in Turkish ankylosing spondylitis patients.
PMCID: PMC3611897  PMID: 23644848
Ankylosing Spondylitis; Endothelial Nitric Oxide Synthase; Nitric Oxide; Inflammation; Atherosclerosis
5.  Allelic polymorphism in the promoter (T–786→C), but not in exon 7 (G894→T) or the variable number tandem repeat in intron 4, of the endothelial nitric oxide synthase gene is positively associated with acute coronary syndrome in the Ukrainian population 
Different allelic variants of endothelial nitric oxide synthase (eNOS) can have different effects on the development of certain forms of ischemic heart disease, depending on the specific human population. The frequency of the polymorphism T–786 → C in the promoter, G894→T in exon 7 and the variable number tandem repeat in intron 4 were assayed in patients with acute coronary syndrome compared with clinically healthy individuals in the Ukrainian population.
Polymerase chain reaction and restriction fragment length polymorphism analysis were used to detect the above mentioned variants of the eNOS gene in 221 patients with acute coronary syndrome and in 83 control subjects.
It was shown that the percentage of normal homozygotes, heterozygotes and pathological homozygotes for the T–786→C promoter polymorphism was 47.5%, 36.2% and 16.3%, respectively (controls: 48.2%, 45.8% and 6.0%; P<0.05 by χ2 test); for the G894→T polymorphism in exon 7, the percentages were 34.4%, 57.9% and 7.7%, respectively (controls: 28.9%, 67.5% and 3.6%; P>0.05); and, for the 4a/4b polymorphism in intron 4, the percentages were 64.7%, 31.2% and 4.1%, respectively (controls: 62.7%, 32.5% and 4.8%; P>0.05).
The C/C promoter variant of eNOS can be considered a risk factor for acute coronary syndrome in the Ukrainian population.
PMCID: PMC2274837  PMID: 18651012
Acute coronary syndrome; Allelic polymorphism; Endothelial NO synthase
6.  The Role of −786T/C Polymorphism in the Endothelial Nitric Oxide Synthase Gene in Males with Clinical and Biochemical Features of the Metabolic Syndrome 
Background. Extensive evidence, arising from models of endothelial nitric oxide synthase gene (NOS3)-knockout mice supports the role of endothelial malfunction in the pathogenesis of the metabolic syndrome (MS). Aims. The aim of this study was to evaluate the role of −786T/C polymorphism in the etiology of MS and assess previously reported interaction with cigarette smoking. Methods. Based on International Diabetes Federation 2005 criteria, we recruited randomly 152 subjects with MS and 75 subjects without MS. Results. Allelic and genotype frequencies did not differ significantly between both groups. Total cholesterol level (CHOLT) and intima-media thickness of carotid arteries were significantly higher in −786CC homozygotes, in comparison with −786TC and −786TT patients. Regarding current smoking status, −786C allele was associated with higher CHOLT than −786T allele. Conclusion. Our study indicates the putative role of −786T/C polymorphism in the development of hypercholesterolemia, in patients with MS, which might be enhanced by cigarette smoking.
PMCID: PMC3227465  PMID: 22164159
7.  The 27-bp repeat polymorphism in intron 4 (27 bp-VNTR) of endothelial nitric oxide synthase (eNOS) gene is associated with albumin to creatinine ratio in Mexican Americans 
Molecular and cellular biochemistry  2009;331(1-2):201-205.
The T-786C, Glu298Asp, and 27 bp variable number of tandem repeats (27 bp-VNTR-a/b) polymorphsims of the endothelial nitric oxide synthase (eNOS) gene are thought to alter nitric oxide production and contribute to the development of vascular and renal disease risk. The objective of this study is to investigate whether these three polymorphisms examined previously by others are associated with cardiovascular and renal disease risk in Mexican Americans. Study participants (N = 848; 21 families) were genotyped for T-786C, Glu298Asp, and 27 bp-VNTR-a/b polymorphisms by PCR followed by restriction digestion. Association analyses were performed by a measured genotype approach implemented in the program SOLAR. Of the phenotypes (type 2 diabetes, hypertension, body mass index, waist circumference, total cholesterol, high density lipoprotein cholesterol, triglycerides, systolic and diastolic blood pressure, albumin to creatinine ratio (ACR), and estimated glomerular filtration rate) examined for association, the 27 bp-VNTR-a/b variant exhibited statistically significant association with ACR (P = 0.047) after accounting for the trait specific covariate effects. In addition, the promoter variant (T-786C) showed a significant association with triglycerides (P = 0.034) after accounting for covariate influences. In conclusion, the present study adds evidence to the role of eNOS candidate gene polymorphisms in modulating the risk factors related to cardiovascular-renal disease in Mexican Americans although the magnitude of the genetic effect is small.
PMCID: PMC3428139  PMID: 19468830
eNOS; Genetic polymorphisms; Association analyses; ACR; Triglycerides; Mexican Americans
8.  Genotype-Dependent Expression of Endothelial Nitric Oxide Synthase (eNOS) and Its Regulatory Proteins in Cultured Endothelial Cells 
DNA and cell biology  2005;24(4):218-224.
DNA polymorphisms in endothelial nitric oxide synthase (eNOS) gene have been shown to be associated with constitutive eNOS expression and coronary artery disease (CAD). In the present study we explored the hypothesis whether genotype-dependent effects can be maintained in vitro during replication, or the effect is conditional on in vivo biological environments. Human umbilical vein endothelial cells (HUVEC) were collected and cultured from 89 normal deliveries of Mexican Americans. The cells were treated with or without cigarette smoking extracts (CSE) and genotypes of eNOS polymorphisms were determined by PCR. We measured the levels of eNOS by ELISA and its binding proteins including heat-shock protein 90 (Hsp-90) and caveolin-1 by Western blotting. The rare C allele for the promoter T786C polymorphism (0.2), and the rare 4 × 27-bp repeat allele in the intron 4 (0.30) were different from those reported in other populations. Yet, the rare T allele in the exon 7 (G894T polymorphism) was similar as others. After four passages in vitro, both the in-tron 4 and promoter polymorphisms maintained significant effects on eNOS mRNA levels in HUVECs (P < 0.05). However, the effects on eNOS protein and enzyme activity were less consistent. Although primary smokers had significantly lower eNOS protein levels (P < 0.05), the in vitro CSE treatment on cultured HUVECs only resulted in a significant reduction in NO levels as measured by the stable metabolites of nitrite/nitrate (P < 0.001). Neither Hsp-90 nor caveolin-1—important eNOS regulators—appears to mediate the genotype-smoking effects on eNOS expression although HUVECs did produce more Hsp-90 when exposed to CSE. Our study demonstrates that endothelial cells maintain genotype-dependent expression even after the deprivation of in vivo environment. However, the cigarette smoking–genotype interaction may require such in vivo conditions to be manifested.
PMCID: PMC1350115  PMID: 15812238
9.  Role of Endothelial Nitric Oxide Synthase Gene Polymorphisms in Predicting Aneurysmal Subarachnoid Hemorrhage in South Indian Patients 
Disease markers  2008;24(6):333-339.
Endothelial nitric oxide synthase (eNOS) gene polymorphisms have been implicated as predisposing genetic factors that can predict aneurysmal subarachnoid hemorrhage (aSAH), but with controversial results from different populations. Using a case-control study design, we tested the hypothesis whether variants in eNOS gene can increase risk of aSAH among South Indian patients, either independently, or by interacting with other risk factors of the disease. We enrolled 122 patients, along with 224 ethnically matched controls. We screened the intron-4 27-bp VNTR, the promoter T-786C and the exon-7 G894T SNPs in the eNOS gene. We found marked interethnic differences in the genotype distribution of eNOS variants when comparing the South Indian population with the reported frequencies from Caucasian and Japanese populations. Genotype distributions in control and patient populations were found to be in Hardy-Weinberg equilibrium. In patients, the allele, genotype and estimated haplotype frequencies did not differ significantly from the controls. Multiple logistic regression indicated hypertension and smoking as risk factors for the disease, however the risk alleles did not have any interaction with these risk factors. Although the eNOS polymorphisms were not found to be a likely risk factor for aSAH, the role of factors such as ethnicity, gender, smoking and hypertension should be evaluated cautiously to understand the genotype to phenotype conversion.
PMCID: PMC3850602  PMID: 18688082
eNOS; SNP; aneurysm; subarachnoid hemorrhage; India
10.  Association of endothelial nitric oxide synthase promoter region (T-786C) gene polymorphism with acute coronary syndrome and coronary heart disease 
Nitric oxide (NO) is an endothelium derived relaxing factor (EDRF) which has an important role for regulating the heart-vessel physiology. The objective of this study was to evaluate the effects of the eNOS T-786C polymorphism on lipid parameters and the development of acute coronary syndrome (ACS) and coronary heart disease (CHD) for the first time in a Turkish study group. We have analyzed the genotype frequencies of the T-786C polymorphism of the eNOS gene in 10 ACS patients (5 men, 5 women), 20 CHD patients (14 men, 6 women), and 31 controls (10 men, 21 women), who were angiographically proven to have normal coronaries.
The demographic, biochemical and left ventricule systolic dysfunction data of the ACS, CHD patients and controls were analyzed as a function of eNOS T-786C genotypes. The eNOS gene T-786C polymorphism frequencies for T/T, C/T and C/C genotypes were respectively 10%, 40%, 50% in subjects with ACS; 75%, 20%, 5% in subjects with CHD and 67.7%, 25.8%, 6.5% in the control group. Significant difference was observed in genotype frequencies between the study groups for T-786C polymorphism (p = 0.001). The CC genotype frequency was found to be the most prevalent in ACS group in comparison to CHD and control groups (p = 0.001). TT was the most frequently observed genotype in both CHD patients and controls (p = 0.001). Left ventricule systolic dysfunction frequency was found to be highest in C/T genotype carriers (66.7%) in patients (ACS+CHD). None of the patients with LVSD were carrying the normal genotype (T/T). The eNOS T-786C polymorphism was not found to be effective over any analyzed lipid variable in patients (ACS+CHD). The HDL-cholesterol levels were found to be lower in CHD group were compared to controls (p < 0.01), whereas glucose and leucocyte levels of the ACS and CHD groups were both higher than controls (p < 0.001).
The significantly high frequency of eNOS -786C/C genotype in ACS patients than in those of controls, indicate the genotype association with ACS. The finding of significantly high frequency of T/T genotype in the CHD group, may support the relationship of CC genotype with ACS without CHD. The high frequency of the mutant (C/C) and heterozygous (C/T) genotypes found may be linked to left ventricule remodeling after MI.
PMCID: PMC2267783  PMID: 18298848
11.  A Promoter Polymorphism of the Endothelial Nitric Oxide Synthase Gene is Associated with Reduced mRNA and Protein Expression in Failing Human Myocardium 
Journal of cardiac failure  2010;16(4):314-319.
Alterations of endothelial nitric oxide synthase (eNOS) enzyme activity via eNOS gene polymorphisms have been associated with significant cardiovascular morbidity and mortality. Both the thymidine to cytosine transition mutation (T−786→C) in the promoter region and the missense mutation in the exon 7 coding region of the eNOS gene (G894→T) have been associated with several cardiovascular disease states. We hypothesized that heart transplant recipients who carried at least one allele of either of the polymorphisms would have reduced myocardial tissue expression of eNOS measured in the explanted heart.
Genomic DNA was isolated from myocardial tissue samples obtained from 43 explanted human hearts using standard methods. Regions of the eNOS gene were amplified from genomic DNA with a polymerase chain reaction using specific primers. Protein expression of eNOS was measured by Western blot analysis. There was a statistically significant decrease in mean eNOS expression in samples containing at least one allele for the T−786→C promoter polymorphism (p = 0.04) compared to patients homozygous for the T allele. There was no change in eNOS expression associated with the G894→T exonic polymorphisms. Conclusions: Our data show in failing human myocardium that the T−786→C promoter polymorphism is associated with reduced eNOS expression whereas the G894→T polymorphism of exon 7 is not associated with change in either eNOS mRNA or protein expression. Reduced eNOS expression associated with the promoter polymorphism may contribute to the vascular, contractile, and autonomic responses to ventricular failure.
PMCID: PMC2848179  PMID: 20350698
Nitric Oxide; Genetics; Cardiomyopathy; Pharmacogenetics
12.  A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays 
BMC Medical Genetics  2012;13:34.
Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation.
The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing.
The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS −786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele).
Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants.
PMCID: PMC3511178  PMID: 22594584
13.  Endothelial nitric oxide synthase gene polymorphisms and cardiovascular damage in hypertensive subjects: an Italian case-control study 
Nitric oxide (NO) synthesized by endothelial nitric oxide synthase (eNOS) plays an important role in regulation of endothelial function and in the control of blood pressure. However, the results from some studies on the association between three clinically relevant eNOS gene polymorphisms (G894T, T786C and intron 4b/a) and essential hypertension are unclear. We designed a case-control study to evaluate the influence of eNOS polymorphisms on target organ damage in 127 hypertensives and 67 normotensives. Clinical evaluation, biochemical parameters, Urinary Albumin Excretion (UAE) and echocardiogram were performed to characterize target organ damage. eNOS polymorphism were recognized by PCR method.
The distribution of eNOS genotypes was similar in hypertensives and normotensives but 4aa was present in the 2.5% of hypertensives and completely absent in normotensives. Subjects with 4bb, G894T, and T786C genotypes showed an increased prevalence of target organ damage. Moreover prevalence of G894T and introne 4 variants was significantly higher in hypertensives than in normotensives both with cardiovascular damage. Logistic regression analysis didn't show any association between eNOS polymorphisms, Body Mass Index (BMI), hypertension, gender and cardiovascular damage. Only the age (OR 1.11; IC 95% 1.06–1.18) was predictive of cardiovascular damage in our population.
Our results seem to indicate a lack of association with eNOS variants and cardiovascular damage onset.
PMCID: PMC2430541  PMID: 18510731
14.  Promoter Polymorphisms in the Nitric Oxide Synthase 3 Gene Are Associated With Ischemic Stroke Susceptibility in Young Black Women 
Background and Purpose
Endothelial nitric oxide exerts a variety of protective effects on endothelial cells and blood vessels, and therefore the nitric oxide synthase 3 gene (NOS3) is a logical candidate gene for stroke susceptibility.
We used the population-based Stroke Prevention in Young Women case-control study to assess the association of five NOS3 polymorphisms in 110 cases (46% black) with ischemic stroke and 206 controls (38% black), 15 to 44 years of age. Polymorphisms included 3 single nucleotide polymorphisms (SNPs) in the promoter region (−1468 T>A, −922 G>A, −786 T>C), 1 SNP in exon 7 (G894T), and 1 insertion/deletion polymorphism within intron 4.
Significant associations with both the −922 G>A and −786 T>C SNPs with ischemic stroke were observed in the black, but not the white, population. This association was attributable to an increased prevalence of the −922 A allele (OR=3.0, 95% CI=1.3 to 6.8; P=0.005) and the −786 T allele (OR=2.9, 95% CI=1.3 to 6.4; P=0.005) in cases versus controls. These 2 SNPs were in strong linkage disequilibrium (D′=1.0), making it impossible to determine, within the confines of this genetic study, whether 1 or both of these polymorphisms are functionally related to NOS3 expression. Two sets of haplotypes were also identified, 1 of which may confer an increased susceptibility to stroke in blacks, whereas the other appears to be protective.
Promoter variants in NOS3 may be associated with ischemic stroke susceptibility among young black women.
PMCID: PMC1494105  PMID: 16100023
genetics; nitric oxide; women and minorities; young, stroke in
15.  Endothelial nitric oxide synthase gene polymorphisms and risk of diabetic nephropathy: a systematic review and meta-analysis 
BMC Medical Genetics  2014;15:9.
Nitric oxide (NO) has numerous functions in the kidney, including control of renal and glomerular hemodynamics, by interfering at multiple pathological and physiologically critical steps of nephron function. Endothelial NOS (eNOS) gene has been considered a potential candidate gene to diabetic nephropathy (DN) susceptibility. Endothelial nitric oxide synthase gene (eNOS-3) polymorphisms have been associated with DN, however some studies do not confirm this association. The analyzed polymorphisms were 4b/4a, T-786C, and G986T.
The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement was used in this report. Case–control studies that had diabetic patients with DN as cases and diabetic patients without nephropathy as controls, as well as that evaluated at least one of the three polymorphisms of interest were considered eligible. All studies published up until December 31st, 2012 were identified by searching electronic databases. Hardy-Weinberg equilibrium assessment was performed. Gene-disease association was measured using odds ratio estimation based on the following genetic contrast/models: (1) allele contrast; (2) additive model; (3) recessive model; (4) dominant model and (4) co-dominant model.
Twenty-two studies were eligible for meta-analysis (4b/a: 15 studies, T-786C: 5 studies, and G984T: 12 studies). Considering 4b/a polymorphism, an association with DN was observed for all genetic models: allele contrast (OR = 1.14, CI: 1.04-1.25); additive (OR = 1.77, CI: 1.37-2.28); recessive (OR = 1.77, CI: 1.38-2,27); dominant (OR = 1.12, CI: 1.01-1.24), with the exception for co-dominance model. As well, T-786C polymorphism showed association with all models, with exception for co-dominance model: allele contrast (OR = 1.22, CI: 1.07-1.39), additive (OR = 1.52, CI: 1.18-1.97), recessive (OR = 1.50, CI: 1.16-1.93), and dominant (OR = 1.11, CI: 1.01-1.23). For the G894T polymorphism, an association with DN was observed in allelic contrast (OR = 1.12, CI: 1.03-1.25) and co-dominance models (OR = 1.13, CI: 1.04-1.37).
In the present study, there was association of DN with eNOS 4b/a and T-786C polymorphism, which held in all genetic models tested, except for co-dominance model. G894T polymorphism was associated with DN only in allele contrast and in co-dominance model. This data suggested that the eNOS gene could play a role in the development of DN.
PMCID: PMC3900462  PMID: 24433471
16.  eNOS Gene Variants and the Risk of Premature Myocardial Infarction 
Disease markers  2013;34(6):431-436.
BACKGROUND: Endothelial nitric oxide synthase (eNOS) as well as nitric oxide play an important role in the regulation of cardiovascular function. There are limited and controversial data regarding the impact of polymorphisms of eNOS gene that is implicated in the vasoconstrictive properties of the endothelium in the pathogenesis of premature myocardial infarction (MI).
OBJECTIVE: We examined whether two common polymorphisms of eNOS gene (G894T and T786C) are associated with the development of premature MI.
METHODS: We recruited 107 patients with premature MI and compared them to 103 age- and sex- matched controls. All patients underwent coronary angiogram and were classified into the subgroup of patients with ‘normal’ or ‘near normal’ coronary arteries and the subgroup of patients with significant coronary artery disease (≥ 50% stenosis in lumen diameter of coronary arteries). The genetic polymorphisms of eNOS gene were assayed with polymerase chain reaction and reverse hybridization.
RESULTS: Nineteen patients (17.8%) had ‘normal’ or ‘near normal’ coronary arteries. A significantly higher frequency of homozygosity for the 786C (32%) and the 894T (21%) alleles of the eNOS gene in patients who develop early MI in the setting of angiographically 'normal' or 'near normal' coronary arteries were found.
CONCLUSIONS: Our data suggest that the T786C and the G894T genetic polymorphisms are associated with the development of MI in very young individuals, whose coronary arteries are characterized by very small atheromatic burden.
PMCID: PMC3809742  PMID: 23594558
Cardiovascular disease; genetic polymorphisms; early myocardial infarction; endothelial nitric oxide synthase
17.  Endothelial nitric oxide synthase polymorphism (-786T>C) and increased risk of angiographic vasospasm after aneurysmal subarachnoid hemorrhage 
Background and Purpose
Vasospasm following aneurysmal subarachnoid hemorrhage (SAH) remains a leading cause of death and disability after aneurysm rupture. Decreased availability of nitric oxide (NO) may be crucial in the pathogenesis. We hypothesize that endothelial NO synthase (eNOS) polymorphisms may determine susceptibility to vasospasm in SAH patients.
We conducted a prospective cohort study of SAH patients, and determined vasospasm by cerebral angiography. We genotyped three eNOS polymorphisms: intron 4 variable-number-tandem-repeat (VNTR), promoter single-nucleotide-polymorphism (-786T>C SNP), and coding SNP in exon 7 (894G>T encoding E298D). Using multivariable logistic regression, we quantified the association of eNOS polymorphisms in patients with vasospasm confirmed by cerebral angiogram.
For the eNOS promoter -786T>C SNP, the presence of the CC genotype compared to any T genotype (CT or TT) was associated with increased odds of vasospasm (OR 2.97, 95%CI 1.32-6.67, p=0.008). No association with vasospasm was observed for the eNOS 894G>T or VNTR polymorphisms.
These findings suggest that genetic variation influencing NO regulation contributes to risk of angiographic vasospasm in patients with SAH. The specific role of the promoter SNP (-786T>C) may determine the effect of NO regulated by this pathway distinct from other known eNOS polymorphisms.
PMCID: PMC2650384  PMID: 18309169
Endothelial nitric oxide; Genetics; Subarachnoid hemorrhage; Vasospasm
18.  Genetic and Pharmacogenetic Associations Between NOS3 Polymorphisms, Blood Pressure, and Cardiovascular Events in Hypertension 
American journal of hypertension  2009;22(7):748-753.
Polymorphisms in the endothelial nitric oxide synthase (NOS3) gene increase susceptibility to hypertension and cardiovascular disease. We examined genetic and pharmacogenetic associations between NOS3 polymorphisms, blood pressure (BP) control, and cardiovascular events in elderly, hypertensive coronary artery disease (CAD) patients.
Patients with CAD were randomly assigned to either verapamil SR– or atenolol-based antihypertensive treatment and followed for cardiovascular events. Cases (all-cause death, nonfatal myocardial infarction (MI), or nonfatal stroke) and an age-, sex-, race/ethnicity-matched control population were genotyped for the -786T>C and Glu298>Asp polymorphisms in NOS3. On-treatment BP and BP control were compared across genotype groups. Logistic regression was performed to estimate odds ratios (ORs) for the -786T>C and Glu298>Asp polymorphisms in the combined population and in randomized treatment groups.
Genotype data were available for 256 cases and 769 controls. Among controls, mean on-treatment BP differed according to -786T>C genotype (T/T 137/78 mm Hg, T/C 133/76 mm Hg, C/C 133/75 mm Hg; P = 0.0007 for systolic, P = 0.09 for diastolic) which corresponded to differing rates of BP control (T/T 63%, T/C 72%, C/C 88%; P = 0.002). Neither polymorphisms was associated with case status, with or without regard to assigned treatment.
The -786T>C, but not the Glu298>Asp variant of NOS3, may correlate with BP but do not appear to be associated with incident cardiovascular events in patients with established cardiovascular disease. The antihypertensive treatment approach did not appear to alter the genetic contribution to either BP control or cardiovascular events.
PMCID: PMC2803093  PMID: 19407804
19.  Endothelial Nitric Oxide Synthase (eNOS) Gene Polymorphism is Associated with Age Onset of Menarche in Sickle Cell Disease Females of India 
Background and Objective
Females with sickle cell disease (SCD) often show late onset of menarche. In transgenic sickle cell mouse, deficiency of gene encoding endothelial nitric oxide synthase (eNOS) has been reported to be associated with late onset of menarche. Thus to explore the possible association of eNOS gene polymorphism with age of onset of menarche in SCD females, 3 important eNOS gene polymorphisms- eNOS 4a/b, eNOS 894G>T (rs1799983) and eNOS-786 T>C (rs2070744) and plasma nitrite levels were tested among three groups of females- SCD late menarche, SCD early menarche and control females.
About 39 SCD females comprising of 18 SCD early menarche and 21 SCD late menarche groups were studied along with 48 control females. Genotyping of eNOS gene polymorphisms were done by PCR-RFLP and quantification of plasma nitrite level was performed by ELISA based commercial kits.
SCD late menarche females showed significantly higher prevalence and higher association of heterozygous genotypes, higher frequency of mutant alleles .4a., .T. and .C. as compared to that of control group and SCD early menarche group. The frequency of haplotype .4a-G-C. and haplotype .4b-G-C. (alleles in order of eNOS 4a/b, eNOS 894G>T and eNOS-786 T>C respectively) were found to be significantly high in SCD late menarche compared to combined groups of SCD early menarche and controls. SCD late menarche group had significantly low level of plasma nitrite concentration for all 3 eNOS gene polymorphisms as compared to controls and SCD early menarche females.
eNOS gene polymorphism may influence age of onset of menarche in SCD females.
PMCID: PMC3684346  PMID: 23795274
20.  Association of the genetic polymorphisms of the ACE gene and the eNOS gene with lupus nephropathy in northern Chinese population 
Li, X | An, J | Guo, R | Jin, Z | Li, Y | Zhao, Y | Lu, F | Lian, H | Liu, P | Zhao, Y | Jin, X
BMC Medical Genetics  2010;11:94.
It has been reported that some single nucleotide polymorphisms (SNPs) of the angiotensin converting enzyme (ACE) gene and the endothelial nitric oxide synthase (eNOS) gene are associated with the development of systemic lupus erythematosus (SLE) and the progression of nephropathy. The aim of this study was to evaluate the possible association between six SNPs (A-5466C, T-3892C, A-240T, C1237T, G2215A and A2350G) of the ACE gene and two SNPs (T-786C and G894T) of the eNOS gene with lupus nephropathy in a northern Chinese population.
In this study, 225 patients with lupus nephropathy were compared to 232 healthy controls, matched by gender, age and ethnicity. Following the extraction of genomic DNA from the leukocytes in the peripheral blood, the genotypes of the eight selected SNPs were determined by the method of PCR-RFLP; the haplotypes were inferred using PHASE 2.1. The associations between the SNPs and the risk of lupus nephropathy were analyzed using Chi-square test and Logistic regression with SPSS13.0 software.
Statistically significant differences of the allele frequency distribution of three SNPs (A-5466C, A2350G and G894T) were observed between cases and controls (P < 0.05). Among the 53 haplotypes identified, the frequencies of five haplotypes (CTTCGA, ACTTAA, ACATGG, ACACGG and ATTCGA) were significantly different between cases and controls (P < 0.05).
Our study indicated an association between the risk of lupus nephropathy and the sequence variations of both the ACE gene and the eNOS gene, which may play an important role in the pathogenesis of lupus nephropathy in the northern Chinese population. Further studies are warranted to validate our findings.
PMCID: PMC2903533  PMID: 20540812
21.  An intron 4 VNTR polymorphism of the endothelial nitric oxide synthase gene is associated with early-onset colorectal cancer 
Endothelial derived nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), may play an important role in colorectal carcinogenesis. However, the putative contribution of common eNOS genetic polymorphisms to colorectal cancer risk remains unknown. We genotyped 3 polymorphisms of eNOS (T-786C, G894T, and intron4b/a) in 727 colorectal adenocarcinoma cases and 736 age- and sex-matched healthy controls in Taiwan. Genotypes of the T-786C and G894T polymorphisms were determined by fluorescence polarization assays and the 27-bp variable number of tandem repeat (VNTR) polymorphism in intron 4 (intron4b/a) was analyzed by PCR. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Among younger participants (≤ 60 yrs), the intron4a variant genotype was associated with a significantly increased risk of colorectal cancer, compared with the intron4bb genotype (OR = 1.60, 95% CI = 1.04-2.46). In addition, those young individuals bearing a greater number of high-risk genotypes (OR > 1, i.e. CT+TT for T-786C, ba+aa for intron4b/a, and GG for G894T) of eNOS had a higher colorectal cancer risk (P trend = 0.039). Compared with younger individuals without any putative high-risk genotypes, those with three high-risk genotypes had a significantly greater cancer risk (OR = 1.89, 95% CI = 1.04-3.43). Our results suggest that the eNOS intron4b/a polymorphism may contribute to early-onset colorectal cancer risk in the Taiwanese population.
PMCID: PMC2653782  PMID: 19115208
colorectal cancer; endothelial nitric oxide synthase; polymorphisms; early-onset
22.  Role of Genetic Changes in the Progression of Cardiovascular Diseases 
This review aims to investigate the role of genetic changes in the development of cardiovascular diseases [CVD]. Oxidation of Low density Lipoprotein (LDL) and mutations in LDL receptors gene are a trigger for numerous of atherogenic events. Also, endothelial nitric oxide synthase (eNOS) plays an important role in vasodilatation of blood vessels through synthesis of nitric oxide. Three single base pair changes, 786T/C, 922A/G, and 1468T/A, have been identified in the promoter region of the eNOS gene and are associated with coronary spasm. Moreover, two distinct variable nucleotide tandem repeats (VNTRs) in introns 4 and 13 have been detected. The presence of a minimum of 38 CA repeats in intron 13 has been associated with an independent 2.2-fold increase in the risk of coronary artery disease [CAD]. Plasma glutathione peroxidase (GPx-3) maintains the vascular bioavailability of nitric oxide (NO), through depletion of reactive oxygen species. Mutation(s) or polymorphism(s) in the plasma GPx-3 gene promoter may predispose to a thrombotic disorder, and constitute a genetic risk factor for thrombotic cerebrovascular disease. Hyperhomocysteinemia is another independent risk factor for atherosclerosis and arterial thrombosis. Severe hyperhomocysteinemia could be caused by cystathionine-β-synthase enzyme deficiency but it could be due to homozygosity of a common 677C/T point mutation in the coding region of the methylenetetrahydrofolate reductase (MTHFR) gene as a 3-fold increase in risk of CAD is associated with the MTHFR 677TT genotype. A second common variant in MTHFR 1298A/C is associated with decreased enzyme activity in vitro and in vivo, especially when occurring simultaneously with the 677 C/T polymorphism. Elevated fibrinogen, an essential component of the coagulation system, has been most consistently associated with arterial thrombotic disorders. Several polymorphisms (148C/T, 455G/A, and -854G/A) have been identified in the genes encoding the 3 pairs of fibrinogen polypeptide chains. The -455G/A, and -854G/A substitutions are the most physiologically relevant mutations. In addition the -455A allele has been associated with the progression of atheroma, and also with a 2.5-fold increase in risk of multiple lacunar infarcts in a cohort of elderly patients with stroke. It is concluded that genetic changes in the previously mentioned genes could play a significant role in the initiation and progression of CVD. This review provides useful information for both physicians and medical students whom are interested in human genetics which is related to cardiovascular diseases.
PMCID: PMC3614847  PMID: 23675242
cardiovascular diseases; genetic polymorphisms of nitric oxidesynthase; hyperlipidemia; hyperhomocysteinemia polymorphisms; paraoxonase polymorphisms
23.  Endothelin-1 but not Endothelial Nitric Oxide Synthase Gene Polymorphism is Associated with Sickle Cell Disease in Africa 
Sickle cell disease shows marked variability in severity and pathophysiology among individuals, probably linked to differential expression of various adhesion molecules. In this study, we investigated the differential distribution, genomic diversity and haplotype frequency of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) polymorphisms, recently implicated as important in modification of disease severity. One hundred and forty five sickle cell disease patients (HbSS) and 244 adult and pediatric controls, without sickle cell disease (HbAA), were recruited from Mali. Genotypic analysis of the functionally significant eNOS variants (T786C, G894T and intron 4) and endothelin-1 (G5665T) was carried out with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Our results show that the wild type alleles are the most frequent for all eNOS variants between cases and controls. Allelic and genotypic frequencies of eNOS polymorphic groups are not significantly different between cases and controls (P > 0.05). In addition, there is no association between eNOS variants and sickle cell disease, contrary to published reports. On the other hand, we report that endothelin-1 (G5665T) mutant variant had the lowest allelic frequency, and is significantly associated with sickle cell disease in Africa (P < 0.05). Similarly, haplotype frequencies were the same between cases and controls, except for the haplotype combining all mutant variants (T, C, 4a; P = 0.01). eNOS polymorphic variants are less frequent, with no significance with sickle cell disease in Africa. On the other hand, endothelin-1 is associated with sickle cell disease, and has the capacity to redefine pathophysiology and possibly serve as modulator of disease phenotype.
PMCID: PMC4051816  PMID: 24932102
endothelial nitric oxide synthase; endothelin-1; sickle cell disease; polymorphisms; pathophysiology
24.  Endothelial nitric oxide synthase gene T−786C and 27-bp repeat gene polymorphisms in retinopathy of prematurity 
Molecular Vision  2008;14:286-290.
Retinopathy of prematurity (ROP), which is associated with abnormal retinal vessel development, is the leading cause of visual loss in preterm infants. Endothelial nitric oxide synthase (eNOS) is believed to play a central role in both retinal angiogenesis and vasculogenesis. The aim of this study was to investigate functional genetic polymorphisms of eNOS in the pathogenesis of ROP.
eNOS T−786C and 27-bp repeat (eNOS, b: wild-type, a: mutant) genotypes were determined using allele-specific polymerase chain reaction in 105 low birth weight (LBW) preterm infants treated for ROP (treated group). A control group was set up and composed of 127 LBW infants with stage 1 or 2 ROP that did not not require treatment (untreated group).
The genotype distribution of eNOS 27-bp repeat polymorphism was found to significantly differ (p=0.015) between the two groups, whereas the genotype distribution of eNOS T−786C did not differ (p=0.984) between the groups. There was no difference in the distribution of either the “a” allele (p=0.153) nor of the C allele (p=0.867) in a groups comparison. Multiple logistic regression analysis revealed that male gender (p=0.046) and eNOS aa genotype (p=0.047 versus ab genotype and p=0.022 versus bb genotype) were significantly associated severe ROP that required treatment. The haplotype estimations based on the detected genotype distributions showed that the prevalence of aT and bT haplotypes was significantly increased in the group treated for ROP.
Functional eNOS 27-bp repeat polymorphism might be associated with the risk of severe ROP, however we found no association between the eNOS T−786C and the pathogenesis of ROP.
PMCID: PMC2263012  PMID: 18334945
25.  Overexpression of Endothelial Nitric Oxide Synthase Prevents Diet-Induced Obesity and Regulates Adipocyte Phenotype 
Circulation research  2012;111(9):1176-1189.
Endothelial dysfunction is a characteristic feature of diabetes and obesity in animal models and humans. Deficits in nitric oxide production by endothelial nitric oxide synthase (eNOS) are associated with insulin resistance, which is exacerbated by high fat diet. Nevertheless, the metabolic effects of increasing eNOS levels have not been studied.
The current study was designed to test whether overexpression of eNOS would prevent diet-induced obesity and insulin resistance.
Methods and Results
In db/db mice and in high fat-fed wild-type (WT) C57BL/6J mice, the abundance of eNOS protein in adipose tissue was decreased without significant changes in eNOS levels in skeletal muscle or aorta. Mice overexpressing eNOS (eNOS-TG mice) were resistant to diet-induced obesity and hyperinsulinemia, although systemic glucose intolerance remained largely unaffected. In comparison with WT mice, high fat-fed eNOS-TG mice displayed a higher metabolic rate and attenuated hypertrophy of white adipocytes. Overexpression of eNOS did not affect food consumption or diet-induced changes in plasma cholesterol or leptin levels, yet plasma triglycerides and fatty acids were decreased. Metabolomic analysis of adipose tissue indicated that eNOS overexpression primarily affected amino acid and lipid metabolism; subpathway analysis suggested changes in fatty acid oxidation. In agreement with these findings, adipose tissue from eNOS-TG mice showed higher levels of PPAR-α and PPAR–γ gene expression, elevated abundance of mitochondrial proteins, and a higher rate of oxygen consumption.
These findings demonstrate that increased eNOS activity prevents the obesogenic effects of high fat diet without affecting systemic insulin resistance, in part, by stimulating metabolic activity in adipose tissue.
PMCID: PMC3707504  PMID: 22896587
Diabetes; mitochondria; metabolism; adipose tissue; cardiovascular disease; obesity; endothelial nitric oxide synthase

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