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Clinical Chemistry 51: 1159-1164, 2005. First published May 19, 2005; 10.1373/clinchem.2005.048850
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(Clinical Chemistry. 2005;51:1159-1164.)
© 2005 American Association for Clinical Chemistry, Inc.

Hemostasis and Thrombosis

Endothelial Nitric Oxide Synthase –786T>C, but Not 894G>T and 4a4b, Polymorphism Influences Plasma Homocysteine Concentrations in Persons with Normal Vitamin Status

Cinzia Fatini1,3,4,a, Francesco Sofi1,3,4, Anna Maria Gori1,3,4, Elena Sticchi1,3,4, Rossella Marcucci1,3,4, Meri Lenti1,3,4, Alessandro Casini2, Calogero Surrenti2, Rosanna Abbate1,3,4 and Gian Franco Gensini1,3,4

1 Department of Medical and Surgical Critical Care, 2 Department of Clinical Pathophysiology, Unit of Nutrition, and 4 Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, Florence, Italy.
3 Thrombosis Centre, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy.

aAddress correspondence to this author at: Department of Medical and Surgical Critical Care, Thrombosis Centre, University of Florence, Viale Morgagni 85, 50134 Florence, Italy. Fax 39-05-54279417; e-mail cinziafatini{at}hotmail.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Nitric oxide (NO) plays a relevant role in various events during atherogenesis. In vitro data suggest that NO may modulate homocysteine (Hcy) concentrations. The aim of this study was to investigate the role of endothelial nitric oxide synthase (eNOS) –786T>C, 894G>T, and 4a4b polymorphisms in influencing Hcy concentrations.

Methods: Blood samples were obtained from 1287 unrelated persons. Plasma Hcy was measured by fluorescence polarization immunoassay, folate and vitamin B12 by RIA, vitamin B6 by HPLC, and eNOS and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms by PCR with restriction fragment length polymorphism analysis.

Results: MTHFR 677C>T polymorphism significantly influenced Hcy concentrations after adjustment for all confounding variables (P <0.0001 for trend). Univariate analysis showed that the eNOS –786T>C polymorphism, but not 894G>T and 4a4b, was significantly associated with the risk of having Hcy in the third tertile [>13.4 µmol/L; odds ratio (OR) = 1.2; 95% confidence interval (CI), 1.02–1.5; P = 0.03]. After adjustment for all variables known to influence Hcy, the –786T>C polymorphism still influenced Hcy concentrations (OR = 1.9; 95% CI, 1.1–3.2; P = 0.01). By analyzing the influence of eNOS polymorphisms on plasma Hcy concentrations according to vitamin concentrations (folate, vitamin B6, and vitamin B12), age, and smoking habits, we found a significant association between the eNOS –786T>C polymorphism and Hcy in nonsmokers, in persons with normal vitamin status, and in persons <60 years.

Conclusion: The eNOS –786T>C polymorphism, but not 894G>T and 4a4b, influences plasma Hcy concentrations mildly but significantly and independently.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO),1 an endothelium-derived relaxing factor, is one of the most relevant molecules involved in biological systems. NO is synthesized from L-arginine by at least 3 isoforms of NO synthase (inducible, neuronal, and endothelial) (1), contributes to vascular tone regulation, and plays a role in various important events during atherogenesis. In addition to relaxing vascular smooth muscle cells, NO inhibits platelet aggregation (2) and leukocyte adhesion to vascular endothelium (3), inhibits smooth muscle cell migration and proliferation (4), and limits the oxidation of atherogenic LDL (5). Impaired availability of endothelium-derived NO has been identified as a mediator of atherosclerosis (6).

High plasma homocysteine (Hcy) concentrations have been shown to play a role in atherosclerosis (7). Several factors, such as circulating vitamins and their intake, smoking habits, and genetic components involved in methionine metabolism, are known to influence plasma Hcy concentrations.

In vitro data suggested that NO may modulate Hcy concentration either by direct inhibition of methionine synthase or via an indirect effect on folate catabolism (8)(9). Common polymorphisms in the gene encoding for endothelial nitric oxide synthase (eNOS) influence the production and functional activity of the enzyme. A substitution of guanine to thymine at nucleotide 894 in exon 7 of the eNOS gene (894G>T), which leads to an amino acid change from Glu to Asp at codon 298 (also called Glu298Asp) is associated with reduced basal NO production (10). Evidence that this polymorphism is associated with atherosclerosis has been found (11)(12)(13). A clinical study indicated that the eNOS 894G>T polymorphism may represent a risk factor for increased plasma Hcy in healthy persons (14), whereas other recent studies did not confirm a role for this polymorphism in affecting Hcy concentrations (15)(16).

Another polymorphism, resulting from a thymine being replaced by a cytosine at nucleotide –786 (–786T>C) and responsible for a reduction in the eNOS gene promoter activity, has been associated with atherosclerotic disease (17)(18). Finally, a variable number of tandem repeats (VNTR) 27-bp repeat polymorphism (4a4b) is associated with altered plasma NO concentrations (19) and has been found to be responsible for variations in the genetic control of plasma nitrite and nitrate concentrations (20). No data are available on the influence of the eNOS –786T>C and 4a4b polymorphisms on Hcy concentrations.

We undertook this study to determine whether plasma Hcy concentrations are influenced by these 3 eNOS gene polymorphisms in addition to the methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism and circulating concentrations of vitamins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
study design
Blood samples were obtained from 1287 unrelated persons (708 males and 584 females; median age, 60 years; range, 20–78 years) recruited between 2001 and 2003 from the staff of the University of Florence, from their partners and friends (n = 544), and from a population study, "Progetto Nutrizione per la Salute e la Prevenzione di Malattia" (21), conducted between 2002 and 2004 and aimed at evaluating lifestyle and dietary habits of clinically healthy persons living in Florence, Italy (n = 743). A detailed interview concerning personal and family histories was performed by expert physicians in conjunction with a physical examination to identify symptom-free individuals and to exclude those suspected of having any kind of vascular disease. Current smoking status was determined at the time of blood collection.

The participants were considered to have hypertension if they had been diagnosed according to the guidelines of the European Society of Hypertension/European Society of Cardiology (22) or were taking antihypertensive drugs. Dyslipidemia was defined according to the Third Report of the National Cholesterol Education Program (23) and diabetes according to the guidelines of the American Diabetes Association (24).

All participants were Caucasian and from the same general area (central Italy); all gave informed consent. The study complies with the Declaration of Helsinki and was approved by the local ethics committee of the Faculty of Medicine, University of Florence.

laboratory measurements
Venous blood was collected from all participants after an overnight fast; blood was collected from an antecubital vein, without venous stasis, by use of a 19-gauge needle into Vacutainer Tubes (Becton Dickinson) containing 0.129 mol/L sodium citrate (final ratio to blood, 1:10). Serum creatinine was measured by a conventional method. To measure Hcy concentrations, whole venous blood was collected into tubes containing EDTA (0.17 mol/L), which were immediately placed in ice and centrifuged within 30 min at 4 °C (1500g for 15 min). The supernatant was stored in aliquots at –80 °C until assay. Plasma total Hcy (free and protein bound) was measured by fluorescence polarization immunoassay (IMx; Abbot Laboratories).

Serum folate, vitamin B6, and vitamin B12 were measured in blood samples collected into evacuated tubes without anticoagulant and centrifuged at 2000g for 10 min; serum was stored at –20 °C until assay. Serum folate and vitamin B12 concentrations were measured by RIA (ICN Pharmaceuticals) and serum vitamin B6 by HPLC (Immundiagnostik).

detection of gene polymorphisms
eNOS and MTHFR 677C>T polymorphisms were analyzed from genomic DNA extracted from peripheral blood leukocytes by a QIAamp Blood Kit (Qiagen). The MTHFR 677C>T polymorphism was analyzed as described previously (25).

ENOS –786T>C polymorphism
The eNOS –786T>C polymorphism was analyzed by PCR with restriction fragment length polymorphism analysis. PCR was performed with the primers 5'-GTGTACCCCACCTGCATTCT-3' (sense) and 5'-CCCAGCAAGGATGTAGTGAC-3' (antisense), and DNA (100 ng) was amplified in a final volume of 20 µL at an annealing temperature of 60 °C in a DNA thermal cycler. The PCR product (306 bp) was digested with 1 U of NaeI restriction enzyme (Turbo Nae I; Promega) at 37 °C for 4 h to obtain fragments of 225 and 81 bp. The PCR digestion products were then subjected to electrophoresis on 2% agarose gels.

ENOS 4A4B polymorphism
The eNOS 4a4b polymorphism was analyzed by PCR amplification. PCR was performed with the primers 5'-CTATGGTAGTGCCTTGGCTGGAGG-3' (sense) and 5'-ACCGCCCAGGGAACTCCGCT-3' (antisense), and DNA was amplified in a final volume of 20 µL at an annealing temperature of 58 °C. The amplified products were analyzed by electrophoresis on a 3.5% agarose gel. The common allele (4b) amplicon size was 210 bp, whereas the rare allele (4a) amplicon size was 183 bp.

ENOS 894G>T polymorphism
eNOS 894G>T polymorphism detection was performed by real-time fluorescence PCR on a LightCycler instrument (Roche Diagnostics).

PCR and melting curve determinations were performed in a final volume of 20 µL with 10 pmol of each primer [NOSF (5'-CACTCCCCACAGCTCTGCAT-3') and NOSR (5'-CAATCCCTTTGGTGCTCACG-3')] and 4 pmol of each probe (anchor and sensor probes). The anchor probe (5'-LC Red 640-CCTTCTGCCCCCCGAGCTGGTCC-3'-P) was 5'-labeled with the LC-Red 640 fluorophore and phosphorylated (P) at its 3' end to prevent probe elongation by the Taq polymerase. The sensor probe, which matches the rare allele sequence (5'-CCCCAGATGATCCCCCAGAACTC-3'FLU; the underlined base indicates the polymorphism), was labeled with fluorescein. DNA was amplified at an annealing temperature of 62 °C. The amplicon size was 206 bp.

The typical melting curve pattern for the mutant is a single melting peak at a temperature of 65.5 °C. For the wild type, the plot gives a single melting peak at a temperature of 60.5 °C. Heterozygous patients have 2 melting peaks, at 60.5 and 65.5 °C (26).

statistical analysis
Data are reported as the mean (SD) or median and range. Statistical analysis was performed with the SPSS statistical package (Ver. 11.5) for Windows (SPSS Inc.). The {chi}2 test was used to test for deviation of genotype distributions from Hardy–Weinberg equilibrium.

Log-transformed values for Hcy, serum creatinine, and circulating vitamin B6, B12, and folate were used in the analysis and were back-transformed for data presentation. We used a dominant model of inheritance, which assumed that the rare allele was dominant (e.g., eNOS 4a4a + 4a4b vs 4b4b) because no data are available about a dose-dependent effect of the rare eNOS gene variants on eNOS activity. To evaluate the relationship between the MTHFR 677C>T polymorphism and Hcy concentrations, we used a general linear models analysis.

The study population was divided into tertiles of circulating Hcy (<10.2, 10.2–13.4, and >13.4 µmol/L), folate (<6.1, 6.1–10.7, and >10.7 nmol/L), vitamin B12 (<226.6, 226.6–325.1, and >325.1 pmol/L), and vitamin B6 (<23.1, 23.1–38.5, and >38.5 nmol/L). A multinomial logistic regression analysis, with Hcy groups as polychotomous dependent variable, was used to evaluate the risk of having Hcy in the third tertile for all eNOS polymorphisms. Variables showed a significant association with Hcy concentrations in the univariate analysis (age, gender, folate, vitamin B6, vitamin B12, MTHFR TT genotype, serum creatinine, and eNOS –786T>C polymorphism). An initial model (model 1) was developed using only variables known to influence plasma Hcy concentrations (age, gender, serum creatinine, circulating vitamins, and MTHFR TT genotype). In model 2, the eNOS –786T>C polymorphism was added. A P value < 0.05 was considered to indicate statistical significance. Odds ratios (ORs) are expressed with their 95% confidence intervals (CIs).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Of the participants 385 (29.9%) were smokers; 328 (25.5%) had hypertension, 249 (19.3%) had dyslipidemia, and 57 (4.4%) had diabetes. The values for analytes related to Hcy metabolism are reported in Table 1 .


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  		Table 1. Values for analytes related to Hcy metabolism in study participants (n = 1287).

Genotype distributions and allele frequencies for the eNOS –786T>C, 894G>T, and 4a4b and the MTHFR 677C>T polymorphisms (Table 2 ) were in Hardy–Weinberg equilibrium.


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  		Table 2. Genotype distributions and allele frequencies of the eNOS polymorphisms in the study participants (n = 1287).

The eNOS –786C variant, but not the 894T and 4a rare variants, influenced plasma Hcy concentrations. The mean plasma Hcy values according to –786T>C genotypes are shown in Fig. 1 .



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  		Figure 1. Plasma Hcy concentrations by eNOS –786t>C genotype.

Mean (SD; error bars) Hcy concentrations: 12.7 (6.2) µmol/L in the –786TT group; 12.8 (5.6) µmol/L in the –786TC group; and 14.6 (7.4) µmol/L in the –786CC group. P = 0.04 for –786TC + CC vs –786TT.

To evaluate the possible influence of eNOS polymorphisms on the presence of increased Hcy, we analyzed tertiles of Hcy. Univariate multinomial logistic regression analysis showed that persons carrying the eNOS –786C allele, but not the eNOS 894T and 4a alleles, had a significant risk of having Hcy concentrations in the third tertile (Table 3 ). After adjustment for variables known to influence plasma Hcy concentrations (age; sex; serum creatinine; circulating folate, vitamin B6, and vitamin B12 concentrations; and MTHFR 677TT genotype), the eNOS –786T>C polymorphism remained associated with Hcy concentrations (Table 3 ). The MTHFR 677C>T polymorphism significantly influenced plasma Hcy after adjustment for all confounding variables (age; sex; serum creatinine; circulating folate, vitamin B6, and vitamin B12 concentrations; and smoking habits).


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  		Table 3. Multinomial logistic regression analysis for highest tertile [third tertile (>13.4 µmol/L)] of plasma Hcy.1

The presence of both the eNOS –786T>C and the MTHFR 677C>T polymorphisms did not have an additive effect in increasing the risk of having Hcy in the third tertile (OR = 2.9; 95% CI, 1.4–6.2; P = 0.005). Similarly, no significant influence of the concurrent presence of –786T>C and the 2 other eNOS polymorphisms on Hcy was demonstrated [–786T>C/4a4b, OR = 1.3 (95% CI, 0.7–2.2; P = 0.4); –786T>C/894G>T, OR = 1.4 (95% CI, 0.8–2.3; P = 0.2)].

Because of the relevant roles of circulating vitamins and the MTHFR 677C>T polymorphism in Hcy metabolism, we subgrouped participants into tertiles of circulating folate, B6, and B12 concentrations and analyzed the influence of eNOS gene polymorphisms in relation to these variables. We found that the eNOS –786T>C polymorphism, but not 894G>T and 4a4b, influences plasma Hcy concentrations in persons with circulating vitamin concentrations within the relevant reference intervals (Table 4 ). We observed that the eNOS –786C variant did influence Hcy in persons carrying the TC and CC genotypes, but not homozygotes for the rare MTHFR T variant (Table 4 ).


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  		Table 4. Multinomial logistic regression analysis for highest tertile [third tertile (>13.4 µmol/L)] of plasma Hcy with respect to circulating vitamins.1

We also evaluated the association between eNOS polymorphisms and Hcy, stratifying the whole population by smoking habits. The eNOS –786T>C polymorphism, but not 894G>T and 4a4b, influenced Hcy concentrations in nonsmokers [for –786T>C, OR = 2.2 (95% CI, 1.2–3.9; P = 0.009); for 894G>T, OR = 0.7 (95% CI, 0.4–1.3; P = 0.3); for 4a4b, OR = 1.7 (95% CI, 0.9–3.2; P = 0.1)]. For smokers, we found no influence of any of the eNOS polymorphisms on having Hcy in the third tertile. As far as age is concerned, we observed that eNOS –786T>C significantly influenced Hcy concentrations in younger persons (age <60 years; OR = 2.9; 95% CI, 1.3–6.3; P = 0.009), but not in older ones.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we document that the eNOS –786T>C polymorphism, but not 894G>T and 4a4b, represents a new determinant of Hcy concentrations in nonsmokers, in normal vitamin status, in persons <60 years of age, and in persons not carrying the MTHFR TT genotype, in the absence of factors known to modulate Hcy.

Actually, the eNOS –786C rare variant of this polymorphism in the promoter region has a functional role by lowering the transcriptional rate of the gene >50% (17). The presence of this variant is associated with decreased NO concentrations.

Data from the literature suggest that NO may modulate Hcy concentrations directly by inhibiting methionine synthase, which synthesizes methionine from homocysteine and 5-methyltetrahydrofolate (8), and indirectly via folate catabolism by inhibiting the synthesis of ferritin (9), which promotes the oxidative cleavage of folate.

Our findings show that eNOS polymorphisms do not affect Hcy concentrations in persons with low concentrations of circulating vitamins, suggesting that the major modulator of Hcy is an environmental factor, such as vitamin status, in addition to the MTHFR 677C>T polymorphism and that the eNOS gene accounts for high Hcy concentrations when the other variables known to influence Hcy are not present. Similarly, with regard to another environmental factor, smoking habits, we found that eNOS polymorphisms did not modulate Hcy concentrations in smokers.

Our results showed no influence of the 894G>T polymorphism in exon 7 and of 4a4b in intron 4, which have been proposed to be functionally involved in the transcriptional pathway by modulating plasma Hcy concentrations. Interestingly, we did not find a role for the eNOS 4a4b polymorphism in influencing Hcy concentrations, although it is in close linkage dysequilibrium with the –786T>C polymorphism and may have a cis-acting effect on eNOS gene transcription (27).

Regarding the 894G>T polymorphism, our findings are in line with those reported by Heil et al. (15). and by Chrysohoou et al. (16), but are at variance with those reported by Brown et al. (14), although the prevalence of the rare 894T allele is almost similar. These different results might be attributable to the different folate concentrations and, most importantly, to the adjustment for vitamin B6, serum creatinine, and smoking habits in our analysis. Furthermore, experimental studies showed that endothelial production of NO is modified by age (27), which affects folate (28) and Hcy concentrations (29). We observed that the –786T>C polymorphism does not influence Hcy concentrations in older persons (>60 years), which suggests that age has a more relevant role than the eNOS gene in modulating Hcy concentrations. To date, this is the first study in which the influence of 3 eNOS polymorphisms on Hcy has been analyzed, thus providing an extended evaluation of the genetic influence of the eNOS gene in modulating Hcy.

Our results provide evidence that, in addition to the MTHFR 677C>T polymorphism, which is involved in the methionine pathway and is known to influence Hcy concentrations (30), the eNOS gene may represent a new modifier gene modulating plasma Hcy in nonsmokers, in normal vitamin status, and persons <60 years of age.

The study has two limitations. The first is the lack of information about the methionine synthase A2756G and methionine synthase reductase A66G polymorphisms involved in methionine metabolism. However, their interaction with the eNOS gene in affecting Hcy concentrations was not observed by Brown et al. (14). The second limitation is that the association between the eNOS –786C variant and Hcy concentrations was not statistically significant because the cohort was divided into 4 or 5 groups based on Hcy concentrations. This could be explained by the only mild biological relevance of these polymorphisms on influencing plasma Hcy concentrations, as otherwise seen by the modest association revealed in the statistical analysis.

In conclusion, our findings highlight another mechanism that can influence the complex methionine pathway. The role of the eNOS –786T>C polymorphism, as a determinant, emerges when other conditions known to influence Hcy concentrations are not present.


   Acknowledgments
 
This work was supported by a grant from the Italian Ministry of Health.


   Footnotes
 
1 Nonstandard abbreviations: NO, nitric oxide; Hcy, homocysteine; eNOS, endothelial nitric oxide synthase; MTHFR, methylenetetrahydrofolate reductase; OR, odds ratio; and CI, confidence interval.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. J. Glueck, R. A. Freiberg, J. Oghene, R. N. Fontaine, and P. Wang
Association Between the T-786C eNOS Polymorphism and Idiopathic Osteonecrosis of the Head of the Femur
J. Bone Joint Surg. Am., November 1, 2007; 89(11): 2460 - 2468.
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C. J. Glueck, M. Haque, M. Winarska, S. Dharashivkar, R. N. Fontaine, B. Zhu, and P. Wang
Stromelysin-1 5A/6A and eNOS T-786C Polymorphisms, MTHFR C677T and A1298C Mutations, and Cigarette-Cannabis Smoking: A Pilot, Hypothesis-Generating Study of Gene-Environment Pathophysiological Associations With Buerger's Disease.
Clinical and Applied Thrombosis/Hemostasis, October 1, 2006; 12(4): 427 - 439.
[Abstract] [PDF]

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