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Association between Endothelial Nitric Oxide Synthase Polymorphisms and Risk of Metabolic Syndrome

BACKGROUND: Previous studies inferring that the NOS3 gene was associated with the pathogenesis of metabolic syndrome (MetS) had inconsistent findings. We investigated the role of three NOS3 polymorphisms (T-786C, intron 4b/a, and G894T) in the risk of MetS using a hospital-based case-control study. METHODS: We recruited 339 MetS cases and 783 non-MetS controls at a central Taiwanese hospital. Information on sociodemographic and lifestyle factors was obtained using a self-administered questionnaire. Genotypes of NOS3 polymorphisms were compared between cases and controls. Effects of interactions between gene polymorphisms and smoking and between gene polymorphisms and drinking on the risk of MetS were also determined. RESULTS: The T-786C TC+CC genotype was significantly associated with a decreased risk of MetS (odds ratio (OR), 0.63; 95% confidence interval (CI), 0.43–0.91), compared to the T-786C TT genotype, according to a logistic regression analysis. This beneficial effect was much greater for those who had ever smoked cigarettes (OR, 0.47; 95% CI, 0.26–0.87) or those who had not consumed alcohol (OR, 0.45; 95% CI, 0.26–0.77). In addition, the intron 4b/a variant genotype was marginally associated with a reduced risk of MetS (OR, 0.68; 95% CI, 0.47–1.00), compared to the intron 4b/a bb genotype, particularly for never alcohol consumers (OR, 0.56; 95% CI, 0.33–0.95). In the haplotype analysis, there was a 53% decrease in the MetS risk among C4bG haplotype carriers (OR, 0.47; 95% CI, 0.25–0.90), compared to those with the most common T4bG haplotype. CONCLUSIONS: Our results suggest that the NOS3 T-786C and intron 4b/a polymorphisms may contribute to the risk of MetS. Further studies are needed to confirm the findings.


Introduction
Metabolic syndrome (MetS) is a cluster of manifestations including abdominal obesity, an increased serum dividuals with MetS are at high risk of developing type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) [5].
In addition to environmental factors such as a poor diet and physical inactivity [6], several studies suggested a genetic etiology of MetS [7,8]. Animal models indicated that a lack of endothelial nitric oxide synthase (eNOS) is related to hypertension, hypertriglyceridemia, and insulin resistance [9]. eNOS involves the production of nitric oxide (NO), which is a ubiquitous molecule responsible for maintaining normal endothelial function. NO is an important molecule involved in vasodilatation, neuronal transmission, smooth muscle relaxation, and immunity [10]. Studies clearly demonstrated that reduced NO-dependent endothelial vasodilatation is an early functional disturbance in the development of atherosclerotic lesions [11,12]. In addition, NO modulation of peripheral and hepatic glucose metabolism and insulin secretion suggests that NO alterations play important roles in the evolution of insulin resistance and T2DM [13]. Moreover, studies reported high NO levels and endothelial dysfunction in T2DM patients, their first-degree relatives [14], and subjects with insulin-resistance syndrome [15].
The aim of the present study was to test the hypothesis that the three NOS3 polymorphisms may play important roles in the risk of MetS in an ethnic Chinese population (i.e., Taiwanese) in Taiwan. In addition, because tobacco smoke and ethanol are involved in modulating eNOS activity [38][39][40], we also investi-gated how cigarette smoking and alcohol consumption interact with the relationship between NOS3 polymorphisms and MetS risk.

Participants
We

Data scope and collection
Anthropometric measurements were obtained during a complete physical examination. Weight and height were measured on an autoanthropometer (Super-view, HW-666, Taipei, Taiwan), with the subjects shoeless and wearing light clothing. The BMI was derived from the formula of weight/height 2 (kg/m 2 ). With the participant standing, the WC was measured at the midway point between the inferior margin of the last rib and the crest of the ilium in a horizontal plane. Blood pressure was measured at the right brachial artery using a random-zero sphygmomanometer with the participant in a sitting position.
Data on sociodemographic characteristics, including gender, age, educational attainment, marital status, household income, smoking, drinking, physical activity, occupational activity, menopausal status, dietary habits, family history of cardiovascular-related diseases, physician-diagnosed diseases, and medication history were collected using a standard questionnaire when the participant underwent the complete physical exam [41]. Smoking status and alcohol consumption history were classified into 3 groups: current users, nonusers, and ex-users.

Laboratory examination
Five mL blood was drawn with minimal trauma from the antecubital vein in tubes without anticoagulants in the morning, after a 12-h overnight fast, and was kept at room temperature and sent for analysis within 4 h of collection. Serum biochemical markers such as HDL-C, TGs, urine albumin, uric acid, and creatinine were analyzed using a biochemical autoanalyzer (Synchron LX-20; Beckman Coulter, Brea, CA, USA) at the Clinical Laboratory Department of China Medical University Hospital. The fasting plasma glucose level was determined by a glucose oxidase method (Astra-8, Beckman Instruments, Fullerton, CA, USA), and the serum insulin level was measured with a commercial enzymelinked immunosorbent assay (ELISA) kit (Diagnostic Products, Los Angeles, CA, USA).

Genotyping
Genomic DNA was extracted from another 5 mL blood samples collected in tubes containing the anticoagulant EDTA using a Gentra Puregene Blood Kit (Gentra Systems, Minneapolis, MN, USA). Quantitation of DNA and the purity index were evaluated by NanoDrop ND-1000 spectrophotometry (NanoDrop Technologies, Wilmington, DE, USA) at an absorbance ratio of 260/280 nm. The extracted DNA was dissolved in Tris-EDTA buffer to a concentration of 500 µg/mL and kept in a −80 • C freezer for further genotyping assays.
Polymorphisms of T-786C (rs2070744) and G894T (rs1799983) in the NOS3 gene were determined by a 5'-nuclease assay with allele-specific TaqMan probes. TaqMan single-nucleotide polymorphism (SNP) genotyping assays were purchased from Applied Biosystems (Foster City, CA, USA) with assay IDs of AHVI3OZ and C 3219460 20. Genotyping was performed using an allelic discrimination assay in the StepOne Real-Time polymerase chain reaction (PCR) System (Applied Biosystems), and genotypes were distinguished using automated software (SDS 2.3,Applied Biosystems). Reactions were run in 5-µL volumes using an amplification protocol of 95 • C for 10 min, followed by 40 cycles of 92 • C for 15 s and 60 • C for 1 min.
The 27-bp intron 4b/a VNTR polymorphism was analyzed using a primer pair (forward, 5'-AGG CCC TAT GGT AGT GCC TT-3'; reverse, 5'-TCT CTT AGT GCT GTG GTC AC-3') [42]. A 25-µL reaction volume was used for the PCR, including 0.5 µL (5 pM) of each primer, 0.2 µL (2.0 mM) dNTPs, 1.0 U of Taq polymerase, and 2.5 µL 10 × PCR buffer together with 25 ng DNA. The thermocycling procedure consisted of initial denaturation at 94 • C for 5 min, 35 cycles of denaturation at 94 • C for 30 s, annealing at 63 • C for 45 s, and extension at 72 • C for 1 min, followed by a final extension at 72 • C for 5 min. The PCR products were separated by 6% acrylamide gel electrophoresis and visualized by ethidium bromide staining. In addition to the wild-type allele (4b), which has five 27-bp repeats, we detected two variants for the intron 4b/a polymorphism. Variants 4a and 4c, which respectively corresponded to four and six 27-bp repeats, were previously observed [42]. For quality control, 10% random samples were repeated and showed 100% concordance for all polymorphisms.

Statistical analysis
In total, 147 subjects were excluded because of few blood samples or loss of DNA extraction (N = 105) and genotyping failure for any polymorphisms of the NOS3 gene (N = 42), leaving 339 MetS cases and 783 non-MetS controls for the final analyses. The demographic characteristics of excluded subjects did not differ from those of included subjects. Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) (termed D' and r 2 ) of the SNPs were assessed using THESIAS among the non-MetS group [43]. A twosample t-test was used to compare differences in quantitative traits between the MetS and non-MetS groups, while Pearson's Chi-square test was used to compare discrete traits. Due to the relatively low allelic frequency of the variant alleles for all NOS3 polymorphisms, participants were classified as carriers vs. non-carriers of the variant alleles. A simple logistic regression model was used to calculate the odds ratios (ORs) and their 95% confidence intervals (CIs) to assess the univariate association between each polymorphisms and MetS risk. A multiple logistic regression model was applied to estimate the age-and gender-adjusted ORs and their 95% CIs. Because tobacco smoke and ethanol play important roles in modulating eNOS activity [38][39][40], cigarette smoking and alcohol consumption (never and had ever) were included in the multiple logistic regression models.
Based on the multiplicative scale, a likelihood ratio test was used to evaluate the effect of the geneenvironment interaction on MetS risk. Interactions between polymorphisms and cigarette smoking or alcohol consumption were assessed by including cross-product  terms and the main effect term in the multiple logistic regression models. For example, the gene-smoking interaction model included the following covariates: interaction term between genotypes and cigarette smoking, genotypes, alcohol consumption, age, and gender. ORs for differences in MetS risk due to environmental factors (i.e., cigarette smoking and alcohol consumption) were estimated using stratified analyses with the wild-type genotypes as the reference in each stratum. All statistical analyses were performed using the Statistical Analysis Software (SAS) package (vers. 9.1.3 for windows; SAS Institute, Cary, NC, USA), and twosided P values of < 0.05 were considered significant.

Results
Demographic characteristics of participants in the study are summarized in Table 1. Compared to the non-MetS controls, MetS cases were more likely to be older, less educated, married, cigarette smokers, and alcohol drinkers (P < 0.05). The study MetS cases also had a much larger mean BMI (27.4 ± 3.56 vs. 22.9 ± 2.97 kg/m 2 ). Table 2 shows the genotypic distributions of the three NOS3 polymorphisms for both the non-MetS and MetS groups. Frequencies of the T-786C C allele, intron 4b/a 4a allele and 4c allele, and G894T T allele among the non-MetS subjects were 10.1%, 8.4%, 0.89%, and 10.3%, respectively. These allele frequencies were in HWE. After adjusting for age, gender, cigarette smoking, and alcohol consumption, the T-786C TC+CC genotype was associated with a significantly reduced risk of MetS, compared to the T-786C TT genotype (OR, 0.63; 95% CI, 0.43-0.91). In addition, the intron 4b/a variant genotype was marginally associated with a reduced risk of MetS, compared to the intron 4b/a bb genotype (OR, 0.68; 95% CI, 0.47-1.00). However, the G894T polymorphism was not significantly associated with MetS risk.
Six haplotypes were observed among twelve possible haplotypes defined by the three NOS3 polymorphisms in our study population ( Table 3). The most common T4bG haplotype, which served as the reference haplo-   Furthermore, we evaluated the association between NOS3 polymorphisms and the risk of MetS stratified by the cigarette smoking status and alcohol consumption status. Table 4 shows the gene-smoking interaction on the risk of MetS. Although the association between the three NOS3 polymorphisms and MetS risk was not significantly modified by the cigarette smoking status, we found that the protective effect of the T-786C TC+CC genotype was significantly stronger among those who had ever smoked cigarettes (OR, 0.47; 95% CI, 0.26-0.87).
A moderate gene-environment interaction between the T-786C polymorphism and alcohol consumption on the risk of MetS was observed as shown in Table 5 (P for interaction = 0.07). Among those who had never consumed alcohol, individuals carrying the T-786C TC+CC genotype had a decreased risk of 55% for MetS (OR, 0.45; 95% CI, 0.26-0.77), compared to those carrying the TT genotype. Although there was no significant association between the intron 4b/a and G894T polymorphisms and MetS risk measured for either drinkers or non-drinkers, among never alcohol consumers, the intron 4b/a variant (ba+aa+bc) genotype was significantly associated with a decreased risk OR: odds ratio; CI: confidence interval. a Numbers not being equal to the total number were due to missing data. b "Never" indicates non-smokers and "Had ever" is a combination of current smokers and ex-smokers. c Adjusted for age, gender, and alcohol consumption. d Based on the likelihood ratio test and adjusted for age, gender, and alcohol consumption. * P < 0.05.  (Table 6). Subjects with the T-786C TC+CC and intron 4b/a ba+aa+bc genotypes had a significantly lower BMI (P = 0.009 and P = 0.02), compared to those with the T-786C TT and intron 4b/a bb genotypes, respectively. Additionally, the variant intron 4b/a genotypes were marginally associated with Table 6 Anthropometric and metabolic components in relation to the three NOS3 polymorphisms of the study population lower waist circumference (P = 0.07). However, the T-786C and intron 4b/a polymorphisms were not associated with other components of MetS. For the G894T polymorphism, the GT+TT genotype (compared to the GG genotype) showed borderline significance for the association with higher LDL-C but lower TGs (P = 0.06 and P = 0.09, respectively). No other metabolic components differed with G894T genotypes. Nevertheless, after adjusting for age, gender, cigarette smoking, and alcohol consumption, both the BMI and waist circumference remained significantly different according to the T-786C genotypes (both P = 0.001) and the intron 4b/a genotypes (P = 0.011 and P = 0.014, respectively).

Discussion
In this large hospital-based case-control study, we found a significantly negative relationship between the T-786C TC+CC genotype of the NOS3 gene and the risk of MetS (OR = 0.63). This protective genetic effect was prominent for cigarette smokers (OR = 0.47) and those who had never consumed alcohol (OR = 0.45). Similarly, the NOS3 intron 4b/a variant genotype was marginally associated with a reduced risk of MetS (OR = 0.68), particularly for never alcohol consumers (OR = 0.56). In addition, the C4bG haplotype for the NOS3 T-786C, intron 4b/a, and G894T polymorphisms was significantly associated with a 53% decreased risk of MetS (OR = 0.47), compared to the most common T4bG haplotype.
To the best of our knowledge, our study is the first to evaluate three NOS3 polymorphisms and their constructed haplotypes in relation to the risk of MetS in an ethnic Chinese (i.e., Taiwanese) population. The variant NOS3 G894T polymorphism was associated with a risk of MetS in five studies [28][29][30][31][32], but not in six other studies [25,27,[33][34][35][36]. In addition, a positive association between the T-786C polymorphism and MetS was shown in five studies [26][27][28]34,36], but not in three other studies [33,35,37]. However, three studies were unable to demonstrate an association between the intron 4b/a polymorphisms and MetS risk [34][35][36].
Discrepancies across those studies might have occurred because of the relatively small sample size in previous studies or because of ethnic differences in allelic frequencies of these polymorphisms. Frequencies of the T-786C C allele, intron 4b/a a allele, and G894T T allele among non-MetS controls in this study (10.1%, 8.4%, and 10.3%, respectively) were similar to controls in a previous study conducted in Taiwan [42]. The frequency of the NOS3 gene in our controls was also comparable to populations in Japan [28,44], but much lower than those reported for Caucasian populations (39.7%∼45.3%, 18.1%, and 30.5%∼33.6%, respectively) [25,27,35].
In the present study, we observed that the variant T-786C C allele (TC+CC genotype) was significantly associated with a 37% decrease in the risk of MetS, which was only apparent for the specific haplotype harboring the variant C allele (C4bG vs. T4bG OR, 0.47; 95% CI, 0.25-0.90). In contrast to our study, Gonzalez-Sanchez et al. [26] recently reported that only the haplotype containing the variant T-786C C allele was associated with an increased risk of MetS (OR, 1.81; 95% CI, 1. 15-2.84). In addition, Fernandez et al. [27] also reported a positive association between the −786C/894G haplotype and MetS in hypertensive patients (P = 0.011). Alkharfy et al. [36] also identified G-a-C as the risk haplotype for MetS susceptibility (OR, 1.54; 95% CI, 1.1-2.3). However, two studies failed to find significant differences in the distribution of NOS3 haplotypes [34,35]. The T-786C polymorphism in the 5'-flanking region of the NOS3 gene was associated with reduced eNOS activity in an in vitro assay [45]. Lower eNOS mRNA and serum nitrite/nitrate levels were found in individuals with the T-786C C variant in one [46] but not in another study [47]. Although our results suggest a protective effect of the T-786C C allele, the underlying mechanism is not clear. We hypothesize that the reduced eNOS level has a protective role in MetS development. Additional research is needed to confirm our observation.
We also observed that the intron 4b/a variant (ba+aa+bc) genotype was slightly associated with decreased risk of MetS (OR = 0.68, P = 0.051). Three studies evaluated the association of MetS with the intron 4b/a polymorphism, but they found no significant association [34][35][36]. Although earlier study suggested that individuals with the 'a' allele of the intron 4b/a polymorphism have decreased plasma NO metabolites than the subjects with the 'b' allele [48], recent studies have shown no significant difference observed in plasma NO concentrations between the intron 4b/a genotypes [49,50]. This may be because the intron 4b/a 4a allele has been reported in linkage disequilibrium with the T-786C C allele [51], and they regulates transcription efficiency in a haplotype-specific way [52]. In a pGL3 reporter system, Wang et al. indicated that the −786C promoter with the 5x27-bp (intron 4b/a 4b allele) insertion had the highest transcription efficiency [52]. Consistent with this, in the present study significant reduced MetS risks were observed for individuals who carrying the NOS3 intron 4b/a variant genotype and C4bG haplotype.
The present study is the first to report that the MetS risk is reduced for individuals with the variant genotypes in the T-786C and intron 4b/a polymorphisms and those who had never consumed alcohol. Endothelial cell experimental studies demonstrated that both acute and chronic ethanol exposure may increase NOS3 gene transcription and increase NO production [38,39]. Alcohol consumption may thus overwhelm the genetic advantage of the T-786C and intron 4b/a substitutions. Therefore, the protective effects of the T-786C TC+CC genotype and the intron 4b/a ba+aa+bc genotype on the risk of MetS were prominent among those who had never consumed alcohol. In contrast, because tobacco smoke causes an irreversible inhibition of eNOS activity and reductions in eNOS protein and mRNA lev-els [40,53], smokers carrying the T-786C C allele may acquire a lower risk of MetS.
This study further demonstrated a significant relationship between the variant genotypes in the NOS3 T-786C and intron 4b/a polymorphisms and a lower BMI and waist circumference. Although previous studies were unable to find any significant association between the T-786C genotype and BMI [28,54], a recent study reported that some specific NOS3 haplotypes were associated with three obesity measures (BMI, waist circumference, and percent body fat) [55]. Studies have shown that increased NO production, preferably by eNOS, may cause decreased subcutaneous adipose tissue lipolysis in obesity [56,57]. Our hypothesis could be further speculated that the T-786C and intron 4b/a polymorphisms might prevent MetS by decreasing the obesity.
There are some limitations inherent in this hospitalbased study. Participants were recruited from one hospital; therefore, the results may mainly refer to local populations in central Taiwan. Because this quitesizable hospital is a prominent choice for medical services in the local area, our findings may reflect the situation of a good portion of the general population in central Taiwan. The other potential limitation is the absence of functional assays. Future research is needed to determine how NOS3 polymorphisms functionally underlie a mechanism leading to MetS. In addition, recall bias is another source of information bias in this study. However, the recall bias may be similar between cases and controls using the standardized study procedure. Finally, this study is a cross-sectional study, whether NOS3 T-786C and intron 4 b/a polymorphisms indeed reduces risk of MetS requires longitudinal follow-up study.

Conclusions
We found a significant negative association between MetS and the NOS3 C4bG haplotype, which harbors only the variant allele of T-786C for the studied NOS3 polymorphisms. In addition, the T-786C TC+CC genotype was associated with a decreased risk of MetS in those who had never consumed alcohol as well as in those who had ever smoked cigarettes. Similarly, the intron 4b/a variant genotype was significant associated with a reduced risk of MetS in never alcohol consumers. Our results suggest that NOS3 polymorphisms may contribute to a risk of metabolic syndrome, but further studies are needed to confirm the findings.