Endocrine Journal 2013, 60 (7), 893-901

Original

Association of endothelial nitric oxide synthase gene polymorphisms with type 2 diabetes mellitus: A meta-analysis Zhaotong Jia1), Xiaoqian Zhang 2), Shan Kang 3) and Yili Wu3) 1)

Department of Endocrinology, The Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, China 09 Grade 10 Class of Medical College, Qingdao University, Qingdao 266021, China 3) Department of Epidemiology and Health Statistics, Qingdao University Medical College, Qingdao 266021, China 2)

Abstract. A meta-analysis was performed to assess the association of endothelial nitric oxide synthase (NOS3) gene polymorphisms [4b4a VNTR and G894T (rs1799983)] with type 2 diabetes mellitus (T2DM). A comprehensive search was conducted to identify all eligible articles. Fixed or random effect pooled measure was selected based on homogeneity test. Heterogeneity among studies was evaluated using the I 2. Meta-regression was used to explore potential sources of between-study heterogeneity. Publication bias was estimated using Egger’s linear regression test. 19 articles involving 8,009 subjects for 4b4a VNTR and 19 articles involving 8,600 subjects for G894T were included. After excluding articles that deviated from Hardy–Weinberg equilibrium in controls and sensitivity analysis, significant association was found between 4a and increased risk of T2DM considering dominant model [OR (95%CI)=1.32 (1.17-1.48)], a vs. b [OR (95%CI)=1.34 (1.21-1.48)], and aa vs. bb [OR (95%CI)=1.52 (1.05-2.22)]. The 894T allele was also found associated with increased risk of T2DM considering dominant model [OR (95%CI)=1.14 (1.03-1.26)], recessive model [OR (95%CI)=1.28 (1.06-1.54)], T vs. G [OR (95%CI)=1.18 (1.09-1.27)], and TT vs. GG [OR (95%CI)=1.33 (1.09-1.62)]. The findings were consistent in Asian population. The meta-analysis indicated that NOS3 gene 4b4a VNTR and G894T polymorphisms might be associated with T2DM risk. Key words: Endothelial nitric oxide synthase, Polymorphism, Type 2 diabetes mellitus, Meta-analysis

Nitric oxide (NO), which is synthesized by the nitric oxide synthase (NOS) isoenzymes gene [endothelial (NOS3), neuronal (nNOS), and cytokine-inducible (iNOS)], had been associated with development of insulin resistance and type 2 diabetes [1]. Systemic inhibition of NO synthesis was found significantly deteriorating the glucose tolerance by increasing insulin clearance and inhibiting insulin secretion in non-diabetic individuals [2], and small increase in NO production might have beneficial effects in regulating skeletal muscle glucose uptake [3-4]. The gene encoding NOS3 is located on chromosome 7q35-36 and comprises 26 exons spanning 21 kilobases [5], ranging from 150688083 to 150711676 in chromosome 7. A 27-base pair (bp) variable number of tandem repeat (VNTR) in intron 4 (called 4b4a VNTR), and the guanine to thymine at nucleotide 894 in exon 7 (G894T, rs1799983, the position is 150696111) Submitted Dec. 28, 2012; Accepted Mar. 12, 2013 as EJ12-0463 Released online in J-STAGE as advance publication Apr. 7, 2013

Correspondence to: Zhaotong Jia, Department of Endocrinology, the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, China. E-mail: [email protected] ©The Japan Endocrine Society

resulting in the amino acid substitution of Glu with Asp in the 298 site (also called Glu298Asp) had been shown associated with oxidative stress and basal NO production [6-9]. A growing number of studies have been conducted to investigate the associations of type 2 diabetes mellitus (T2DM) with the NOS3 gene (4b4a VNTR and G894T) polymorphisms, however, the results are controversial. Hence we conducted a meta-analysis to (1) assess the effect of NOS3 gene (4b4a VNTR and G894T) polymorphisms on the risk of T2DM, (2) evaluate the potential heterogeneity among studies, and (3) explore the potential publication bias.

Materials and Methods Search strategy A comprehensive search was conducted in the following databases to 2012: Pubmed, Web of knowledge, CBM (China biology medical literature database), CNKI (China National Knowledge Infrastructure), VIP (Database of Chinese Scientific and Technical Periodicals), and Google scholar using the following keywords: ‘NOS3’,

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‘endothelial nitric oxide synthase,’ ‘nitric oxide synthase’ and ‘polymorphism,’ ‘mut*,’ ‘varia*’ and ‘diabet*’ without language restriction (*, the Wildcard). Reference lists of the identified literatures were also reviewed carefully to identify additional eligible studies. Inclusion criteria The inclusion criteria were as follows: (1) studies were published as an original study to evaluate the association between (4b4a VNTR and G894T) polymorphisms in NOS3 gene and risk of T2DM; (2) genotype distributions in both patients and controls were available; (3) the largest population was selected if the data was multiple published. Two investigators carefully reviewed the identified articles to determine whether an individual study was eligible for inclusion in this meta-analysis. Data extraction Two investigators collected the data independently and reached a consensus on all items. The following basic information was extracted from the eligible studies: first author, year of publication, ethnicity of population studied, genotype distributions, mean age and male percentage in cases and controls. Statistical analysis The inverse-variance weighted mean of the logarithm of OR with 95% CI was used to assess the strength of association between NOS3 gene polymorphisms and T2DM risk. Hardy-Weinberg equilibrium (HWE) was tested by Chi square analysis with exact probability. This meta-analysis assessed the association between NOS3 gene polymorphisms and T2DM risk with dominant model (aa + ba vs. bb for 4b4a VNTR and TT+GT vs. GG for G894T), recessive model (aa vs. ba +bb for 4b4a VNTR and TT vs. GT +GG for G894T), allele comparison (a vs. b for 4b4a VNTR and T vs. G for G894T) models, and genotype comparison (aa vs. bb for 4b4a VNTR and TT vs. GG for G894T), respectively. The I 2 of Higgins and Thompson was used to quantitatively assess heterogeneity among studies (I 2 values of 0, 25%, 50%, and 75% represents no, low, moderate, and high heterogeneity, respectively) [10]. I 2 describes the proportion of total variation attributable to between-study heterogeneity as opposed to random error or chance [11]. If the heterogeneity was moderate or lower (I 2 3.0, only low or moderate heterogeneity was found in all models for 4b4a VNTR and most of models for G894T. Influence analysis No individual study was found to have excessive influence on the pooled effect for 4b4a VNTR and G894T polymorphisms in any of the above-mentioned models (Fig. 3 presents the influence analysis for 4b4a VNTR in dominant model after excluding two articles that deviated from HWE in controls and after sensitivity analysis). Publication bias evaluation No significant publication bias was detected for 4b4a VNTR and G894T polymorphisms in any of the abovementioned inherited models, respectively.

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Fig. 3 Influence analysis of the risk of type 2 diabetes mellitus in dominant model (aa + ba vs. bb) of NOS3 gene 4b4a VNTR restricting studies that were in Hardy-Weinberg equilibrium in control group and after sensitivity analysis.

Discussion In this meta-analysis, significant association was found of 4a of 4b4a VNTR and 894T of G894T with increased risk of T2DM. Because of the inconsistent results from relatively small studies underpowered to detect the effect, a meta-analysis is the appropriate approach to obtain an improved provisional conclusion. And this meta-analysis, of 19 articles involving 8,009 subjects for 4b4a VNTR and 19 articles involving 8,600 subjects for G894T, allowed a much greater possibility of reaching reasonably conclusions. Recently, many loci associated with T2DM have been discovered from genome-wide association studies (GWAS) and meta-analysis of GWAS using SNP chips for Europeans [43], South Asians [44] and East Asians [45]. These GWAS involved more T2DM cases (6000-8000) than that included in this meta-analysis, and the two variants (4b4a VNTR and G894T ) have not been represented on GWAS chips, thus the findings of this meta-analysis need to be confirmed in the GWAS. And the reports of T2DM with the genes near the NOS3 gene including ABP1, KCNH2, ATG9B, ABCB8, ASIC3, CDK5 and SLC4A2 are also limited. Results from 3 meta-analysis [46-48] suggested that NOS3 gene polymorphisms (4b4a VNTR and G894T) might be associated with risk of diabetic nephropathy.

The rs1549758, a C/T variation (774T>C) in the NOS3 gene on human chromosome 7, resulting in no substitution amino acid at position 258 of the protein (D258D), is shown in linkage disequilibrium with G894T (pairwise r2 ranges from 0.367-1.000 in different population samples) according to the 1000 Genomes data. However, the reports of rs1549758 with T2DM are also lacking. According to the linkage disequilibrium data by population in the 1000 Genomes data, the G894T and T-786C polymorphism are in weak LD in all population samples, with a biggest value of r2 (0.336) found in a Mexican ancestry population. However, the 4b4a VNTR is not captured in 1000 Genomes data. Between-study heterogeneity is common in metaanalysis for genetic association studies [49], and exploring the potential sources of between-study heterogeneity is the essential component of meta-analysis [50]. The between-study heterogeneity might arise from an indeterminate number of characteristics that vary among studies, e.g. study quality, population stratification, characteristics of the subjects involved, genotyping quality, variation of the covariate, and deviation from HWE in some studies, etc. Thus we used meta-regression to explore the causes of heterogeneity for covariates. However, publication year, ethnicity of population, mean age, sex and sample size were found not to be important sources of disease–effect heterogeneity in

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this meta-analysis. In evaluating associations between genetic variants and common complex diseases, biologically meaningful associations of single genetic variant with common diseases are expected in small effects (risk ratios mostly3.0. And after excluding articles that deviated from HWE in controls and sensitivity analysis, only low or moderate heterogeneity was found in all models for 4b4a VNTR and most of models for G894T. Besides, Diabetes mellitus have a complex aetiology and pathophysiology generated by the combined effects of genes and environment factors. Thus other genetic and environment variables, as well as their possible interaction, may well be potential contributors to the heterogeneity observed. Other limitations involved in this meta-analysis are as follows: First, previous studies showed that haplotype analysis of NOS3 gene polymorphisms (4b4a VNTR and G894T) showed significant associations between NOS3 haplotypes and impaired endogenous NO formation [51-54], supporting the idea that combinations of genetic polymorphisms within haploypes are of major relevance to examine the biological and clinical relevance of polymorphisms. And three studies [17, 29-30] included in this meta-analysis also demonstrated significant association between NOS3 gene variants and T2DM with haplotype analysis. But the limited number of studies precluded a haplotype analysis in this meta-analysis. Second, previous studies suggested that 894T allele for G894T was more common

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in Caucasians than African-Americans or Asians, while the 4a variant for 4b4a VNTR was more common in African-Americans than Caucasians or Asians in the American population [55-56]. However, the limited data precluded a further estimation of the association between NOS3 polymorphisms (4b4a VNTR, G894T) and T2DM risk in other ethnicity besides Asian. Thus although ethnicity of population was not found to be the important source of disease–effect heterogeneity across the studies in this meta-analysis, we cannot rule out the underling ethnicity-specific effect of NOS3 gene polymorphisms on T2DM risk. Finally, in a meta-analysis of published studies, publication bias may be a problem. However, we found no evidence of significant publication bias in this meta-analysis. Nevertheless, it is worthy of note that the validity of our publication bias test is questioned, because of small number of studies [57] in our meta-analysis. In conclusion, this meta-analysis suggested that NOS3 gene 4b4a VNTR and G894T polymorphisms might be associated with T2DM risk. Since potential biases and confounders could not be ruled out completely in this meta-analysis, further studies of NOS3 gene 4b4a VNTR and G894T polymorphisms with T2DM using LD/haplotype-SNP tagging approach are warranted.

Acknowledgements None.

Conflict of Interest None.

References 1. Monti LD, Barlassina C, Citterio L, Galluccio E, Berzuini C, et al. (2003) Endothelial nitric oxide synthase polymorphisms are associated with type 2 diabetes and the insulin resistance syndrome. Diabetes 52: 1270-1275. 2. Natali A, Ribeiro R, Baldi S, Tulipani A, Rossi M, et al. (2013) Systemic inhibition of nitric oxide synthesis in non-diabetic individuals produces a significant deterioration in glucose tolerance by increasing insulin clearance and inhibiting insulin secretion. Diabetologia. 2013 Jan 31.[Epub ahead of print] 3. McConell GK, Rattigan S, Lee-Young RS, Wadley GD, Merry TL (2012) Skeletal muscle nitric oxide signal-

ing and exercise: a focus on glucose metabolism. Am J Physiol Endocrinol Metab 303: E301-307. 4. Chai W, Dong Z, Wang N, Wang W, Tao L, et al. (2012) Glucagon-like peptide 1 recruits microvasculature and increases glucose use in muscle via a nitric oxide-dependent mechanism. Diabetes 61: 888-896. 5. Marsden PA, Heng HH, Scherer SW, Stewart RJ, Hall AV, et al. (1993) Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem 268: 17478-17488. 6. Wang XL, Mahaney MC, Sim AS, Wang J, Blangero J, et al. (1997) Genetic contribution of the endothelial constitutive nitric oxide synthase gene to plasma nitric oxide

900

Jia et al.

levels. Arterioscler Thromb Vasc Biol 17: 3147-3153. 7. Veldman BA, Spiering W, Doevendans PA, Vervoort G, Kroon AA, et al. (2002) The Glu298Asp polymorphism of the NOS 3 gene as a determinant of the baseline production of nitric oxide. J Hypertens 20: 2023-2027. 8. Tsukada T, Yokoyama K, Arai T, Takemoto F, Hara S, et al. (1998) Evidence of association of the ecNOS gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Commun 245: 190-193. 9. Ritt M, Ott C, Delles C, Schneider MP, Schmieder RE (2008) Impact of the endothelial nitric oxide synthase gene G894T polymorphism on renal endothelial function in patients with type 2 diabetes. Pharmacogenet Genomics 18: 699-707. 10. Higgins JP, Thompson SG, Deeks JJ, Altman DG (2003) Measuring inconsistency in meta-analyses. BMJ 327: 557-560. 11. Higgins JP, Thompson SG (2002) Quantifying heterogeneity in a meta-analysis. Stat Med 21: 1539-1558. 12. Tobias A (1999) Assessing the influence of a single study in the meta-analysis estimate. Stata Technical Bulletin 47: 15-17. 13. Sharp S (1998) Meta-analysis regression. Stata Technical Bulletin 42: 16-22. 14. Harbord RM, Egger M, Sterne JA (2006) A modified test for small-study effects in meta-analyses of controlled trials with binary endpoints. Stat Med 25: 34433457. 15. Khoury MJ, Little J, Gwinn M, Ioannidis JP (2007) On the synthesis and interpretation of consistent but weak gene-disease associations in the era of genome-wide association studies. Int J Epidemiol 36: 439-445. 16. Deng WQ, Wei J, Wu YN, Wang FH, Song DY, et al. (2009) association between polymorphism of endothelial nitric oxide synthase gene and type 2 diabetic patients with erectile dysfunction. Lin Chuang Hui Cui 24: 481-485 (In Chinese). 17. Ezzidi I, Mtiraoui N, Mohamed MB, Mahjoub T, Kacem M, et al. (2008) Association of endothelial nitric oxide synthase Glu298Asp, 4b/a, and -786T>C gene variants with diabetic nephropathy. J Diabetes Complications 22: 331-338. 18. Huang HS, Lin LX, Chen M (2002) Association of polymorphism of endothelial nitric oxide synthase gene with essential hypertension and type 2 diabetes mellitus. Zhong Hua Nei Fen Mi Dai Xie Za Zhi 18 (In Chinese). 19. Li C, Dong Y, Lu W (2001) [The association between polymorphism of endothelial nitric oxide synthase gene and diabetic nephropathy]. Zhonghua Nei Ke Za Zhi 40: 729-732 (In Chinese). 20. Lin S, Qu HC, Qiu M (2002) Study on the association between ecNOS4b/a polymorphism and diabetic nephropathy. Zhong Hua Shen Zang Bing Za Zhi 18: 258-261 (In Chinese). 21. Luo H, Ning Y (2003) Association of polymorphism of

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

endothelial nitric synthase gene with diabetic nephropathy. Zhong Guo Tang Niao Bing Za Zhi 11: 317-320 (In Chinese). Neugebauer S, Baba T, Watanabe T (2000) Association of the nitric oxide synthase gene polymorphism with an increased risk for progression to diabetic nephropathy in type 2 diabetes. Diabetes 49: 500-503. Pulkkinen A, Viitanen L, Kareinen A, Lehto S, Vauhkonen I, et al. (2000) Intron 4 polymorphism of the endothelial nitric oxide synthase gene is associated with elevated blood pressure in type 2 diabetic patients with coronary heart disease. J Mol Med 78: 372-379. Sandrim VC, de Syllos RW, Lisboa HR, Tres GS, TanusSantos JE (2006) Endothelial nitric oxide synthase haplotypes affect the susceptibility to hypertension in patients with type 2 diabetes mellitus. Atherosclerosis 189: 241-246. Sun HY, Yang MG, Liu SQ, Wang CJ, Zhang Q, et al. (2004) Association of endothelial nitric oxide synthase gene polymorphisms with type 2 diabetes mellitus and diabetic retinopathy. Zhong Hua Nei Fen Mi Dai Xie Za Zhi 20: 52-53 (In Chinese). Wu Y, Ge J, Sheng J (2007) The ralationship between polymorphism of intron 4 (4b/a) of the endothelial constitutive nitric oxide synthase gene and type 2 diabetes millitus complicated with coronary heart disease. He Bei Yi Yao 29: 539-541 (In Chinese). Yu LH, Tian G, Cai J, Wang X (2009) Study on relationship between the endothelial constitutive nitric oxide synthase gene polymorphism and hyperuricemia in type 2 diabetes mellitus. Tian Jin Yi Yao 37: 347-349 (In Chinese). Zhang M, Song DP, Liu H, Wang YM, Duan Y, et al. (2003) The polymorphism of eNOS gene is associated with higher triglyceride levels. Zhong Guo Tang Niao Bing Za Zhi 11: 34-36 (In Chinese). de Syllos RW, Sandrim VC, Lisboa HR, Tres GS, TanusSantos JE (2006) Endothelial nitric oxide synthase genotype and haplotype are not associated with diabetic retinopathy in diabetes type 2 patients. Nitric Oxide 15: 417-422. Bae J, Kim IJ, Hong SH, Sung JH, Lim SW, et al. (2010) Association of endothelial nitric oxide synthase polymorphisms with coronary artery disease in Korean individuals with or without diabetes mellitus. Exp Ther Med 1: 719-724. Lee YJ, Chang DM, Tsai JC (2003) Association of a 27-bp repeat polymorphism in intron 4 of endothelial constitutive nitric oxide synthase gene with serum uric acid levels in Chinese subjects with type 2 diabetes. Metabolism 52: 1448-1453. Galanakis E, Kofteridis D, Stratigi K, Petraki E, Vazgiourakis V, et al. (2008) Intron 4 a/b polymorphism of the endothelial nitric oxide synthase gene is associated with both type 1 and type 2 diabetes in a genetically

NOS3 gene polymorphisms and T2DM

homogeneous population. Hum Immunol 69: 279-283. 33. Mehrab-Mohseni M, Tabatabaei-Malazy O, HasaniRanjbar S, Amiri P, Kouroshnia A, et al. (2011) Endothelial nitric oxide synthase VNTR (intron 4 a/b) polymorphism association with type 2 diabetes and its chronic complications. Diabetes Res Clin Pract 91: 348-352. 34. Santos KG, Crispim D, Canani LH, Ferrugem PT, Gross JL, et al. (2011) Association of eNOS gene polymorphisms with renal disease in Caucasians with type 2 diabetes. Diabetes Res Clin Pract 91: 353-362. 35. Fu ZJ, Li CG, Wang ZC, Yan S (2007) Coexistence of aidose reductase gene and endothelial nitric oxide synthase polymorphisms associates with diabetic nephropathy. Zhong Guo Zu Zhi Gong Cheng Yan Jiu Yu Lin Chuang Kang F 11: 6893-6896 (In Chinese). 36. Luo R, Fan JY, Zhang P, Liu W, Gao ZH, et al. (2006) A study on relationship between the endothelial constitutive nitric oxide synthase gene polymorphism and the coronary disease in patients with type 2 diabetes. Zhong Guo Yi Shi Za Zhi 8: 5-7 (In Chinese). 37. Ma JR, Yu DM, Liu D (2007) The relationship between the G894T mutation of the endothelial-constitutive nitric oxide synthase (ecNOS) and diabetic microangiopathies. Zhong Guo Tang Niao Bing Za Zhi 15: 471-473 (In Chinese). 38. Odeberg J, Larsson CA, Rastam L, Lindblad U (2008) The Asp298 allele of endothelial nitric oxide synthase is a risk factor for myocardial infarction among patients with type 2 diabetes mellitus. BMC Cardiovasc Disord 8: 36. 39. Ren T, Xiang SS, Liu L (2003) An association of diabetic neuropathy with polymorphisms of eNOS, PON1, RAGE and ALR2 genes. Shang Hai Yi Yao 26: 24-27 (In Chinese). 40. Shin Shin Y, Baek SH, Chang KY, Park CW, Yang CW, et al. (2004) Relations between eNOS Glu298Asp polymorphism and progression of diabetic nephropathy. Diabetes Res Clin Pract 65: 257-265. 41. Ukkola O, Erkkila PH, Savolainen MJ, Kesaniemi YA (2001) Lack of association between polymorphisms of catalase, copper-zinc superoxide dismutase (SOD), extracellular SOD and endothelial nitric oxide synthase genes and macroangiopathy in patients with type 2 diabetes mellitus. J Intern Med 249: 451-459. 42. Angeline T, Krithiga HR, Isabel W, Asirvatham AJ, Poornima A (2011) Endothelial nitric oxide synthase gene polymorphism (G894T) and diabetes mellitus (type II) among South Indians. Oxid Med Cell Longev 2011: 462607. 43. Voight BF, Scott LJ, Steinthorsdottir V, Morris AP, Dina C, et al. (2010) Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis. Nat Genet 42: 579-589. 44. Kooner JS, Saleheen D, Sim X, Sehmi J, Zhang W, et al.

45.

46.

47.

48.

49. 50. 51.

52.

53.

54.

55.

56.

57.

901

(2011) Genome-wide association study in individuals of South Asian ancestry identifies six new type 2 diabetes susceptibility loci. Nat Genet 43: 984-989. Cho YS, Chen CH, Hu C, Long J, Ong RT, et al. (2011) Meta-analysis of genome-wide association studies identifies eight new loci for type 2 diabetes in east Asians. Nat Genet 44: 67-72. Zeng Z, Li L, Zhang Z, Li Y, Wei Z, et al. (2010) A metaanalysis of three polymorphisms in the endothelial nitric oxide synthase gene (NOS3) and their effect on the risk of diabetic nephropathy. Hum Genet 127: 373-381. Zintzaras E, Papathanasiou AA, Stefanidis I (2009) Endothelial nitric oxide synthase gene polymorphisms and diabetic nephropathy: a HuGE review and metaanalysis. Genet Med 11: 695-706. He Y, Fan Z, Zhang J, Zhang Q, Zheng M, et al. (2011) Polymorphisms of eNOS gene are associated with diabetic nephropathy: a meta-analysis. Mutagenesis 26: 339-349. Munafo MR, Flint J (2004) Meta-analysis of genetic association studies. Trends Genet 20: 439-444. Lau J, Ioannidis JP, Schmid CH (1998) Summing up evidence: one answer is not always enough. Lancet 351: 123-127. Metzger IF, Ishizawa MH, Rios-Santos F, Carvalho WA, Tanus-Santos JE (2011) Endothelial nitric oxide synthase gene haplotypes affect nitrite levels in black subjects. Pharmacogenomics J 11: 393-399. Metzger IF, Sertorio JT, Tanus-Santos JE (2007) Modulation of nitric oxide formation by endothelial nitric oxide synthase gene haplotypes. Free Radic Biol Med 43: 987-992. Sandrim VC, de Syllos RW, Lisboa HR, Tres GS, TanusSantos JE (2007) Influence of eNOS haplotypes on the plasma nitric oxide products concentrations in hypertensive and type 2 diabetes mellitus patients. Nitric Oxide 16: 348-355. Metzger IF, Souza-Costa DC, Marroni AS, Nagassaki S, Desta Z, et al. (2005) Endothelial nitric oxide synthase gene haplotypes associated with circulating concentrations of nitric oxide products in healthy men. Pharmacogenet Genomics 15: 565-570. Tanus-Santos JE, Desai M, Flockhart DA (2001) Effects of ethnicity on the distribution of clinically relevant endothelial nitric oxide variants. Pharmacogenetics 11: 719-725. Marroni AS, Metzger IF, Souza-Costa DC, Nagassaki S, Sandrim VC, et al. (2005) Consistent interethnic differences in the distribution of clinically relevant endothelial nitric oxide synthase genetic polymorphisms. Nitric Oxide 12: 177-182. Sterne JA, Gavaghan D, Egger M (2000) Publication and related bias in meta-analysis: power of statistical tests and prevalence in the literature. J Clin Epidemiol 53: 1119-1129.