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

Or i g i n a l Association of endothelial nitric oxide synthase polymorphisms with type 2 diabetes mellitus: A meta-analysis

Zhaotong Jia1), Xiaoqian Zhang2), Shan Kang3) and Yili Wu3)

1) Department of Endocrinology, The Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, China 2) 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

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 resulting in the amino acid substitution of Glu with Asp nitric oxide synthase (NOS) isoenzymes gene [endothe- in the 298 site (also called Glu298Asp) had been shown lial (NOS3), neuronal (nNOS), and cytokine-inducible associated with oxidative stress and basal NO produc- (iNOS)], had been associated with development of insu- tion [6-9]. A growing number of studies have been con- lin resistance and type 2 diabetes [1]. Systemic inhibi- ducted to investigate the associations of type 2 diabe- tion of NO synthesis was found significantly deteriorat- tes mellitus (T2DM) with the NOS3 gene (4b4a VNTR ing the glucose tolerance by increasing insulin clearance and G894T) polymorphisms, however, the results are and inhibiting insulin secretion in non-diabetic individu- controversial. Hence we conducted a meta-analysis to als [2], and small increase in NO production might have (1) assess the effect of NOS3 gene (4b4a VNTR and beneficial effects in regulating skeletal muscle glucose G894T) polymorphisms on the risk of T2DM, (2) eval- uptake [3-4]. The gene encoding NOS3 is located on uate the potential heterogeneity among studies, and (3) 7q35-36 and comprises 26 exons spanning explore the potential publication bias. 21 kilobases [5], ranging from 150688083 to 150711676 in . A 27- (bp) variable num- Materials and Methods ber of tandem repeat (VNTR) in intron 4 (called 4b4a VNTR), and the guanine to thymine at nucleotide 894 in Search strategy exon 7 (G894T, rs1799983, the position is 150696111) A comprehensive search was conducted in the follow- ing databases to 2012: Pubmed, Web of knowledge, CBM Submitted Dec. 28, 2012; Accepted Mar. 12, 2013 as EJ12-0463 (China biology medical literature database), CNKI (China Released online in J-STAGE as advance publication Apr. 7, 2013 Correspondence to: Zhaotong Jia, Department of Endocrinology, National Knowledge Infrastructure), VIP (Database the Affiliated Hospital of Medical College, Qingdao University, of Chinese Scientific and Technical Periodicals), and Qingdao 266003, China. E-mail: [email protected] Google scholar using the following keywords: ‘NOS3’,

©The Japan Endocrine Society 894 Jia et al.

‘endothelial nitric oxide synthase,’ ‘nitric oxide synthase’ DerSimonian and Laird random effect model (REM) and ‘polymorphism,’ ‘mut*,’ ‘varia*’ and ‘diabet*’ with- was adopted. Influence analysis [12] was conducted to out language restriction (*, the Wildcard). Reference describe how robust the pooled estimator is to removal lists of the identified literatures were also reviewed care- of individual studies, and an individual study is sus- fully to identify additional eligible studies. pected of excessive influence if the point estimate of its omitted analysis lies outside the 95% CI of the com- Inclusion criteria bined analysis. Meta-regression with restricted max- The inclusion criteria were as follows: (1) studies imum likelihood estimation [13] was performed to were published as an original study to evaluate the assess the potentially important covariates that might association between (4b4a VNTR and G894T) poly- exert significant impact on between-study heteroge- morphisms in NOS3 gene and risk of T2DM; (2) gen- neity. Publication bias was estimated using modified otype distributions in both patients and controls were Egger’s linear regression test as proposed by Harbord available; (3) the largest population was selected if et al. [14]. In evaluating associations between genetic the data was multiple published. Two investigators variants and common complex diseases, biologically carefully reviewed the identified articles to determine meaningful associations of single genetic variant with whether an individual study was eligible for inclusion common diseases are expected in small effects (risk in this meta-analysis. ratios mostly<2.0) [15], and very big effect estimates, which might cause significant findings by chance, Data extraction could be caused by unstable effect estimations result- Two investigators collected the data independently ing from low cell counts within each single study and and reached a consensus on all items. The following possible genotyping errors. And considering the fact basic information was extracted from the eligible stud- that original studies with relatively small participants ies: first author, year of publication, ethnicity of pop- might be underpowered to detect the effect, thus sen- ulation studied, genotype distributions, mean age and sitivity analysis was also performed excluding results male percentage in cases and controls. with OR>3.0. Subgroup analyses were conducted in Asian. All statistical analyses were performed with Statistical analysis STATA version 10.0 (Stata Corporation, College The inverse-variance weighted mean of the logarithm Station, TX, USA). All reported probabilities (P val- of OR with 95% CI was used to assess the strength of ues) are two-sided, with P<0.05 considered statisti- association between NOS3 gene polymorphisms and cally significant. T2DM risk. Hardy-Weinberg equilibrium (HWE) was tested by Chi square analysis with exact probability. Results This meta-analysis assessed the association between NOS3 gene polymorphisms and T2DM risk with domi- Characteristics of studies nant model (aa + ba vs. bb for 4b4a VNTR and TT+GT 19 articles [16-34] involving 8,009 subjects (4,966 vs. GG for G894T), recessive model (aa vs. ba +bb for cases and 3,043controls) for 4b4a VNTR and 19 arti- 4b4a VNTR and TT vs. GT +GG for G894T), allele cles [1, 9, 16-17, 19, 23-25, 29-30, 34-42] involving comparison (a vs. b for 4b4a VNTR and T vs. G for 8,600 subjects (4,795 cases and 3,805 controls) for G894T) models, and genotype comparison (aa vs. bb G894T. The genotype distribution of controls in two for 4b4a VNTR and TT vs. GG for G894T), respec- articles [16, 21] for 4b4a VNTR and one article [16] tively. The I 2 of Higgins and Thompson was used to for G894T was not in HWE. Detailed information for quantitatively assess heterogeneity among studies (I 2 the published articles included in this meta-analysis is values of 0, 25%, 50%, and 75% represents no, low, showed in Table 1. moderate, and high heterogeneity, respectively) [10]. I 2 describes the proportion of total variation attribut- Quantitative synthesis able to between-study heterogeneity as opposed to ran- Detailed information for the combined results is dom error or chance [11]. If the heterogeneity was shown in Table 2. 2 moderate or lower (I <50%), the fixed effect model Association of 4b4a VNTR with T2DM risk (FEM) was used as the pooling method, otherwise, the Significant association was found between 4a and NOS3 gene polymorphisms and T2DM 895

Table 1 Characteristics of NOS3 gene 4b4a VNTR and 894T polymorphisms genotype distributions in this meta-analysis Genotypes % of male Agea Author [ref.] Year Ethnicity Case Number Control Number PHWE case/control case/control 4b4a VNTR bb/ba/aa bb/ba/aa Neugebauer [22] 2000 Japanese 172/36/7 133/22/0 1.00 61/75 57.9/43.0 Pulkkinen [23] 2000 Finnish 155/85/11 79/26/5 0.16 66/76 64.0/51.0 Li [19] 2001 Chinese 103/40/0 62/22/1 1.00 38/41 63.4/61.2 Lin [20] 2002 Chinese 112/14/1 64/6/0 1.00 52/49 57.5/38.6 Huang [18] 2002 Chinese 66/16/3 61/7/0/ 1.00 57/53 53.0/56.0 Lee [31] 2003 Chinese 674/112/14 340/57/1 0.71 46/71 61.7/61.8 Luo [21] 2003 Chinese 43/6/35 34/1/2 0.00 57/53 53.0/56.0 Zhang [28] 2003 Chinese 111/19/2 68/12/0 1.00 38/40 56.0/55.0 Sun [25] 2004 Chinese 241/57/4 93/18/2 0.27 50/47 59.8/56.0 de syllos [29] 2006 Brazilian 122/43/5 79/20/4 0.09 40/58 57.5/51.9 Sandrim [24] 2006 Brazilian 131/44/4 79/19/4 0.07 58/59 57.5/52.1 Wu [26] 2007 Chinese 138/33/1 96/23/0 0.60 50/56 56.6/50.9 Ezzidi [17] 2008 Tunisian 548/305/50 511/217/20 0.70 46/50 59.3/58.7 Galanakis [32] 2008 Greek 74/29/10 120/39/1 0.47 51/nab 70.5/na Deng [16] 2009 Chinese 146/43/13 75/16/9 0.00 100/100 56.1/54.2 Yu [27] 2009 Chinese 181/17/2 87/12/1 0.39 59/52 52.6/51.2 Bae [30] 2010 Korean 61/26/2 248/51/0 0.15 61/53 61.5/59.7 Mehrab [33] 2010 Iranian 157/54/9 80/16/0 1.00 29/57 61.5/59.7 Santos [34] 2011 Brazilian 405/158/16 67/30/3 1.00 na/na na/na G894T polymorphism GG/GT/TT GG/GT/TT Pulkkinen [23] 2000 Finnish 136/97/18 54/45/11 0.82 66/76 64.0/51.0 Li [19] 2001 Chinese 93/49/1 63/21/1 1.00 38/41 63.4/61.2 Ukkola [41] 2001 Finnish 106/93/17 112/92/18 1.00 53/53 58.3/51.3 Monti [1] 2003 Italian 52/63/44 86/82/29 0.21 65/61 57.4/54.5 Ren [39] 2003 Chinese 159/28/3 67/15/0 1.00 59/69 61.1/52.0 Shin Shin [40] 2004 Korean 147/30/0 116/13/0 1.00 39/50 60.8/51.6 Sun [25] 2004 Chinese 239/54/6 84/28/1 0.69 50/47 59.8/56.0 de syllos [29] 2006 Brazilian 82/78/10 54/45/4 0.20 40/58 57.5/51.9 Luo [36] 2006 Chinese 121/47/4 98/19/2 0.29 50/56 na/na Sandrim [24] 2006 Brazilian 86/82/11 53/45/4 0.20 58/59 57.5/52.1 Fu [35] 2007 Chinese 97/42/0 51/12/0 1.00 39/38 64.0/64.0 Ma [37] 2007 Chinese 240/59/0 86/14/0 1.00 49/57 62.0/59.0 Ezzidi [17] 2008 Tunisian 350/442/122 335/334/69 0.31 46/50 59.3/58.7 Odeberg [38] 2008 Swedish 222/153/28 407/322/70 0.61 50/49 70.0/60.1 Ritt [9] 2008 Germany 37/36/11 38/33/13 0.23 73/73 59.5/47.8 Deng [16] 2009 Chinese 139/46/17 80/14/6 0.00 100/100 56.1/54.2 Bae [30] 2010 Korean 75/14/0 245/53/1 0.49 61/53 61.5/46.4 Angeline [42] 2011 Indian 25/55/20 113/47/0 0.88 100/100 na/na Santos [34] 2011 Brazilian 294/261/54 47/48/5 0.14 49/50 61.0/46.4 a, mean age; b, not available increased risk of T2DM considering dominant model, (OR=1.52, 95%CI=1.05-2.22). And significant asso- recessive model, a vs. b and aa vs. bb, respectively. ciation was found between 4a and increased risk of After excluding articles that deviated from HWE in T2DM for Asian before and after excluding articles controls and sensitivity analysis excluding the results that deviated from HWE in controls as well as sensitiv- with OR > 3.0, significant association was also found in ity analysis, respectively. dominant model (OR=1.32, 95%CI=1.17-1.48, Fig. 1), Association of G894T with T2DM risk a vs. b (OR=1.34, 95%CI=1.21-1.48) and aa vs. bb The 894T allele was found significantly associated 896 Jia et al.

Table 2 Pooled measures on the relation of NOS3 gene 4b4a VNTR and G894T polymorphisms with type 2 diabetes mellitus risk All articles Articles in HWE and after sensitivity analysis Inherited Data 2 2 model REM FEM P I REM FEM I Cases/ OR (95% CI) OR (95% CI) (%) OR (95% CI) OR (95% CI) P (%) N controls 4b4a Total Dominant 1.34 (1.15-1.57) 1.33 (1.19-1.49) 5e-7 37.0 1.31 (1.16-1.49) 1.32 (1.18-1.48) 3e-6 9.1 17 4680/2906 VNTR Recessive 1.84 (1.09-3.09) 1.68 (1.22-2.31) 2e-3 44.3 1.37 (0.92-2.04) 1.42 (0.98-2.06) 0.08 3.3 11 3826/2048 a vs. b 1.38 (1.16-1.64) 1.35 (1.22-1.49) 3e-4 58.8 1.33 (1.16-1.52) 1.34 (1.21-1.48) 3e-8 30.2 17 4680/2906 aa vs. bb 1.96 (1.16-3.32) 1.80 (1.31-2.49) 3e-4 44.9 1.45 (0.97-2.18) 1.52 (1.05-2.22) 5e-3 4.5 11 3826/2048 Dominant 1.40 (1.09-1.79) 1.33 (1.13-1.57) 9e-3 50.2 1.31 (1.06-1.62) 1.29 (1.09-1.54) 4e-3 23.3 11 2485/1583 Recessive 2.63 (1.15-6.05) 1.93 (1.12-3.33) 0.02 45.3 1.38 (0.52-3.67) 1.38 (0.52-3.67) 0.78 0.0 6 1774/885 Asian a vs. b 1.49 (1.13-1.97) 1.38 (1.19-1.61) 5e-3 67.0 1.37 (1.11-1.70) 1.35 (1.15-1.59) 3e-4 36.3 11 2485/1583 aa vs. bb 2.78 (1.20-6.45) 2.05 (1.18-3.55) 0.01 44.5 1.39 (0.52-3.71) 1.39 (0.52-3.71) 0.69 0.00 6 1774/885 G894T Total Dominant 1.25 (1.06-1.48) 1.20 (1.09-1.32) 0.01 60.7 1.15 (0.99-1.32) 1.14 (1.03-1.26) 0.01 40.6 17 4493/3445 Recessive 1.36 (1.05-1.74) 1.36 (1.13-1.63) 8e-4 29.4 1.25 (1.00-1.57) 1.28 (1.06-1.54) 4e-4 14.2 14 3890/3171 T vs. G 1.22 (1.06-1.41) 1.19 (1.10-1.28) 7e-3 64.6 1.20 (1.04-1.40) 1.18 (1.09-1.27) 0.04 65.3 18 4593/3705 TT vs. GG 1.48 (1.06-2.06) 1.47 (1.21-1.78) 0.02 52.3 1.28 (0.96-1.71) 1.33 (1.09-1.62) 0.01 29.9 14 3890/3171 Dominant 1.49 (1.08-2.05) 1.50 (1.24-1.82) 0.01 63.8 1.28 (0.95-1.71) 1.26 (1.01-1.57) 0.04 43.0 8 1508/990 Recessive 2.14 (1.32-3.49) 2.14 (1.32-3.49) 2e-3 0.00 1.40 (0.68-2.92) 1.40 (0.68-2.92) 0.60 0.00 5 905/716 Asian T vs. G 1.42 (1.10-1.84) 1.50 (1.27-1.77) 7e-3 56.1 1.39 (1.04-1.86) 1.48 (1.24-1.76) 0.02 60.6 9 1608/1250 TT vs. GG 2.85 (1.68-4.84) 2.88 (1.72-4.84) 6e-5 1.60 1.58 (0.55-4.53) 1.58 (0.55-4.53) 0.40 0.00 5 905/716 HWE, Hardy–Weinberg equilibrium; FEM, fixed effect model; REM, random effect model; P, P value for significance test of OR=1 in FEM if I 2 <50%, otherwise in REM (I 2 >50%)

Fig. 1 Forest plot of odds ratios (ORs) for type 2 diabetes mellitus in dominant model (aa + ba vs. bb) for NOS3 gene 4b4a VNTR re- stricting studies that were in Hardy-Weinberg equilibrium in control group and after sensitivity analysis. Open diamond denotes the pooled ORs including fixed (denoted as I-V) and random (denoted as D+L) effects. Black squares indi- cate the OR in each study, with square sizes inversely proportional to the standard error of the OR. Horizontal lines represent 95% confidence intervals. Weights are from random effects analysis NOS3 gene polymorphisms and T2DM 897

Fig. 2 Forest plot of odds ratios (ORs) for type 2 diabetes mellitus in dominant model (TT+GT vs. GG) for NOS3 gene G894T re- stricting studies that were in Hardy-Weinberg equilibrium in control group and after sensitivity analysis. Open diamond denotes the pooled ORs including fixed (denoted as I-V) and random (denoted as D+L) effects. Black squares indicate the OR in each study, with square sizes inversely proportional to the standard error of the OR. Horizontal lines repre- sent 95% confidence intervals. Weights are from random effects analysis

with increased risk of T2DM in all of the above-men- found having a significant impact on between-study tioned models. After excluding articles that deviated heterogeneity in any of the above-mentioned inherited from HWE in controls as well as sensitivity analy- models. However, after excluding articles that devi- sis, significant association was also found in domi- ated from HWE in controls as well as sensitivity analy- nant model (OR=1.14, 95%CI=1.03-1.26, Fig. 2), sis excluding results with OR > 3.0, only low or mod- recessive model (OR=1.28, 95%CI=1.06-1.54), T erate heterogeneity was found in all models for 4b4a vs. G (OR=1.18, 95%CI=1.09-1.27), and TT vs. GG VNTR and most of models for G894T. (OR=1.33, 95%CI=1.09-1.62). And significant asso- ciation was found between 894T allele increased risk Influence analysis of T2DM for Asian before and after excluding articles No individual study was found to have excessive that deviated from HWE in controls as well as sensitiv- influence on the pooled effect for 4b4a VNTR and ity analysis, respectively. G894T polymorphisms in any of the above-mentioned models (Fig. 3 presents the influence analysis for 4b4a Between-study heterogeneity and meta-regression VNTR in dominant model after excluding two articles analysis that deviated from HWE in controls and after sensitiv- Univariate meta-regression analysis was performed ity analysis). to explore the sources of between-study heterogeneity, with the covariates of publication year, ethnicity (cat- Publication bias evaluation egorized as Asian and non-Asian), mean age (ratio of No significant publication bias was detected for 4b4a mean age in case group to that in control group), sex VNTR and G894T polymorphisms in any of the above- (ratio of male percent in case group to that in control mentioned inherited models, respectively. group) and sample size. However, no covariate was 898 Jia et al.

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

Discussion The rs1549758, a C/T variation (774T>C) in the NOS3 gene on chromosome 7, resulting in no substitu- In this meta-analysis, significant association was tion amino acid at position 258 of the (D258D), found of 4a of 4b4a VNTR and 894T of G894T with is shown in linkage disequilibrium with G894T (pair- increased risk of T2DM. Because of the inconsistent wise r2 ranges from 0.367-1.000 in different popula- results from relatively small studies underpowered to tion samples) according to the 1000 Genomes data. detect the effect, a meta-analysis is the appropriate However, the reports of rs1549758 with T2DM are also approach to obtain an improved provisional conclu- lacking. According to the linkage disequilibrium data sion. And this meta-analysis, of 19 articles involving by population in the 1000 Genomes data, the G894T 8,009 subjects for 4b4a VNTR and 19 articles involv- and T-786C polymorphism are in weak LD in all popu- ing 8,600 subjects for G894T, allowed a much greater lation samples, with a biggest value of r2 (0.336) found possibility of reaching reasonably conclusions. in a Mexican ancestry population. However, the 4b4a Recently, many loci associated with T2DM have VNTR is not captured in 1000 Genomes data. been discovered from genome-wide association stud- Between-study heterogeneity is common in meta- ies (GWAS) and meta-analysis of GWAS using SNP analysis for genetic association studies [49], and explor- chips for Europeans [43], South Asians [44] and East ing the potential sources of between-study heterogene- Asians [45]. These GWAS involved more T2DM cases ity is the essential component of meta-analysis [50]. (6000-8000) than that included in this meta-analysis, The between-study heterogeneity might arise from and the two variants (4b4a VNTR and G894T ) have an indeterminate number of characteristics that vary not been represented on GWAS chips, thus the find- among studies, e.g. study quality, population stratifica- ings of this meta-analysis need to be confirmed in the tion, characteristics of the subjects involved, genotyping GWAS. And the reports of T2DM with the near quality, variation of the covariate, and deviation from the NOS3 gene including ABP1, KCNH2, ATG9B, HWE in some studies, etc. Thus we used meta-regres- ABCB8, ASIC3, CDK5 and SLC4A2 are also limited. sion to explore the causes of heterogeneity for covari- Results from 3 meta-analysis [46-48] suggested that ates. However, publication year, ethnicity of popula- NOS3 gene polymorphisms (4b4a VNTR and G894T) tion, mean age, sex and sample size were found not to might be associated with risk of diabetic nephropathy. be important sources of disease–effect heterogeneity in NOS3 gene polymorphisms and T2DM 899 this meta-analysis. In evaluating associations between in Caucasians than African-Americans or Asians, while genetic variants and common complex diseases, biolog- the 4a variant for 4b4a VNTR was more common in ically meaningful associations of single genetic variant African-Americans than Caucasians or Asians in the with common diseases are expected in small effects (risk American population [55-56]. However, the limited ratios mostly<2.0) [15], and very big effect estimates, data precluded a further estimation of the association which might cause significant findings by chance, could between NOS3 polymorphisms (4b4a VNTR, G894T) be caused by unstable effect estimations resulting from and T2DM risk in other ethnicity besides Asian. Thus low cell counts within each single study and possible although ethnicity of population was not found to be genotyping errors. Thus sensitivity analysis was also the important source of disease–effect heterogene- performed excluding results with OR>3.0. And after ity across the studies in this meta-analysis, we can- excluding articles that deviated from HWE in controls not rule out the underling ethnicity-specific effect of and sensitivity analysis, only low or moderate heteroge- NOS3 gene polymorphisms on T2DM risk. Finally, in neity was found in all models for 4b4a VNTR and most a meta-analysis of published studies, publication bias of models for G894T. Besides, Diabetes mellitus have may be a problem. However, we found no evidence a complex aetiology and pathophysiology generated by of significant publication bias in this meta-analysis. the combined effects of genes and environment factors. Nevertheless, it is worthy of note that the validity of Thus other genetic and environment variables, as well our publication bias test is questioned, because of small as their possible interaction, may well be potential con- number of studies [57] in our meta-analysis. tributors to the heterogeneity observed. In conclusion, this meta-analysis suggested that NOS3 Other limitations involved in this meta-analysis are gene 4b4a VNTR and G894T polymorphisms might as follows: First, previous studies showed that hap- be associated with T2DM risk. Since potential biases lotype analysis of NOS3 gene polymorphisms (4b4a and confounders could not be ruled out completely in VNTR and G894T) showed significant associations this meta-analysis, further studies of NOS3 gene 4b4a between NOS3 haplotypes and impaired endogenous VNTR and G894T polymorphisms with T2DM using NO formation [51-54], supporting the idea that com- LD/haplotype-SNP tagging approach are warranted. binations of genetic polymorphisms within haploypes are of major relevance to examine the biological and Acknowledgements clinical relevance of polymorphisms. And three stud- ies [17, 29-30] included in this meta-analysis also dem- None. onstrated significant association between NOS3 gene variants and T2DM with haplotype analysis. But the Conflict of Interest limited number of studies precluded a haplotype analy- sis in this meta-analysis. Second, previous studies sug- None. gested that 894T allele for G894T was more common

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