Endothelial Nitric Oxide Synthase Gene Polymorphisms and the Risk ...

GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 16, Number 6, 2012 ª Mary Ann Liebert, Inc. Pp. 574–579 DOI: 10.1089/gtmb.2011.0218

Endothelial Nitric Oxide Synthase Gene Polymorphisms and the Risk of Diabetic Nephropathy in Type 2 Diabetes Mellitus Amira Shoukry,1 Sally M. Shalaby,2 Shymaa Abdelazim,3 Marwa Abdelazim,3 Ayman Ramadan,1 Mabrouk I. Ismail,1 and Mohamed Fouad1

Endothelial dysfunction plays an important role in the pathogenesis of diabetic vascular disease, including diabetic nephropathy (DN). Endothelial nitric oxide synthase (eNOS) gene polymorphisms that affect eNOS activity are associated with endothelial dysfunction. The aim of this study was to evaluate the association of three polymorphisms of the eNOS gene (894G > T, - 786T > C, and 27-bp-VNTR) with the risk of DN among type 2 diabetic patients. A total of 400 type 2 diabetic patients were enrolled in this study. The DN group comprised 200 patients; the group of diabetics without nephropathy comprised another 200 patients. Genetic analysis for eNOS gene polymorphisms was done in all subjects. Measurement of nitric oxide levels was estimated. The C allele for - 786T > C and the T allele for 894G > T were significantly more frequent in diabetics with nephropathy than in diabetics without nephropathy ( p < 0.001; odds ratio [OR] and 95% confidence interval [CI] for the C allele = 1.64 [1.24–2.17] and p < 0.001; OR and 95% CI = 1.7 [1.27–2.26] for the T allele). The haplotypes CTa (with all the mutant alleles) and CTb were significantly more common in patients with DN ( p = 0.01 and 0.003, respectively). These results suggested that the eNOS polymorphisms might represent genetic determinants for developing DN in type 2 diabetic Egyptians.

Introduction

D

iabetic nephropathy (DN) is a chronic microangiopathic complication of both type 1 and type 2 diabetes mellitus and is the primary cause of end-stage renal disease (Gross et al., 2005). Vascular endothelial dysfunction resulting from impaired nitric oxide synthase (NOS) activity in the endothelial cells of blood vessels has been suggested as playing an important role in the pathogenesis of DN (Nakagawa et al., 2007). It regulates endothelial function and maintains endothelial-dependent vasodilation in multiple organs, including the kidney (Moncada and Higgs, 2006). Nitric oxide (NO) is synthesized from l-arginine catalyzed by NOS. NO diffuses from the endothelium to the vascular smooth muscle cells, where it increases the concentration of cGMP by stimulating soluble guanylate cyclase, leading to vascular relaxation (Moncada and Higgs, 1993). There are three isoforms of NOS: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) (Noiri et al., 2002). eNOS is found in the glomerular afferent and efferent arterioles (Star, 1997). Endothelium-derived NO plays a key role in the regulation of vascular tone (Loscalzo and Welch, 1995),

and has vaso-protective effects by scavenging superoxide radicals and suppressing platelet aggregation, leukocyte adhesion, and smooth muscle cell proliferation (Lefer, 1997). Altered NO levels have been described in patients with DN, including increased NO expression in early DN, followed by a marked down regulation (Zanchi et al., 2000). However, advanced nephropathy leading to severe proteinuria, declining renal function, and hypertension is associated with a state of progressive NO deficiency (Nakagawa et al., 2007). Susceptibility to DN involves many elements, among which the genetic factor has been shown to be significant (Canani et al., 1999). So, polymorphisms of individual genes are expected to confer a modest risk to susceptibility of DN (Zintzaras et al., 2009). The eNOS gene is located on chromosome 7q35–36, and it comprises 26 exons and 25 introns, with an entire length of 21 kb (Zanchi et al., 2000). Variants of the eNOS gene contribute to endothelial dysfunction and attenuate the NO production (Ezzidi et al., 2008). The most clinically relevant polymorphisms that have been described in the eNOS gene are the following: (i) a 894G > T substitution in exon 7, which is a single-nucleotide polymorphism (SNP) that results in a Gluto-Asp substitution at codon 298 (Yoshimura et al., 1998); (ii) a

Departments of 1Internal Medicine, 2Medical Biochemistry, and 3Microbiology and Immunology, Faculty of Medicine, Zagazig University, Zagazig, Egypt.

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T-786C substitution, another SNP in the promoter region (Asakimori et al., 2002); and (iii) the 27-bp variable number tandem repeat (VNTR) polymorphism in intron 4 of eNOS, which is also designated b/a insertion-deletion in intron 4 consisting of two alleles (the allele designated ‘‘a-deletion’’ has four tandem 27-bp repeats, and the allele designated ‘‘binsertion’’ has five repeats) (Nadaud et al., 1994). Two meta-analyses have discussed the association of eNOS gene polymorphisms with the risk of DN, the first one supported lack of association between them (Zintzaras et al., 2009). Although the second meta-analysis supports the effects of the three polymorphisms (894G > T, 4b/a, and T-786C) in the eNOS gene on the risk of DN, it reported that none of them has been convincingly proved as determinants responsible for the development of DN, and suggested that their associations are still very likely to be due to that these polymorphisms might be in linkage disequilibrium with other unidentified functional causative mutations that exist in the eNOS gene and affect the susceptibility to DN (Zeng et al., 2010). So, this work was carried out to study the association of three polymorphisms of the eNOS gene (- 786T > C, 894G > T, and 27-bp-VNTR) with the risk of DN among type 2 diabetic subjects. Also, association of these polymorphisms with serum NO levels in these subjects were examined.

1. Case group: 200 patients with DN who had persistent proteinuria, which was defined as albumin/creatinine ratio (ACR) in spot urine collection is ‡ 300 mg/g (Lamb et al., 2009). 2. Control group: 200 patients without nephropathy who had T2DM for at least 10 years or more after diagnosis and whose ACR was < 30 mg/mg (Lamb et al., 2009).

Methods Patients This case–control study was started in February 2009 to December 2010. It included 400 type 2 diabetic patients (T2DM): 200 patients with nephropathy as cases and 200 without nephropathy as controls. They were recruited from Endocrinology and Nephrology outpatient clinics of the Internal Medicine Department, Zagazig University hospitals. All subjects were Egyptians from Sharkia–Egypt and they belonged to the same ethnic group: mixed. A written informed consent was obtained from all patients. The study was approved by our institute’s ethics committee. Type 2 diabetes was defined by the 1999 criteria of the World Health Organization (i.e., fasting glucose level > 126 mg/dL and/or 2-h postprandial glucose level > 200 mg/dL) (World Health Organization, 2006). Patients who did not meet these criteria as under treatment but who gave a history of T2DM were also included in the study. Patients were divided into two groups:

Diabetic subjects without proteinuria but on antihypertensive drug treatment were excluded from the study group in order to avoid misclassification of phenotype. Laboratory tests Serum NO was measured as nitrite/nitrate levels in subjects using Griess reagent (a 1:1 mixture of 1% sulfanilamide in 5% H3PO4 and 0.1% N-1-napthyl-ethylenediamine) (Torre et al., 1996). Genetic analysis Genomic DNA was isolated using the Wizard Genomic DNA Purification Kit purchased from Promega. Two eNOS SNPs, namely, - 786T > C (rs 2070744) and 894G > T (Glu298Asp; rs 1799983), were genotyped using the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). Primers for - 786T > C SNP were the same as previously used by Thameem et al. (2008), whereas primers for 894G > T SNP were the same as previously used by Miyamoto et al. (1998). The 27-bp-VNTR is characterized by presence of either four 27-bp repeats (a allele) or five 27-bp repeats (b allele) and was genotyped using a PCR assay with primers as previously used by Wang et al. (1996). Details, including location of SNPs in the respective genes, primer sequences, PCR conditions, and restriction enzyme with product sizes, are presented in Table 1. The PCR was done using Taq PCR Master Mix kit (Qiagen, GmbH) as following: 25 mL of Taq PCR master mix was dispensed into each PCR tube, and then the following materials were added to each tube containing 100 ng of extracted DNA, 25 mM forward primer, and 25 mM reverse primer (Operon Biotechnologies, Inc.), and then 19 mL dd H2O was added giving a final volume of 50 mL. The digested PCR products were resolved on 3% agarose gels stained with ethidium bromide. Thirty percent of the RFLP assays were repeated and no discrepancy was observed.

Table 1. Standard Polymerase Chain Reaction Conditions Used in Genotyping - 786T > C, 894G > T, and 27VNTR Single-Nucleotide Polymorphisms of the eNOS Gene SNP

Primers

Amplicon Annealing (bp) temperature (C)

894G > T

P1: 5¢-AAG GCA GGA GAC AGT GGA TGG A-3¢ P2: 5¢-CCC AGT CAA TCC CTT TGG TGC TCA-3¢

248

62

- 786T > C

P1: 5¢-TGG AGA GTG CTG GTG TAC CCC A-3¢

180

61

393/420

59

P2: 5¢-GCC TCC ACC CCC ACC CTG TC-3¢ 27-bp-VNTR P1: 5¢-AGG CCC TAT GGT AGT GCC TTT-3¢ P2: 5¢-TCT CTT AGT GCT GTG GTC AC-3¢ SNP, single nucleotide polymorphism.

Restriction enzyme/ allele size BanII (New England Biolabs) G = 163, 85 bp T = 248 bp MspI (New England Biolabs) T = 140, 40 bp C = 90, 50, 40 bp b allele = 420 bp a allele = 393 bp

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Statistical analysis The appropriate sample size and power of the study were determined using PAWE-3D. PAWE-3D calculations showed that the sample size, together with the specified study design, allele frequencies, the incidence of nephropathy among T2DM patients (30%), and allowable error rates, can give as high as 90% power and can detect variant allele frequency of at least 0.05 and genotype relative risk of ‡ 1.8 at 80% power. The results for continuous variables are expressed as means – standard deviation. The statistical significances of differences in frequencies of variants between the groups were tested using the chi-square (v2) test. In addition, the odds ratios (ORs) and 95% confidence intervals (CIs) were calculated as a measure of the association of the eNOS alleles with groups. Haplotypes were determined based on Bayesian alogorithm using the Phase program. The Fisher Exact Probability test was performed to determine significance of the haplotypes between case and control groups. p-values were considered significant when p < 0.05. Results Demographic, clinical, and laboratory characteristics of the studied groups are shown in Table 2. The genotype and allele frequencies of the eNOS polymorphisms in diabetic patients without nephropathy and diabetics with nephropathy are shown in Table 3. Genotype frequencies of all eNOS polymorphisms were in agreement with Hardy–Weinberg equilibrium in each study group. Our results showed significant differences between patients with DN and patients without DN regarding the genotype and allele distributions of the - 786T > C SNP Table 2. Demographic, Clinical, and Laboratory Characteristics of the Studied Groups Parameter Age (years) Male/female BMI (kg/m2) Duration of diabetes (years) SBP (mmHg) DBP (mmHg) 2 h postprandial glucose levels (mg/dL) HbA1c (%) Total cholesterol (mg/dL) HDL (mg/dL) Triglycerides (mg/dL) Blood urea (mg/dL) Serum creatinine (mg/dL)

Patients without DN

Patients with DN

p

54.6 – 5.2 116/84 27.9 – 3.7 13.8 – 3.2

55.3 – 5.8a 108/92b 27.4 – 3.2a 14.5 – 4.3a

0.21 0.5 0.15 0.07

134.3 – 16.7 82.3 – 6.2 205.7 – 33.2

141.7 – 19.6a 87.6 – 9.3a 208.6 – 35.5a

< 0.001 < 0.001 0.39

7.3 – 1.5 201.5 – 41.3

7.5 – 1.4a 209.3 – 43.6a

0.17 0.07

58.2 – 10.5 159.9 – 31.5

56.4 – 12.4a 162.3 – 34.8a

0.12 0.47

38.4 – 14.5 1.12 – 0.13

92.6 – 29.4a 2.86 – 1.15a

< 0.001 < 0.001

Values are mean – standard deviation. a Calculated by t-test. b Calculated by Pearson’s chi square test. DN, diabetic nephropathy; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high density lipoprotein.

Table 3. Genotype and Allele Frequencies of the eNOS Gene Polymorphisms in Studied Groups

- 786T > C SNP Genotype TT TC CC Allele T C 894G > T Genotype GG GT TT Allele G T 27VNTR Genotype bb ab aa Allele b a a

Patients without DN, n (%)

Patients with DN, n (%)

84 (42) 83 (41.5) 33 (16.5)

57 (28.5) 89 (44.5) 54 (27)

0.005 0.55 0.01

251 (62.7) 149 (37.3)

203 (50.7) 197 (49.3)

< 0.001

99 (49.5) 77 (38.5) 24 (12)

66 (33) 94 (47) 40 (20)

275 (68.75) 125 (31.25)

226 (56.5) 174 (43.5)

131 (65.5) 60 (30) 9 (4.5)

124 (62) 64 (32) 12 (6)

0.47 0.31 0.5

322 (80.5) 78 (19.5)

312 (78) 88 (22)

0.09

pa

0.001 0.09 0.03 < 0.001

Pearson’s chi square test.

polymorphism. The CC genotype for the - 786T > C polymorphism was significantly more frequent in diabetics with nephropathy than in diabetics without nephropathy (27% vs. 16.5%, p = 0.01), and also the C allele was more frequent in the DN group as compared to diabetics without nephropathy ( p < 0.001), OR, and 95% CI for the C allele of - 786T > C = 1.64 (1.24–2.17). For the 894G > T SNP, the TT genotype was significantly more frequent in diabetics with nephropathy than in diabetics without nephropathy (20% vs. 12%, p = 0.03); similarly, the T allele was more frequent in the DN group than in diabetics without nephropathy ( p < 0.001): OR and 95% (CI) for the T allele of 894G > T = 1.7 (1.27–2.26). The results showed no significant differences in allele or genotype frequencies between patients with DN and patients without DN for 27-bp-VNTR polymorphism ( p > 0.05). The haplotype TGb (with all the wild alleles) was significantly higher in diabetics without nephropathy than in diabetics with nephropathy ( p = 0.008). Conversely, the haplotypes CTa (with all the mutant alleles) and CTb were more common in patients with DN than in patients without DN ( p = 0.01 and 0.003 respectively; Table 4). Serum NO levels were significantly decreased in the DN group (0.5 – 0.12 nmol/mL) as compared to diabetes without nephropathy (0.81 – 0.20 nmol/mL; p < 0.001). Regarding association between the eNOS polymorphisms and serum NO levels (Table 5), the results showed that serum NO levels were significantly decreased in the ( - 786T/C) CC and TC genotypes relative to the TT genotype in the DN group and in diabetics without nephropathy ( p < 0.05 for each). Similarly, NO levels were significantly decreased in the 894G > T TT and GT genotypes relative to the GG genotype in


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Table 4. Haplotype Frequencies of eNOS Gene Polymorphisms in Studied Groups

Haplotypea

Patients without DN, n (%)

TGb TGa CGb TTb CTb TTa CTa CGa

180 92 47 39 22 16 3 1

(45) (23) (11.75) (9.75) (5.5) (4) (0.75) (0.25)

Patients with DN, n (%) 142 85 46 41 46 22 14 4

(35.5) (21.25) (11.5) (10.25) (11.5) (5.5) (3.5) (1)

pb 0.008 0.61 1.0 0.91 0.003 0.41 0.01 0.38

a Order of SNPs in eNOS haplotypes: - 786T > C, 894G > T, and 27VNTR. b p calculated by Fisher Exact test. eNOS, endothelial nitric oxide synthase.

both groups ( p < 0.05 for each). Concerning the 27-bp-VNTR polymorphism, there were no significant changes in NO levels in the different genotyping groups of the DN group and in the diabetics without nephropathy group ( p > 0.05 for each). When comparing NO levels in carriers versus noncarriers of eNOS haplotype CTa and CTb, there was a significant decrease of NO levels in carriers of CTa and CTb compared to the noncarriers (data not shown). Discussion Why some diabetics develop nephropathy, whereas others do not, despite having a long-term hyperglycemia remains an unresolved question. Because known environmental factors do not fully explain this, researchers have sought the answer at the genetic background of the host. Polymorphisms in the eNOS gene have been implicated with DN. The mechanism responsible for this potential association is not known yet. However, variants of the eNOS gene may cause defective NO synthesis and decreased NO levels, enhancing the susceptibility to glomerular disease and deteriorating the renal function (Shin et al., 2004; Ahluwalia et al., 2008). Therefore, this metabolic pathway of diabetes may be involved in renal complications of diabetes. In the present study, we examined the associations of two potentially functional SNPs, 894G > T and - 786T > C, along with a 27-bp-VNTR polymorphism of the eNOS gene with DN among T2DM patients. Our study revealed that the TT genotype and the T allele of eNOS 894G > T polymorphism were

significantly more frequent in diabetics with nephropathy than in diabetics without nephropathy. Our results were supported by Ahluwalia et al. (2008) in a cohort of T2DM patients of Mixed North Indian ethnicity. Also, Nagase et al. (2003) found a higher prevalence of 894T in Japanese patients with diabetes mellitus as a cause of renal failure, and added that this polymorphism has been proposed as a candidate factor for the accelerated nephropathy in type 2 diabetes mellitus. Ezzidi et al. (2008) found increased TT genotype in DN compared to diabetics without nephropathy in Tunisian patients. As regard - 786T > C SNP, our results also demonstrated that the CC genotype and the C allele were significantly more frequent in diabetics with nephropathy than in diabetics without nephropathy. These results coincided with Ahluwalia et al. (2008), who observed that excess risk of DN was specifically associated with the ( - 786) CC genotype. Moreover, association of the ( - 786) C allele with a higher risk of DN was reported by Zanchi et al. (2000) in Caucasian type 1 diabetic patients and by Asakimori et al. (2002) in Japanese type 2 diabetic patients. Regarding the 27-bp-VNTR polymorphism in intron 4 (4b/a), our results showed no significant differences in allele or genotype frequencies between patients with DN and patients without DN. Our results were supported by Lin et al. (2002), who concluded that a-allele in intron 4 of the eNOS gene did not increase the risk of DN in type 2 diabetes of the Chinese population, also Fujita et al. (2000) and Shimizu et al. (2002) found lack of association between the b-insertion/a-deletion polymorphism in intron 4 of the eNOS gene and DN in type 2 diabetic East Asian and Japanese patients, respectively. However, other studies concluded that eNOS intron 4 polymorphism is associated with an increased risk for progression to DN in type 2 diabetes (Neugebauer et al., 2000; Asakimori et al., 2001). The frequency of 894T allele was more common in Caucasian (28.7%–036.6%) than in East Asian (5.9%–11.7%) patients without DN (Zeng et al., 2010). The frequency of 894T allele (31.25%) in Egyptians was similar to that reported in other mixed populations (12.3%–34.5%). The - 786C variant was found to be more frequent in controls in Caucasians (35.2%– 39.7%) than in the East Asian and Mixed populations (17.3%– 25.7%) (Zeng et al., 2010). In the current study, it was 37.3% in Egyptians. The frequency of the 4a allele varied widely across the Caucasian (13.6%–22.4%), the East Asian (6.9%–14.3%), and the Mixed population (10.7%–23.2%) (Zeng et al., 2010). In our sample, the frequency of 4a variant (19.5%) was comparable to that found in the Mixed control subjects. Such ethnic

Table 5. The Relation Between Endothelial Nitric Oxide Synthase Genotypes and Nitric Oxide Levels eNOS polymorphisms - 786T > C 894G > T 27-bp-VNTR

a

eNOS genotypes Patients with DN Patients without DN eNOS genotypes Patients with DN Patients without DN eNOS genotypes Patients with DN Patients without DN

NO levels (nM)

NO levels (nM)

NO levels (nM)

TT 0.54 – 0.13 0.83 – 0.23 GG 0.56 – 0.12 0.82 – 0.25 bb 0.51 – 0.22 0.79 – 0.23

TC 0.48 – 0.15a 0.75 – 0.22a GT 0.5 – 0.13a 0.71 – 0.23a ba 0.5 – 0.26 0.78 – 0.24

CC 0.45 – 0.12a 0.73 – 0.18a TT 0.49 – 0.14a 0.7 – 0.15a aa 0.49 – 0.25 0.77 – 0.23

Significant difference from the homozygous wild genotype within a group.

578 differences in the occurrence of genetic variants at the eNOS locus may relate to the ethnic-specific predisposition to DN. A recent meta-analysis assessed the association between the alleles of the eNOS gene 4b/a, 894G/T, and - 786T/C polymorphisms and DN and a subgroup analysis was performed based on ethnicity. The evidence accumulated suggested that 4b/a and G894T polymorphisms in the eNOS gene were associated with susceptibility to DN in Asian populations but not in Caucasian populations (He et al., 2011). Another meta-analysis found 24 genetic variants associated with DN; two of them were - 786T > C and 4b/a of the eNOS gene (Mooyaart et al., 2011). In a trial to investigate the functional aspects of the three polymorphisms, we measured NO levels for the patients in both groups of the study. We found that serum NO levels were significantly decreased in diabetics with nephropathy as compared to diabetics without nephropathy. Moreover, serum NO levels were significantly decreased in 894G > T GT and TT genotypes than in GG genotype, and also in the (- 786T/C) TC and CC genotypes than in the TT genotype in the DN group and in the diabetics without nephropathy group. Concerning the 27-bp-VNTR polymorphism, there were no significant changes in serum NO levels in different genotyping groups. In support of our results Nakayama et al. (1999) and Miyamoto et al. (2000) found significant diminished serum NO products levels among the - 786C allele carriers. Moreover, lower eNOS mRNA and serum nitrite/ nitrate levels have been found in individuals with the - 786C variant in various studies (Hibi et al., 1998; Yoshimura et al., 2000). However, in contrast to our results, Ritt et al. (2008) found that 894G > T had no impact on basal NO activity in the renal circulation of patients with or without DM, and Moon et al. (2002) revealed that there was no substantial effect of 894G > T polymorphism on the variance of plasma NO levels in a healthy Korean population. Tsukada et al. (1998) detected a significant decrease in plasma NO metabolites levels in 4a allele carriers in healthy subjects. There are a number of studies that have previously investigated the functional aspects of the three polymorphisms. The protein product of eNOS 894T is more susceptible to selective proteolytic cleavage in endothelial cells and vascular tissues (Tesauro et al., 2000), which could lead to reduced vascular NO generation. However, other expression studies have demonstrated no difference in NO generation between G894 (298Glu) and 894T (298Asp) (Bank and Aynedjian, 1993; Fairchild et al., 2001). Regarding the - 786T > C polymorphism, the ( - 786) C variant has been found to be associated with a significant reduction in the eNOS gene promoter activity (Nakayama et al., 1999). Other studies identified a protein (replication protein A1, RPA1) that might function as a repressor of transcriptional activity of the eNOS gene with the - 786C allele (Miyamoto et al., 2000); consequently, this could lead to diminished serum NO products levels among the - 786C allele carriers. The graded differences in NO generation could play a significant role in the pathophysiological condition at the microvascular level. Decreased renal NO accelerates the progression of DN in the rat model, presumably through mechanisms, such as increased renal vascular tone and potentiation of angiotensin II effects (Zanchi et al., 1999). Thus, reduced eNOS activity associated with - 786T > C and 894G > T polymorphism could play a role in downregulation of eNOS expression and endothelial dysfunction.

SHOUKRY ET AL. In this study, the interactions of the three polymorphisms within haplotypes were assessed. We found that the haplotype TGb (carrying all wild-type alleles) was more prevalent in diabetics without nephropathy as compared to diabetics with nephropathy. Thus, this haplotype may be considered to be protective for developing DN. Conversely, the haplotypes CTa and CTb were significantly higher in patients with DN than in patients without DN. Our results coincided with Ahluwalia et al. (2008), who found that the haplotype TGb was associated with a low risk of DN, and that the haplotype with all the three variant alleles CTa was associated with nearly 2.5-fold increase in the risk of DN. Concerning susceptible haplotype CTb in our Egyptian sample, Ahluwalia et al. (2008) showed no differences in its frequency between patients with DN and the patients without DN. Also, Ezzidi et al. (2008) observed that the haplotypes Asp298/4b/ - 786T and the Asp298/4a/ - 786C were present at higher frequencies among diabetics with nephropathy than among diabetics without nephropathy. All these results suggest that co-occurrence of variant/wild alleles of 894G > T, - 786T > C, and 27-bp-VNTR polymorphisms may modulate risk of nephropathy in diabetic patients. There are a few limitations of our study. Identifying significant associations of genetic variants with complex qualitative trait, such as DN, may need a larger sample size. However, the present study fulfills most of the criteria of a good genetic association study (Hattersley and McCarthy, 2005). Further, all the study groups were in Hardy–Weinberg equilibrium. In conclusion, of the three eNOS polymorphisms examined, the 894G > T and - 786T > C SNPs were associated with increased risk of DN among type 2 diabetic subjects, whereas no association was found between the 27-bp-VNTR polymorphism and the risk of DN in Egyptians. Disclosure Statement No competing financial interests exist. References Ahluwalia TS, Ahuja M, Rai TS, et al. (2008) Endothelial nitric oxide synthase gene haplotypes and DN among Asian Indians. Mol Cell Biochem 314:9–17. Asakimori Y, Yorioka N, Taniguchi Y, et al. (2002) T( - 786)/C polymorphism of the endothelial nitric oxide synthase gene influences the progression of renal disease. Nephron 91:747–751. Asakimori Y, Yorioka N, Yamamoto I, et al. (2001) Endothelial nitric oxide synthase intron 4 polymorphism influences the progression of renal disease. Nephron 89:219–223. Bank N, Aynedjian HS. (1993) Role of EDRF (nitric oxide) in diabetic renal hyperfiltration. Kidney Int 43:1306–1312. Canani LH, Gerchman F, Gross JL. (1999) Familial clustering of DN in Brazilian type 2 diabetic patients. Diabetes 48:909–913. Ezzidi I, Mtiraoui N, Mohamed MB, et al. (2008) Association of endothelial nitric oxide synthase Glu298Asp, 4b/a, and - 786T > C gene variants with DN. J Diabetes Complications 22:331–338. Fairchild TA, Fulton D, Fontana JT, et al. (2001) Acidic hydrolysis as a mechanism for the cleavage of the Glu298Asp variant of human endothelial nitric oxide synthase. J Biol Chem 276: 26674–26679. Fujita H, Narita T, Meguro H, et al. (2000) Lack of association between an ecNOS gene polymorphism and DN in T2DM with proliferative diabetic retinopathy. Horm Metab Res 32:80–83.


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Address correspondence to: Sally M. Shalaby, M.D. Department of Medical Biochemistry Faculty of Medicine Zagazig University Zagazig Egypt E-mail: [email protected]