Clinical Endocrinology (2008) 68, 542–546 doi: 10.1111/j.1365-2265.2007.03089.x © 2007 The Authors 542 Journal compilation © 2007 Blackwell Publishing Ltd ORIGINAL ARTICLE Blackwell Publishing Ltd Endothelial nitric oxide synthase Glu298Asp, 4b/a, and T-786C polymorphisms in type 2 diabetic retinopathy Intissar Ezzidi*, Nabil Mtiraoui*, Manel Ben Hadj Mohamed*, Touhami Mahjoub*, Maha Kacem† and Wassim Y. Almawi‡ *Research Unit of Haematological and Autoimmune Diseases, Faculty of Pharmacy, Monastir, Center University, Tunisia, †Nephrology and Internal Medicine Service – EPS F. Bourguiba, Monastir, Tunisia, ‡Department of Medical Biochemistry, College of Medicine & Medical Sciences, Arabian Gulf University, Manama, Bahrain Summary Objective The possible association between the endothelial nitric oxide (eNOS) gene T-786C (promoter region), 27-bp repeat 4b/4a (intron 4), and Glu298Asp (exon 7) polymorphisms with diabetic retinopathy (DR) was investigated. Design A retrospective case-control study. Patients A total of 872 type 2 diabetes (T2DM) patients were studied, of whom 383 presented with preproliferative/proliferative retinopathy (DR group), and 489 with absent/mild retinopathy (DWR group). Measurements Glu298Asp and T-786C genotyping was carried out by PCR-RFLP analysis, while 4b/4a was assessed by PCR. Genotype distribution was compared using the χ 2 -test, and the contributions of the polymorphisms to DR were analysed by haplotype analysis and multivariate regression analysis. Results Lower prevalence of mutant 4a (P = 0·011), and het- erozygous 4b/4a (P = 0·042) were seen in the DR compared to the DWR groups; the allele and genotype distribution of the Glu298Asp and T-786C polymorphisms were comparable between DR and DWR groups. Three-loci haplotype analysis demonstrated sig- nificant association between eNOS variants and DR, with protective [haplotype 122 (Glu298/4a/-786C)], and susceptible haplotypes [haplotypes 112 (Glu298/4b/-786C) and 222 (Asp298/4a/-786C)] identified. Multivariate regression analysis confirmed the association between haplotypes 122 (P = 0·015); 112 (P = 0·027), and 222 (P = 0·048) and DR, after controlling for potential covariates (including age, sex, age of disease onset; HbA1c; hypertension, total cholesterol). Conclusions This study identifies genetic variation at the eNOS locus as genetic risk factor for diabetic retinopathy, which may serve as a useful marker of increased susceptibility to the risk of retinopathy. (Received 16 June 2007; returned for revision 17 August 2007; finally revised 29 August 2007; accepted 24 September 2007) Introduction Diabetic retinopathy (DR) is a major cause of blindness among diabetic adults, 1 and is aggravated by poor glycaemic control. 2,3 Several mechanisms are reportedly involved in the DR-associated malfunction of the blood–retinal barrier (BRB), including induction of inflammatory processes, 4 altered endothelial cell junctions and viability, 5 and central retinal venous congestion. 5,6 Endothelial dysfunction induced by reduced nitric oxide (NO) availability, and consequently increased reactive oxygen species production, reportedly impaired ocular haemodynamics, suggesting a role for NO in DR pathogenesis. 7 NO is a pleiotropic molecule, which regulates several aspects of vascular tone, including inhibition of platelet aggregation, down-regulation of leucocyte adherence, 8 and suppression of smooth muscle cell proliferation. 8,9 NO is produced by three nitric oxide synthase (NOS) isoforms: neuronal NOS, inducible NOS, and endothelial NOS (eNOS or NOS3). 8,9 Low NO concentrations induced by eNOS are necessary for maintaining endothelial function, while attenuation of NO production induced by eNOS gene mutations resulted in endothelial dysfunction, and precipitated atherogenic events, including those associated with T2DM. 10,11 Several eNOS gene polymorphisms have been identified, of which the T-786C (promoter region), Glu298Asp (exon 7), and the 27-bp repeat 4b4a (intron 4) polymorphisms are the most investigated, and are associated with cardiovascular diseases, hypertension, and vascular disorders. 10 –13 A limited number of studies have examined the possible association between these eNOS polymorphisms and DR, with inconsistent results. For example, the (mutant)-786C 12,13 and 4a 12 alleles were associated with DR in some studies, while others reported no such association of either T-786C 14,15 or 4b/4a. 14–16 Others suggested that the 4b, but not 4a, allele was associated with a high risk of DR in type 1 diabetes. 17 Here we compared the distribution of the three eNOS polymorphisms in Tunisian T2DM patients with (DR) or without (DWR) retinopahy, together with their possible association Correspondence: Wassim Y. Almawi, Department of Medical Biochemistry, College of Medicine & Medical Sciences, Arabian Gulf University, PO Box 22979, Manama, Bahrain. Tel.: +973 39717118; Fax: +973 17271090; E-mail: wyalmawi@yahoo.co.uk