Endothelial nitric oxide synthase Glu298Asp, 4b ⁄ a, and )786T>C gene polymorphisms and the risk of ischemic stroke Introduction Ischemic stroke (IS) is the leading cause of disability in the world (1). While it has a strong genetic predisposition (2), the genes contributing to IS pathogenesis are still largely unknown (2, 3). Clinical and experimental evidence suggests a role for vascular dysfunction in IS pathogenesis, of which endothelium-derived nitric oxide (NO) plays a pivotal role in vascular protection (1, 4). NO is an intracellular second messenger produced by NO synthase enzyme (NOS), of which three major NOS forms with different calcium dependency and secretion requirements were described: endothelial (eNOS), inducible, and neuronal NOS (5). NO exerts atheroprotective effects, highlighted by its capacity to inhibit platelet aggregation (4), and leukocyte adhesion (6) to vascular endothe- lium, leading to inhibition of vascular smooth muscle cell growth and proliferation (7). Several polymorphisms affecting eNOS production levels or function were described, including )786T>C (promoter), Glu298Asp (G894T; exon 7), and 4b ⁄ 4a variable-number 27-bp tandem repeat Acta Neurol Scand 2010: 121: 114–119 DOI: 10.1111/j.1600-0404.2009.01192.x Copyright Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Munksgaard ACTA NEUROLOGICA SCANDINAVICA Saidi S, Mallat SG, Almawi WY, Mahjoub T. Endothelial nitric oxide synthase Glu298Asp, 4b ⁄ a, and )786T>C gene polymorphisms and the risk of ischemic stroke Acta Neurol Scand: 2010: 121: 114–119. Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Munksgaard. Background and purpose – Endothelial nitric oxide synthase (eNOS) gene polymorphisms were associated with reduced NO production, and were evaluated as risk factors for ischemic stroke (IS). We investigated the association between eNOS gene )786T>C (promoter), 27-bp repeat 4b ⁄ 4a (intron 4), and Glu298Asp (exon 7) polymorphisms with IS in 329 IS patients and 444 controls. Materials and methods – Glu298Asp and )786T>C genotyping was done by PCR-RFLP, 4b ⁄ 4a was assessed by PCR–ASA. The contribution of eNOS polymorphisms to IS was analyzed by haplotype and multivariate regression analysis. Results – Higher frequency of 298Asp allele was seen in IS patients (P= 1.2 · 10 )10 ), which remained independently associated with IS on multivariate analysis after controlling for traditional cerebrovascular risk factors. Allele and genotype distribution of 4b ⁄ 4a and )786T>C polymorphisms were comparable between patient and controls. Significantly higher prevalence of 298Asp ⁄ 4b ⁄ )786T and 298Asp ⁄ 4b ⁄ )786C haplotypes were seen in IS cases, thus conferring a disease susceptibility nature to these haplotypes. Multivariate regression analysis confirmed the association of 298Asp ⁄ 4b ⁄ )786T and 298Asp ⁄ 4b ⁄ )786C haplotypes, and in addition identified 298Asp ⁄ 4a ⁄ )786T haplotype to be independently associated with IS, after controlling for traditional cerebrovascular risk factors. Conclusions – Genetic variation at the eNOS locus represent genetic risk factor for increased susceptibility to IS. S. Saidi 1 , S. G. Mallat 2 , W. Y. Almawi 3 and T. Mahjoub 1 1 Research Unit of Hematological and Autoimmune Diseases, Faculty of Pharmacy, University of Monastir, Tunisia; 2 UniversiteÕ St. Joseph and Hotel Dieu du France Hospital, Beirut, Lebanon; 3 College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain Key words: endothelial nitric oxide synthase; polymorphisms; ischemic stroke W. 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: wassim@agu.edu.bh Accepted for publication March 2, 2009 114