191 Ethnicity & Disease, Volume 15, Spring 2005 ORIGINAL REPORTS:CARDIOVASCULAR HEALTH ENDOTHELIAL NITRIC OXIDE SYNTHASE INTRON 4POLYMORPHISM ISA MARKER FOR CORONARY ARTERY DISEASE IN AFRICAN-AMERICAN AND CAUCASIAN MEN Objectives: We investigated the association of the intron 4 polymorphism of the endothelial nitric oxide synthase (eNOS) gene with coro- nary artery disease (CAD). Background: Genetic alterations in the gene encoding for eNOS could contribute to the de- velopment and progression of CAD. Methods: We genotyped for the eNOS intron 4 polymorphism in 194 subjects undergoing coronary angiography. Genotyping was per- formed with polymerase chain reaction-restric- tion fragment length polymorphism for the var- iable number of tandem repeats in intron 4. Coronary artery disease (CAD) was assessed by quantitative coronary angiography, and endo- thelial function was measured by brachial ul- trasonography. We performed logistic regres- sion analysis for the effect of eNOS intron 4 polymorphism and other coronary risk factors on multi-vessel CAD and endothelial function. Results: The 4a-allele frequency in African Americans was 0.31, while the 4a-allele fre- quency in Caucasians was 0.15 (P.001). The prevalence of the 4a-allele was highest among subjects with multi-vessel disease both for Af- rican Americans and for Caucasians. A race- adjusted comparison of the prevalence of the 4a-allele among subjects with multi-vessel dis- ease vs those without was statistically signifi- cant (P=.03). No correlation was found be- tween the eNOS intron 4 polymorphism and endothelial function. Conclusions: The eNOS intron 4 polymor- phism may be a marker of multi-vessel CAD in African Americans and Caucasians. (Ethn Dis. 2005;15:191–197) Key Words: Coronary Artery Disease, Endo- thelium, Genetic Polymorphisms, Nitric Oxide Synthase From the Division of Cardiology (SR, MND, AMZ), Department of Epidemiology (HA), Emory University, Atlanta; Atlanta Veterans Affairs Medical Center, Decatur (SR, MND, AMZ); Georgia. Address correspondence and reprint re- quests to A. Maziar Zafari, MD, PhD; Emory University School of Medicine; Division of Cardiology; 1639 Pierce Drive, 319 Swapna Rao, BS; Harland Austin, DSc; Madalyn N. Davidoff, MD; A. Maziar Zafari, MD, PhD INTRODUCTION Nitric oxide (NO) is a highly dif- fusible, short-lived molecule that affects many biological pathways. It is pro- duced enzymatically from the amino acid, L-arginine, by a family of three ni- tric oxide synthase (NOS) isoforms: en- dothelial NOS (eNOS or NOS-3), in- ducible NOS (iNOS or NOS-2), and neuronal NOS (nNOS or NOS-1). 1 Physiologically, NO diffuses from the endothelium to the vascular smooth muscle cell (VSMC) where it binds to the heme moiety of soluble guanylate cyclase and activates the conversion of guanosine 5'-monophosphate (GMP) to cyclic GMP (cGMP). 2 This increase in cGMP results in relaxation of vascu- lar smooth muscle, mediating endothe- lium-dependent vasodilation, inhibiting platelet aggregation and monocyte ad- hesion to the endothelium, inhibiting VSMC growth and migration, and fi- nally, inhibiting the oxidation of low- density lipoprotein (LDL) cholesterol. 3 Recent studies have found much ev- idence for the role of oxidative stress in the degradation of NO. This reduction in vascular NO bioavailability has been shown to contribute to altered vaso- motor tone, hypertension, endothelial dysfunction, and development and pro- gression of atherosclerosis. 4,5 The hu- man eNOS gene is located on chro- mosome 7q35–36 and comprises 26 ex- ons that span 21 kb and encode a 135 kD protein. 6 Ten polymorphisms have WMB; Atlanta, GA 30322; 404-712-8338; 404-329-2211 (fax); azafari@emory.edu been reported in the eNOS gene: three in the 5'-flanking region, two in the coding sequence, and five in intronic re- gions. 3 Clinical association studies have investigated the association of various polymorphisms of the eNOS gene to various cardiovascular diseases such as CAD, hypertension, stroke, and deep venous thrombosis (DVT), with con- flicting results. 3 Different ethnic popu- lations have been studied worldwide for the detection and frequency of the 27- bp variable number of tandem repeat (VNTR) polymorphism in the intron 4 of eNOS in relation to CAD. 7–19 Wang et al have reported that the eNOS in- tron 4 polymorphism has a major locus contribution to circulating nitrite and nitrate levels, a surrogate measure of NO. 20 Thus, we hypothesized that the eNOS intron 4a-allele may affect sever- ity of CAD as measured by coronary an- giography. In addition, since endothelial dysfunction plays an important role in atherosclerosis, we also studied endothe- lium-dependent and -independent vas- cular reactivity as an indicator of the functional consequence of the eNOS in- tron 4 polymorphism. METHODS Patient Population Our study sample consisted of a well-characterized group of 194 Cauca- sian and African-American patients who were referred for coronary angiography between March 1999 and August 2000 at the Atlanta Veterans Affairs Medical Center. The study was approved by the institutional review board, and all sub-