Correlation of Glu298Asp eNOS Polymorphism with Serum NO Levels in Egyptian Coronary Artery Disease Patients Sahar Abdel-Maksoud, Sally Ibrahim, Feeby Samir, Khaled Abu-Aisha, Mohamed Z Gad Clinical Biochemistry Unit, Faculty of Pharmacy, The German University in Cairo Abstract Nitric oxide (NO) synthesized by the vascular endothelium is a potent vasodilator. It has been reported that endothelial nitric oxide synthase (eNos) Glu298Asp polymorphism has been associated with coronary heart disease (CAD). In this study this polymorphism was analyzed in 146 age matched male subjects; 77 patients with coronary heart disease (CAD) classified according to severity and 69 normal controls. Serum NO was determined for all subjects. Results showed that the frequencies of the eNOS Glu298Asp genotypes in CAD subjects were G/G (53.49%), G/T (33.72%), and T/T (12.79%) whereas the allele distributions of G and T were and 29.65%, respectively. In the controls, the genotype frequency was 58.42% for G/G, 33.66% for G/T, and 7.92% for T/T, and the frequency of either G or T allele was 75.25% and 24.75%, respectively. There were no significant differences in genotype and allele frequencies between the patients with CAD and the control subjects. The mean serum NO levels of CAD was significantly increased compared to healthy subjects (p=0.0139). Significant association was found between CAD who were homozygous for the G allele and NO serum levels. 77 male CAD Egyptians were recruited in the study with age range (35-50 years). They were admitted to in- and out patient clinics of the National Heart Institute (NHI), Imbaba, Cairo, Egypt and angiographically diagnosed. They were classified according to the severity of coronary insufficiency and type of management into four subclasses; (1) patients under conservative medical treatment (Med, n = 9), (2) patients directed for percutaneous coronary interventions ( PCI, n = 31), (3) patients advised to have a coronary artery bypass graft operation (CABG, n =25 ), and (4) patients suffering from acute myocardial infarction (AMI, n = 12). Genomic DNA was prepared from whole blood using a QIAamp DNA blood mini kit (Qiagen). Genotypes for the Glu298Asp polymorphisms were determined by PCR-restriction fragment length polymorphism (PCR-RELP) analysis using specific oligonucleotide primers: forward 5’-TGAGGGTCACACAGGTTCCT-3’ and reverse 5’- TCCCTGAGGAGGGCATGAGGCT-3’. PCR products were digested by BanII, and separated by electrophoresis using agarose gel (2%) and visualized by eithidium bromide staining. The wild type allele G has BanII cutting site producing smaller fragments (137 and 320 bp). In the case of a G to T substitution, a BanII restriction site is lost. Table 1: Serum NO values vs. gene variants and allelic frequencies of eNOS Glu298Asp in controls and CAD patients Serum NO levels were measured as total nitrite concentration with Griess reagents (2% w/v sulphanilamide in 5% HCL and 0.1% napthylethanolamine in H 2 O),after conversion of nitrate to nitrite by vanadium (III) chloride (VCL 3 ). Calibration curves were plotted for potassium nitrate in distilled H 2 O (0-100 umol/L). Absorbance was measured at 540 nm by spectrophotometer Figure 2: Distribution of Glu298Asp eNOS genotypes among controls and CAD patient subclasses Figure 3: Serum NO levels in controls and CAD patients. Figure 4: Mean serum NO levels of different eNOS genotypes in CAD patients and healthy subjects GroupSubjectsNO (µM) Genotypes (n%) Glu/Glu Glu/Asp Asp/Asp Alleles (n%) Glu (G) Asp (T) Odds ratio CI (95%) p Control ± (53.6%)25 (36.2%)7 (10.2%)99 (71.7%) 39 ( 28.3%)OR= to p=1 CAD patients ± (55.1%)26 (3%)11 (12.9%)110 (71.2%)46 (28.8%) 1) Lack of correlation between eNOS Glu298 Asp polymorphism & CAD 2) Serum NO levels are increased in CAD 3) Lack of association between the genotypes of eNOS Glu298 Asp and serum NO levels in CAD patients and controls Fig 1: Gel electrophoresis of PCR products after incubation with Ban II This study was supported by the Science and Technology Development Fund (STDF ) grant No.2951 Investigate the correlation of the eNOS Glu298Asp polymorphism and Serum NO levels in a group of CAD Egyptians 1- M G Colombo, M G Andreassi, U Paradossi, N Botto, S Manfredi, S Masetti, G Rossi, A Clerico, A Biagni, References for association of a common variant of the endothelial nitric oxide synthase gene (Glu 298  Asp polymorphism) to the presence, extent and severity of coronary artery disease, Heart 2002;87: Tsukada T, Yokoyama K, Arai T,Takemoto F,Hara S, Yamada A, Kawaguchi Y,Hosova T,lgari J, Evidence of association of the ecNOS gene polymorphism with plasma NO metabolite levels in humans. Biochem Biophys Res Commun; 1998 Apr 7;245(1): GGTTGT 457 bp 320 bp 137 bp