Jeerooburkhan, Noor; (2001) Phenotype and genotype relationship in nitric oxide and pterin pathways in man. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In the vascular endothelium, nitric oxide (NO) is synthesised from L-arginine by endothelial nitric oxide synthase (eNOS). Tetrahydrobiopterin (BH4) is an essential cofactor of eNOS and NO production is enhanced by increased concentration of BH4. The latter is synthesised from guanosine triphosphate (GTP) by the rate-limiting enzyme of the pterin pathway, GTP cyclohydrolase I (GTPCH). Neopterin is a side reaction product of the pterin pathway. In the vasculature, NO exerts a tonic vasodilator influence, regulates regional blood flow and systemic blood pressure (BP), and also confers thromboresistant and atheroprotective properties to the endothelium. A reduction in its synthesis or availability might underlie the impaired endothelium-dependent vasodilatation, which is observed in blood vessels from individuals with cardiovascular disease (CVD) risk factors, including active and passive smokers as well as patients with hypertension and hypercholesterolaemia. Furthermore, loss of NO-mediated effects may predispose to the development of atherosclerosis. Conversely an increase in inflammatory NO, paralleled by a rise in BH4 avaibility may cause endothelial damage and dysfunction. We hypothesised that sequence variation in the eNOS gene (NOS3) and/or GTPCH gene (GCH1) would influence the generation of NO in vivo and in so doing would determine both endothelial vasodilator function and the risk of ischaemic heart disease (IHD). Because NO production might be regulated by BH4 availability, which could be genetically determined, we searched for polymorphisms in GCH1 and investigated associations with activity of the pterin pathway, NO production and IHD risk. The relationship between NO and neopterin production, classical CVD risk factors, IHD events and both NOS3 and GCH1 polymorphisms were studied in a cohort of 3179 healthy men aged 50-61 years from the second Northwick Park Heart Study (NPHSII). High throughput assay methods were established for measuring plasma NOx (i.e. NO3- and NO2-) and neopterin, as an index of eNOS and GTPCH activities respectively. Three novel polymorphisms, i.e. -577 (G/A), -741 (T/C) and -796 (G/A), were identified in the promoter of GCHl by Single Strand Conformation Polymorphism (SSCP). Genotyping of GCHl variants at positions -577 and -796 as well as four previously described NOS3 polymorphisms, i.e. -786 (T/C), -922 (A/G), +894 (G/T) and a 27-bp tandem repeats in intron 4 (eNOS4b/4a), were performed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based methods combined with Microplate Array Diagonal Gel Electrophoresis (MADGE) technique. New data on allelic association of NOS3 polymorphisms was obtained. NOx levels were found to be lower in smokers compared to non-smokers but were not influenced by NOS3 genotype. Both -577 (G/A) and -796 (G/A) variants influenced plasma neopterin levels significantly. Neopterin was positively associated with fibrinogen, an acute-phase protein and an established cardiovascular risk factor. This finding supports the view that an inflammatory reaction might be the basis of the pathology of CVD. Over 9 years of follow-up, there was no influence of NOS3 and GCHl polymorphisms on IHD risk. Genetic variations in GCH1 may influence the activity of the pterin pathway and further studies are required to confirm this finding and to determine their influence on vascular function in health and disease.

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