Review articlePolymorphisms in endothelial nitric oxide synthase and atherogenesis: John French Lecture 2000
Introduction
The Human Genome Project (HGP) recently completed its first draft sequence of the human genome [1]. An additional major aim of this huge endeavour is not only to document the consensus sequence of all human DNA but also to identify the common genetic variation that is thought to underlie differences in susceptibility to common diseases [2]. In the words of Frances Collins, Director of the US National Human Genome Research Institute ‘virtually all disease, except some trauma, has a hereditary component’.
Atherosclerosis and its complications are no exception [3]. It has long been known that coronary artery disease (CAD) aggregates in families, Sir William Osler having made reference to its familial tendency as early as 1897. The familial aggregation of this disorder could, in theory, arise from the effects of a shared environment, genetic influences or both. However, studies of twins [4] suggest that the familial risk of CAD correlates with degree of relatedness, an observation that implicates genetic factors, and not merely common familial environment, as a cause.
Section snippets
Magnitude of the genetic contribution to atherosclerosis
Despite clear evidence for the heritability of atherosclerosis and CAD, the nature and number of genes involved remains elusive. Although single gene defects such as familial hypercholesterolaemia can manifest as premature CAD [5], the common form of the disorder is a heterogeneous, late-onset condition with no clear mode of inheritance. It is likely that this pattern of disease results from an interaction of a variety of environmental risk factors with one or more susceptibility genes. This
A model for understanding genetic susceptibility to atherosclerosis
A working model for understanding the genetic basis of atherosclerosis and CAD is illustrated in Fig. 1. In this model, common sequence variation in DNA (polymorphism) is considered to underlie differences in the expression or activity of a gene product relevant to the development of atherosclerosis (e.g. an enzyme that converts substrate X to product Y). Genetically-determined differences in the activity of the enzyme would be entirely compatible with health but, in the presence of adverse
Candidate gene approaches to atherosclerosis
Despite these potential problems, case-control studies have been used extensively to understand the genetic basis of atherosclerosis and its complications. The advantage of this approach is that it does not necessitate the recruitment of family clusters with multiple affected subjects, but instead utilises unrelated cases and controls that are more easily ascertained. Thus far, this type of study design has focused on candidate genes – genes whose protein products are considered relevant to the
Acquired and inherited deficiency in endothelial nitric oxide and atherogenesis
In the last decade there has been much interest in the vascular endothelium as a tissue that maintains the intrinsic thromboresistant, vasodilatory and atheroprotective properties of the vascular wall through the release of mediators such as nitric oxide (NO) [8]. This has led to the proposal that a deficiency in vascular NO, either inherited or acquired, might contribute to the development of atherosclerosis [9], [10]. NO is synthesised in endothelial cells from l-arginine and molecular oxygen
Endothelial nitric oxide synthase as a candidate gene for atherosclerosis
About 5 years ago, it was considered that inherited defects in NO synthesis might predispose to the development of atheroma. Since the regulation of endothelial NO availability occurs at the level of the synthetic enzyme (eNOS) it seemed logical to focus on the gene that encodes eNOS (NOS 3) as a potential candidate atherosclerosis-susceptibility gene. NOS 3 was cloned and sequenced in 1993 and localised to chromosome 7q35-36 [42]. Spanning 4.4 kb of genomic DNA, the gene comprises 26 exons
Endothelial nitric oxide synthase Asp298 as a risk factor for coronary artery disease and myocardial infarction
Since genetic case-control studies can be sensitive to type II statistical error for the reasons stated earlier, a reproducibility study was undertaken in which the frequency of the Glu298Asp polymorphisms was assessed in 249 patients with acute myocardial infarction and a further set of 183 healthy controls. Homozygosity for eNOS Asp298 was once again an independent risk factor for MI with an odds ratio of 2.5 (95% CI: 1.3–4.2; P < 0.01; Table 1B and C) [43]. These findings have been supported
Enzymatic studies of the endothelial nitric oxide synthase Asp298 variant
The observation that individuals homozygous for eNOS Asp298 are over-represented among individuals with CAD led to the speculation that eNOS Asp298 might have reduced enzymatic activity when compared to eNOS Glu298. At the time of our discovery, it was considered that this might arise through reduced binding of arginine or tetrahydrobiopterin to the oxidative domain of eNOS within which residue 298 is located. Enzymatic studies of recombinant eNOS Asp298 and Glu298 were therefore undertaken in
Endothelial nitric oxide synthase Asp298 and vascular response
If NO generation in carriers of the eNOS Asp298 allele is indeed low, this might be reflected in the form of a reduction in NO-mediated vascular responses. No full publication has yet addressed whether endothelium-dependent vasodilation is impaired in carriers of the Asp298 allele, but Philip et al. [47] recently showed that the pressure response to a systemic infusion of phenylephrine in patients on cardiopulmonary bypass differed according to eNOS genotype. The increase in mean arterial blood
Additional polymorphisms at the endothelial nitric oxide synthase locus
Although the work described thus far has focused on the sole amino acid polymorphism in eNOS (Glu298Asp), several additional polymorphisms have been identified in the eNOS gene promoter as well as in a number of its introns (Fig. 5). The intronic polymorphisms are less likely to have a functional role per se than either the promoter or coding region variants, but they may act as markers for unidentified, potentially functional variants elsewhere in the gene. An extensively studied variable
Summary and future directions
In summary, a body of work now exists that supports the view that certain common polymorphisms in the eNOS gene influence the expression and functional activity of the enzyme and, in turn, the susceptibility to atherogenesis. Further in vitro studies will be required to identify the molecular and cellular mechanisms that underlie these observations, and further phenotypic studies in genetically-characterised subjects will be required to delineate precisely the effects of these variants on
Acknowledgements
I would like to thank Professor Morris Brown, Kevin O'Shaughnessy, Chia Fan Liang, Amelia Lyon, Sue Monteith and Haiyan Jia at the Clinical Pharmacology Unit at Addenbrooke's Hospital Cambridge, Professors Patrick Vallance, Steve Humphries and John Deanfield together with Noor Jeerooburkhan and Lisa Jones at University College London, Professor Nigel Benjamin and Michele Cafferkey at St. Bartholomew's Hospital, and Wendy Alderton, Rick Williamson and Richard Knowles at Glaxo Wellcome Medicines
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