EDITORIAL COMMENT
Complex Trait GeneticsThe Role of Mechanistic "Intermediate Phenotypes" and Candidate Genetic Loci*
Fangwen Rao, MD,
Lian Zhang, MD and
Daniel T. O'Connor, MD*
Departments of Medicine and Pharmacology, Center for Human Genetics and Genomics, University of California at San Diego, and Veterans Administration San Diego Healthcare System, San Diego, California.
* Reprint requests and correspondence: Dr. Daniel T. O'Connor, Department of Medicine (0838), UCSD School of Medicine and VASDHS, 9500 Gilman Drive, La Jolla, California 92093-0838. (Email: doconnor{at}ucsd.edu).
Key Words: nitric oxide • phenotype • genomics • haplotype • endothelium
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Coronary Artery Disease Risk
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Although coronary artery disease (CAD) is the most common type of heart disease, its etiology and pathogenesis are not well understood. Hereditary determination of CAD is suggested by epidemiologic studies in which first-degree relatives of coronary patients have a 2.5- to 7-fold increase in risk of death from coronary disease compared with those without a family history of CAD (1). Young adults with a family history of premature CAD are likely to have the strongest genetically determined influence on their risk of subsequent arterial disease (2). Despite increased understanding of rare mendelian contributors to CAD (e.g., familial dyslipidemias [3,4]), the common presentation of CAD is likely to be a complex trait, with both hereditary and environmental determination (2,5).
Experimental and clinical studies suggest that endothelial dysfunction may be a precursor to CAD (2,6). Maintenance of endothelial function is a critical aspect of vascular homeostasis. The gaseous transmitter nitric oxide (NO), derived from 3 nitric oxide synthase (NOS) isoforms, plays a key role in vascular tone, and alterations in NO production influence endothelium-dependent vasodilation and blood pressure. Loss of normal endothelial production of NO is an early and characteristic feature of many vascular disease states and plays a role in disease pathogenesis (7). Alterations of NO availability may be involved in the genesis of atherosclerosis, and thus may contribute to progression of CAD (7).
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Vascular Risk Mechanisms and Heredity
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Several major cardiovascular risk factors, such as hypertension (8,9) and dyslipidemia (3,8), are known to be genetically influenced. In twin studies, human NO production displays substantial heritability and joint genetic determination with autonomic activity (5). In humans, a variant of the endothelial NO synthase gene (eNOS, NOS3) within exon 7 (G>T transversion at nucleotide position 894 [G894T], resulting in a change of Glu298Asp) predicts enhanced responsiveness to alpha-adrenergic stimulation (10). In an examination of the effect of G894T polymorphism on the endothelium in subjects with premature myocardial infarction (11), the 894T (298Asp) allele was associated with impaired vasodilation and higher levels of von Willebrand factor.
In the face of significant heritability yet uncertain mode of inheritance for complex traits such as CAD, a useful strategy may be to establish so-called "intermediate phenotypes" (12): ideally, pathogenic traits with early expression even before disease onset.
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Role of GTP Cyclohydrolase (GCH1) Polymorphism on Endothelial Function
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Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the first and rate-limiting step in tetrahydrobiopterin (BH4) biosynthesis. Tetrahydrobiopterin is an essential cofactor for several transmitter biosynthetic pathway enzymes, including not only the 3 NOS but also tyrosine hydroxylase (TH), phenylalanine hydroxylase, and tryptophan hydroxylase. The human GCH1 locus experiences rare natural inactivating mutations with profound consequences; such mutations cause 2 disorders: autosomal dominant hereditary progressive dystonia/DOPA-responsive dystonia (HPD/DRD) and autosomal recessive GCH1-deficient hyperphenylalaninemia (HPA). Biochemical characterizations of these diseases demonstrate lower cerebrospinal fluid levels of BH4 and the BH4 pathway metabolite neopterin (13). A mouse model for dominantly inherited GCH1 deficiency also showed low brain levels of BH4 (13). Earlier studies in cultured cells showed that GCH1 inhibition lowers BH4 levels, leading to decreased NO production (14). Exogenous BH4 in the spontaneously (genetic) hypertensive rat can prevent development of elevated BP (15).
In states such as atherosclerosis (16), diabetes mellitus, hypertension (17), or insulin resistance, vascular BH4 deficiency may be mediated, at least in part, by increased intracellular oxidation of BH4 to BH2 by reactive oxygen species (such as peroxynitrite) (16).
Other studies suggest that changes in BH4 biosynthesis, perhaps through regulation of the rate-limiting enzyme GCH1 (18) or dihydropteridine reductase (19), may also be important for vascular BH4 bioavailability.
The gaseous transmitter NO plays a critical role in endothelium-dependent vasodilation and BP. Understanding the full spectrum of genes that influence NO production might thus contribute to an improved understanding of CAD. As reported in this issue of the Journal, Antoniades et al. (20) studied endothelium-dependent dilation (to acetylcholine), as well as GCH1 expression, BH4 bioavailability, NO bioavailability, and vascular superoxide production in saphenous vein (SV) and internal mammary artery (IMA) segments from 347 patients with CAD undergoing coronary artery bypass grafting (CABG) surgery in the United Kingdom.
The researchers (20) defined GCH1 haplotypes first by genotyping 347 patients with coronary artery disease and 741 control subjects. Three SNPs spanning the locus (promoter G>A, rs8007267; intron-1 A>T, rs3783641; 3'-UTR C>G, rs10483639) were sufficient to define haplotypes. Common haplotype ATG (the third most common haplotype in the population [21], designated "X" by the authors) accounted for  15% of chromosomes, and did not differ significantly in frequency between CAD and control subjects. However, haplotype X had pronounced effects on several "intermediate phenotypes" for vascular risk, including reduction in GCH1 vascular gene (messenger ribonucleic acid) expression, reduction in plasma and vascular BH4 concentrations, increased vascular superoxide production, and decreased vascular dilation to an endothelium-dependent stimulus (acetylcholine in this case). In multivariate analyses of several risk predictors, GCH1 haplotype was an independent predictor of reduced BH4 levels in both plasma and vascular tissue.
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The Current Results in Context With the Literature
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The impact of common genetic variation in GCH1 for BH4 (and therefore NO) production has only recently been subjected to another study in humans (19), whose results may be complementary to the current report: systematic polymorphism discovery across the GCH1 locus (exons and promoter) revealed 13 common single nucleotide variants. In a series of twin pairs phenotyped for autonomic and renal traits, urinary NO (estimated by nitrate/nitrite) excretion was influenced by GCH1 polymorphism, and the effect was mapped onto a common variant in the 3'-UTR: C+243T (rs841). This same 3'-UTR polymorphism coordinately predicted not only NO production but also baroreflex coupling, heart rate variability, and minimum heart rate. In a primary care population, 3'-UTR variant C+243T influenced both SBP and DBP. One is tempted to speculate that trait-associated allele in the GCH1 3'-UTR (C+243T) might be responsible for the vascular findings from Antoniades et al. (20). However, an additional 13 common SNPs were discovered in this gene, with 3 located in the promoter region (5); therefore, different polymorphic sites might alter function in alternative tissues (coronary artery versus kidney). The precise nature and roles of causative variants in vascular tissues remain to be established.
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Implications of This Research
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The current study (20) represents the first attempt to characterize GCH1 gene variability to detect functionally important changes in patients with CAD. Careful phenotyping allowed the authors to detect the effects of GCH1 common variation on important risk traits, both biochemical and pharmacologic, even though the active haplotype did not associate with the ultimate disease trait in the CAD case/control study. Thus, the importance of mechanistic phenotyping is highlighted by this report.
The results in the GCH1/NO pathway also emphasize the likely utility of this pathway in design of future therapeutics targeting endothelial function (22) and ultimately CAD risk. The GCH1 genotypes might be useful in defining the pathogenesis of risk, as well as quantitatively profiling such risk or the likelihood that pathway-directed therapeutics (e.g., BH4) would be effective in a given individual (i.e., GCH1 pharmacogenetics). Indeed, stable oral BH4 supplementation is now effective in a subset of subjects with autosomal recessive phenylalanine hydoxylase deficiency (23), although the successful use of BH4 supplementation in cardiovascular disease has not yet advanced beyond the experimental animal stage (24).
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Footnotes
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Supported by the Department of Veterans Affairs, National Institutes of Health.
* Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology. 
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References
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Coronary Artery Disease Risk
Vascular Risk Mechanisms and...