Nonconventional Genetic Risk Factors
        for Cardiovascular Disease
  Christopher N. Tymchuk, MD, PhD, Jaana Hartiala,...
Nonconventional Genetic Risk Factors for CVD                 Tymchuk et al.     185

Figure 1. Steps in the progression...
186   Genetics

 Table 1. Chromosomal regions identified for cardiovascular disease traits that do not exhibit
Nonconventional Genetic Risk Factors for CVD           Tymchuk et al.   187

acts in a dominant negative fashion and disr...
188   Genetics

 Table 2. Nonconventional risk factor genes with consistent evidence of association with
 cardiovascular ...
Nonconventional Genetic Risk Factors for CVD          Tymchuk et al.   189

aging of endothelial cells, several associati...
190   Genetics

were among the first to demonstrate the importance of             asthma previously led to the development...
Nonconventional Genetic Risk Factors for CVD                     Tymchuk et al.       191

3.••    Wang Q, Rao S, Shen GQ...
192    Genetics

41.     Subbarao K, Jala VR, Mathis S, et al.: Role of leukotriene B4   46.•• Helgadottir A, Manolescu A...
Upcoming SlideShare
Loading in...5

Nonconventional Genetic Risk Factors for Cardiovascular Disease


Published on

  • Be the first to comment

  • Be the first to like this

No Downloads
Total Views
On Slideshare
From Embeds
Number of Embeds
Embeds 0
No embeds

No notes for slide

Nonconventional Genetic Risk Factors for Cardiovascular Disease

  1. 1. Nonconventional Genetic Risk Factors for Cardiovascular Disease Christopher N. Tymchuk, MD, PhD, Jaana Hartiala, MS, Pragna I. Patel, PhD, Margarete Mehrabian, PhD, and Hooman Allayee, PhD Corresponding author Hooman Allayee, PhD tive oxygen species (Fig. 1). The resulting production of Institute for Genetic Medicine, USC Keck School of Medicine, adhesion molecules, chemokines, and growth factors by 2250 Alcazar Street, IGM 240, Los Angeles, CA 90089-9075, USA. endothelial cells causes inflammatory cells, comprised E-mail: predominantly of monocytes, to adhere to the vessel wall Current Atherosclerosis Reports 2006, 8:184 –192 and migrate into the subendothelial space. In this micro- Current Science Inc. ISSN 1523-3804 environment, the activated monocytes proliferate and Copyright © 2006 by Current Science Inc. differentiate into macrophages, which begin to engulf the oxidized LDL particles and subsequently transform into foam cells (Fig. 1). Accompanied by a progres- Despite numerous advances made in identifying the genes sive increase in extracellular lipids and intimal smooth for rare mendelian forms of cardiovascular disease (CVD), muscle cells that have migrated through the media, the relatively little is known about the common, complex resulting “fatty streaks” develop into advanced lesions as forms at the genetic level. Moreover, most genes that the lipid-laden macrophages undergo apoptosis to form a have been associated with CVD, whether they are single necrotic core (Fig 1). Proinflammatory cytokines that are gene forms or more common forms of the disease, have also expressed in the lesion cause the smooth muscle cells primarily been involved in biochemical pathways related to proliferate and secrete collagen and other extracellular to what are considered “conventional” risk factors. matrix proteins, resulting in the formation of a fibrotic However, recent genetic studies have begun to identify cap. Such advanced lesions become increasingly complex genes and pathways associated with CVD that would with calcification, ulceration at the luminal surface, and not be considered to underlie conventional risk factors. hemorrhage from small vessels that grow into the lesion In this review, we discuss the evidence for this latter from the media, rendering them unstable and prone to notion based on recent linkage and association studies in rupture (Fig. 1). Ultimately, plaque erosion and rupture humans. As an example, we also illustrate how a combi- can lead to clinical events such as myocardial infarction nation of mouse and human genetics led to identification (MI) or stroke. of the 5-lipoxygenase pathway for CVD, with potentially Over the past half century, epidemiologic studies have important implications for its treatment and diagnosis. revealed numerous risk factors for atherosclerosis [2]. We conclude with a discussion of the prospects for These are in large part controlled by traits with genetic identifying CVD genes in the future and for potentially components, including gender, age, hypertension, hyper- developing more effective therapeutic strategies. lipidemia, obesity, and diabetes. Collectively, these have come to be known as “conventional” risk factors. In addi- tion, environmental risk factors such as stress, high-fat Introduction diet, lack of exercise, and smoking are also important Atherosclerosis, the most common cause of cardiovascular contributors to CVD. The importance of genetics and disease (CVD), is a progressive disease characterized by environment in CVD has been examined in many fam- the accumulation of lipids and fibrous elements in large ily and twin studies. Within a population, the observed arteries [1]. It is a complex process involving numerous fac- heritability of atherosclerosis has been large in most tors and mediators and is based on the dynamic exchange studies, frequently exceeding 50%. With the exception of signals between resident cells, such as endothelial of lipoprotein(a) (Lp(a)) levels, which are determined cells, smooth muscle cells, and infiltrating monocyte/ almost entirely by common variations of the LPA gene, macrophages and lymphocytes. The atherogenic process each of the genetic risk factors involves multiple genes. is initiated by the accumulation of low-density lipopro- This complexity can be clearly observed in genetic crosses tein (LDL) particles in the subendothelial layer of the with animals maintained under similar environmental artery wall, where they are oxidized by cell-derived reac- conditions, which have revealed dozens of loci contrib-
  2. 2. Nonconventional Genetic Risk Factors for CVD Tymchuk et al. 185 Figure 1. Steps in the progression of atherosclerosis in the artery wall. I, Low-density lipoprotein (LDL) particles accumulate in the subendothelial cell layer and become trapped within matrix proteins, where they are oxidized (oxLDL) by cell-derived reactive oxygen species. II, Chemotactic and growth factors stimulate monocytes to transmigrate across the endothelial cell layer and differentiate into macrophages. Macrophages begin to engulf the oxLDL, leading to foam cell formation. III, The foam cells produce a variety of mediators and eventually undergo apoptosis or necrosis, contributing their contents to a growing core of cellular debris and cholesterol. This process is accompanied by the migration of smooth muscle cells into the intima from the media and the formation of a fibrotic cap. IV, As the advanced lesion continues to grow, it becomes increasingly unstable and prone to rupture, which can result in a thrombus or clinical event such as myocardial infarction or stroke. uting to atherosclerosis, lipoprotein levels, body fat, Family-based Linkage Studies and other risk factors [3••]. Another level of complex- Since the year 2000, over 10 genome-wide linkage scans ity involves the interactions between genes themselves have been carried out in families to identify chromosomal and with environmental factors. Thus, common forms regions segregating with CVD-related traits. Numerous of CVD result from the interplay between susceptibility loci have been reported, some of which exhibit coincident genes and lifestyle. linkage with conventional risk factors such as high-den- Although the role of conventional risk factors in sity lipoprotein (HDL) levels or metabolic syndrome CVD is generally accepted, it is also likely that there traits. We have summarized these results in Table 1 but are genetic contributions to CVD that are independent have restricted our discussion to emphasize recent studies of known risk factors. For example, certain individu- that have identified loci with major genetic effects and/or als develop premature heart disease but do not exhibit those not exhibiting coincident linkage with known risk high cholesterol or hypertension. This may, in part, be factors. Presumably, these regions harbor susceptibility due to genetic influences that act at the level of vessel genes that could reflect novel pathways for CVD. wall metabolism, which leads to variations in cellular In the past 2 years, two important genome-wide link- function or inflammatory responses among individu- age analyses for MI and/or CAD have been published. als. As discussed in the following text, several recent The first was by Wang et al. [3••] who studied 428 white studies support this concept and emphasize the need families in the United States with at least two members for identifying new genes and pathways for CVD. For affected by premature CAD or MI before the age of 45 the purposes of this review, we refer to CVD as a broad years in men and 55 years in women. Of note, this study spectrum of disease states including MI, coronary artery used more restrictive criteria for ascertaining families by disease (CAD), stroke, and surrogate markers of athero- excluding patients with hypercholesterolemia, diabetes, sclerosis such as carotid intima-media thickness (IMT) or childhood hypertension, and, therefore, specifically and coronary artery calcification (CAC). selected for genetic forms of MI and CAD that are not
  3. 3. 186 Genetics Table 1. Chromosomal regions identified for cardiovascular disease traits that do not exhibit coincident linkage with conventional risk factors Study population Chromosomal (ethnicity) Ascertainment scheme Sample size (n) region (gene) Study Finnish (white) Angiographically proven 156 families (n = 364) 2q21.1-22; Pajukanta et al. [48] premature CAD in proband Xq23-26 German (white) MI or premature CAD before 513 families (n = 1406) 14q32.2 Broeckel et al. [49] age of 60 y in proband Australian MI or unstable angina before 61 sibpairs (n = 122) 2q36-37.3 Harrap et al. [50] (not reported) age of 70 y in proband United States (white) Premature CAD in 2 or 428 families (n = 1163) 1p34-36 Wang et al. [3••] more family members United States, British, Premature CAD in proband 438 families (n = 1168) 5q31 Hauser et al. [5] Welsh, Swiss, and and 2 siblings German (white) United States (white) Family displaying autosomal 1 family (n = 21) 15q26 Wang et al. [6] dominant inheritance of CAD (MEF2A) US Framingham Prospective cohort study; 311 families (n = 1225) 12q24.3 Fox et al. [9] Heart Study (white) linkage analysis with carotid IMT United States CAD in proband; linkage 91 families (n = 413) 2q35 Wang et al. [10] (Mexican-American) analysis with carotid IMT United States (white) 2 or more siblings with 29 sibships (n = 94) 6p21.3; Lange et al. [11] hypertension before age of 60 y; 10q21.3 linkage analysis with CAC Icelandic (white) Stroke or transient ischemic 179 families (n = 914) 5q13 Gretarsdottir et al. [12] attack in proband (PDE4D) Icelandic (white) MI before age of 75 y in proband 296 families (n = 2454) 13q12-13 Helgadottir et al. [43•] (ALOX5AP) CAC—coronary artery calcification; CAD—coronary artery disease; IMT—intima-media thickness; MI—myocardial infarction. associated with known risk factors. A locus for MI was least one member of a nuclear family. Although several identified on chromosome 1p34-36 with a remarkably linked chromosomal regions were identified in the entire high logarithm of the odds (LOD) score of 11.7. Such a dataset as well as the stratified subsets, most of the loci level of significance is very rarely observed for complex overlapped with regions where lipid and metabolic traits diseases and is more typical of linkage results for single had previously been mapped. However, one locus on gene disorders. This may be due, in part, to the stringent the distal arm of chromosome 5 exhibited nominal evi- MI phenotype under study and the exclusion of poten- dence for linkage to CAD (LOD = 1.4), which was derived tially confounding covariates. Interestingly, no significant predominantly from the families excluded for diabetes. results were obtained for CAD, suggesting that CAD Similar to the results described here previously, this locus and MI do not necessarily share the same genetic deter- may represent a gene contributing to CVD independent minants. Connexin 37 (CX37), a potential candidate of known risk factors. gene that resides in the region, is a gap junction protein As is true for other complex diseases, understanding expressed in arterial endothelium and has previously mendelian forms of CVD can be a powerful approach for been associated with MI in a Japanese population [4]. It identifying novel susceptibility genes. Recently, Wang et al. remains to be determined whether this is the causal gene [6] conducted a genome scan of a single family with an for MI in these families. autosomal dominant pattern of CVD consisting of 13 The second genome-wide linkage study was under- individuals affected by CAD or MI. A putative locus was taken by Hauser et al. [5] and studied 438 white families mapped to chromosome 15q26 with a significant LOD from the United States and Europe in which at least two score of 4.2 [6]. This region contained 93 genes, including siblings were affected by premature CAD. The authors the myocyte enhancing factor 2A gene (MEF2A), which further defined three additional phenotypic stratifica- was evaluated as a possible candidate given its role in vas- tions of the families, including 1) MI or unstable angina culogenesis. All 10 living affected members of this family in two or more siblings; 2) the absence of diabetes in were found to carry an in-frame deletion of seven amino any affected family member; and 3) dyslipidemia in at acids. Functional analysis demonstrated that the mutation
  4. 4. Nonconventional Genetic Risk Factors for CVD Tymchuk et al. 187 acts in a dominant negative fashion and disrupts local- is not entirely clear how PDE4D is involved in stroke, ization of MEF2A to the nucleus. Although the molecular the enzyme degrades cyclic adenosine monophosphate, mechanism for increased susceptibility to CAD/MI is and its expression was found to be lower in affected unknown, the authors speculate that abnormal MEF2A individuals who carried the at-risk alleles compared activity affects endothelial cell function, rendering the with control subjects. coronary vessels more susceptible to inflammation and In total, genome scans in multiple study popula- thrombosis [7]. In addition, screening of 207 indepen- tions have mapped over 10 loci for CVD traits. With few dent CAD/MI cases for mutations in the MEF2A gene by exceptions, there is minimal overlap between these loci, the same group identified three rare amino acid substitu- which could be due to differences in the populations, tions whereas none were identified in control subjects [7]. the phenotypes measured, or the statistical methods The significance of these findings has come under debate used for analysis. For example, although MI and CAD recently from a study by another group that screened for are correlated, these phenotypes could represent distinct the MEF2A gene in 300 individuals with premature CAD pathophysiologic processes that are controlled by differ- and 300 elderly control subjects and identified only one ent genetic factors. Moreover, although these loci do not amino acid substitution unique to cases [8]. Moreover, exhibit linkage with known risk factors, it is still possible this latter study did not observe co-segregation of the 7 that the causative genes in these regions could be related amino acid deletion with CAD/MI in one family where to pathways involving lipid metabolism, blood pressure, the proband was a carrier of the mutation [8]. Thus, the or obesity. Nonetheless, identification of the genes could contribution of MEF2A mutations to CAD and MI still also provide novel genetic mechanisms leading to CVD. remains to be conclusively determined. In addition to MI and CAD, genome scans have also been conducted for other CVD-related traits such as Candidate Gene and Whole Genome stroke, CAC, and carotid artery IMT. Carotid IMT is a Association Studies measure of subclinical atherosclerosis and predictive of Although family-based linkage studies have identified subsequent MI and stroke. Two genome-wide linkage numerous loci for CVD and other related traits, the analyses of carotid IMT have been published, including identity of the underlying genes, with rare exception, a study by Fox et al. [9] who found significant linkage has remained elusive. Another approach for identifying of IMT to a locus on chromosome 12 using 311 families genes for CVD has been to conduct association studies of the Framingham Heart Study Offspring cohort. The with candidate genes or, more recently, through a whole other genome scan was conducted in 91 Mexican-American genome analysis. Association studies in general provide families ascertained through a proband with CAD and greater statistical power than linkage analyses for detect- revealed linkage to chromosome 2q35 [10]. CAC is ing genetic effects, and dozens of studies have examined a another marker of atherosclerosis that can be measured variety of candidate genes for association with CVD traits. noninvasively using electron beam computed tomog- Most of these have focused on genes involved in lipid, raphy. In a genome-wide linkage analysis involving 29 blood pressure, or obesity-related pathways. By compari- sibships with two or more siblings affected by hyperten- son, fewer studies have examined genes involved in other sion, Lange et al. [11] demonstrated significant linkage of processes traditionally not considered to be risk factors. CAC to two regions on chromosomes 6p21 and 10q21. Studies in the past few years, however, have reported Similar to the results obtained with more formal measures and confirmed genes that are part of pathways, such as of CVD, these latter loci also lacked coincident linkage inflammation, endothelial cell function, and plaque sta- with known risk factors. Interestingly, the chromosome bility that are associated with CVD traits. Although not 10 locus for CAC contains the 5-lipoxygenase (5-LO) an exhaustive list, several genes and pathways for which gene, which as discussed in more detail in the following there is consistent evidence of association with CVD are text, has recently been associated with atherosclerosis in highlighted in Table 2 and discussed here. mice and humans. Strokes can be caused by hemorrhage of an intra- Inflammatory processes cranial blood vessel, occlusion of large or small arteries Using a whole-genome association study design, Ozaki in the brain, or emboli of cardiac origin as a result of et al. [14••] reported association of the lymphotoxin atherosclerosis. A genome-wide linkage analysis of 179 alpha LTA gene (LTA) with MI in a Japanese population. Icelandic families with one affected member mapped a Specifically, the frequencies of an intronic SNP and an major locus for stroke on chromosome 5q12 [12]. In a amino acid–altering substitution were significantly follow-up study, the same group further examined this higher in cases compared with control subjects, even region using fine mapping and a case-control dataset and after adjustment for diabetes, hypertension, and hyper- obtained evidence for association of stroke with haplo- lipidemia. Both SNPs occur on the same haplotype and types of single nucleotide polymorphisms (SNPs) in the individually result in either increased expression of LTA phosphodiesterase 4D (PDE4D) gene [13]. Although it or its activity. Given its role as a cytokine, these results
  5. 5. 188 Genetics Table 2. Nonconventional risk factor genes with consistent evidence of association with cardiovascular disease traits Gene Type of study (phenotype) Potential mechanism Myocyte enhancer factor 2A (MEF2A) Family-based, case-control (MI, CAD) Vascular endothelium function (?) Phosphodiesterase 4D (PDE4D) Family-based, case-control (stroke) cAMP degradation, cell proliferation (?) Lymphotoxin alpha (LTA) Case-control, cohort study Inflammatory response (MI, coronary atherosclerosis) Galectin 2 (LGALS2) Case-control (MI) Regulation of LTA secretion and inflammatory response Toll-like receptor 4 (TLR4) Cohort study, case-control Receptor signaling (carotid IMT, MI) and innate immunity Thrombospondin gene family Case-control (MI) Endothelial cell function (THBS-1, THBS-2,THBS-4) Connexin 37 (CX37) Case-control (CAD, MI) Endothelial cell function Matrix metalloproteinase gene family (MMP) Case-control, cohort study (coronary Proteolysis and plaque rupture artery aneurysm, MI, carotid IMT) Arachidonate 5-lipoxygenase (ALOX5) Cohort study (carotid IMT) LT biosynthesis and inflammatory response Arachidonate 5-lipoxygenase activating protein Family-based, case-control LT biosynthesis and (ALOX5AP) (MI and stroke) inflammatory response Leukotriene A4 hydrolase (LTA4H) Case-control (MI and CAD) LTB 4 synthesis and inflammatory response CAD—coronary artery disease; IMT—intima-media thickness; LT—leukotriene; MI—myocardial infarction. provide a functional mechanism by which common such as MI [21–23]. Taken together, these studies provide SNPs of the LTA gene in the human population could convincing evidence for the genetic contribution of the lead to increased inflammation in the artery wall, and LTA and TLR4 genes to CVD and are consistent with the subsequently MI. Other studies have replicated the hypothesis that immune responses are an important part association of LTA with CVD traits, including another of atherogenesis. Japanese population [15–17]. Interestingly, the effect of LTA was not observed in a third Japanese popula- Vascular endothelium function tion [18]. Such discrepancies are commonly observed As described previously, endothelial cells can play in the genetics of complex diseases and demonstrate the an important part in the atherogenic process. Recent difficulties in carrying out these studies. More recently, studies support this concept and suggest that variation the same investigators who first identified LTA used an in endothelial cell function can be under genetic con- Escherichia coli two-hybrid system to identify galectin 2 as trol. For example, Topol et al. [24] carried out a large a protein that interacts with LTA and potentially regulates case-control study with 62 vascular biology candidate its secretion from macrophages and smooth muscle cells. genes and obtained the strongest evidence of asso- In a case-control analysis, the T allele of an intronic SNP ciation of familial premature CAD or MI with three (C3279T) in the LGALS2 gene was shown to occur at higher members of the thrombospondin (THBS) gene family frequency in the control group, and thus was protective (THBS-1, THBS-2, and THBS-4). The association between against MI. Additionally, the T allele had 50% decreased THBS-2 and THBS-4 variants with CAD/MI has since transcriptional activity in vitro, which would presumably been replicated in several studies [25,26]. Interest- lead to less LTA secretion and inflammation [19•]. ingly, variation in the THBS-2 gene confers protection Another immune response gene recently associated against CVD whereas SNPs in the THBS-1 and THBS-4 with CVD is toll-like receptor 4 (TLR4). Using a cohort of genes lead to increased risk. Although the exact mech- 810 individuals, Kiechl et al. [20•] demonstrated that car- anism by which the THBS proteins modulate artery riers of an Asp299Gly substitution in TLR4 had decreased wall metabolism remains to be elucidated, there is carotid atherosclerosis and plasma biomarkers of inflam- functional evidence to suggest that they are involved mation but they were more susceptible to severe bacterial in endothelial cell function [27]. infections. Similarly, other investigators have replicated Given the role of CX37 as a gap junction protein these results with more clinically relevant phenotypes involved in the growth, regeneration after injury, and
  6. 6. Nonconventional Genetic Risk Factors for CVD Tymchuk et al. 189 aging of endothelial cells, several association studies have A Combined Approach Identifies a Novel examined the CX37 gene in humans. In a large Japanese Pathway for Cardiovascular Disease case-control study with 112 SNPs in 71 candidate genes, With our ability to perform fine mapping, the power a C1019T substitution in CX37 was one of only three vari- to map their genes for complex traits, and our ability ants associated with MI. This association has also been to genetically manipulate their genome, mice provide observed in an Italian population from Sicily [28], but a number of advantages for gene discovery. In this the mechanism by which this variant alters CX37 func- regard, numerous knockout and transgenic mice have tional is still unknown. been generated with varying effects on atherosclerosis [33]. Although common forms of CVD in humans are Plaque instability and rupture not due to the complete inactivation of a single gene, Plaque instability and rupture is very important from a the ability to genetically modify genes in mice, either clinical standpoint because it can lead to MI or stroke. through overexpression or inactivation, has provided an The matrix metalloproteinases (MMPs) are a family invaluable tool for undertaking experiments that would of over 25 enzymes that degrade extracellular matrix simply not be feasible or ethical in humans. In addition, proteins such as collagen, elastin, and proteoglycans. linkage mapping studies in mice have identified over 20 Several of these enzymes, such as MMP-1, are expressed unique loci for atherosclerosis, a subset of which show in the vulnerable shoulder regions of atherosclerotic conserved synteny with regions of the human genome lesions where they could contribute to plaque rupture. where CVD traits have also been mapped [34,35]. Accordingly, the genes for these enzymes have also been Importantly, many of the loci identified in mice do examined in association studies [29]. In particular, poly- not exhibit linkage with traits such as lipid levels and morphisms of the MMP-1, MMP-3, and MMP-9 genes have metabolic phenotypes, which is consistent with the shown associations across several studies with various observations in humans. This further supports the exis- CVD traits, including coronary artery aneurysms and MI tence of genetic factors that may act at the cellular level, [29–31]. Interestingly, certain variants of the MMP-1 and perhaps influencing the function of vascular endothe- MMP-3 genes increase transcription of the enzymes but lium or leukocytes that infiltrate the vessel wall. Thus, are associated with smaller lesions. A plausible explana- it is possible that the genes segregating with atheroscle- tion for this apparent paradox is that these alleles lead to rosis in mouse crosses represent unknown pathways for less extracellular matrix accumulation and thus, smaller atherosclerosis that can be examined in human popula- plaques, but these plaques are more prone to rupture. tions once the genes have been identified in mice. Although other MMPs have also been investigated in Recent studies have elegantly illustrated this com- genetic studies, the evidence for their contribution is not bined approach and led to the identification of a novel as consistent. Nonetheless, the results are in agreement pathway for CVD with important therapeutic implica- with the notion that genetic-mediated increased proteolysis tions. Initially, an atherosclerosis-susceptibility locus, in the arterial wall may act as a susceptibility factor for Artles, was mapped in an F2 intercross between two CVD, and MI in particular. inbred mouse strains, C57BL/6 and CAST/Ei, and sub- Based on the association studies, there is convincing sequently confirmed in a congenic strain [36]. Among evidence for genetic factors that affect atherosclerosis the candidate genes in the linkage interval was the gene development at the level of the artery wall. As a recent encoding 5-LO (Alox5), the rate-limiting enzyme in the study suggests, CVD can also be associated with com- production of leukotrienes (LTs), which are a class of pletely novel mechanisms for which a pathophysiologic inflammatory molecules derived from arachidonic acid. link is unclear or not obvious. In a gene-centric genome 5-LO is expressed primarily in leukocytes and has been association study, Shiffman et al. [32] demonstrated that studied mainly in the context of acute, not chronic, four genes encoding a cytoskeletal protein, a receptor inflammation, particularly that associated with asthma tyrosine kinase, and two G protein–coupled receptors are [36]. Two types of LTs are generated through the 5-LO associated with MI. These genes would most likely never pathway. The first, LTB 4, is generated from LTA4 through have been selected in candidate gene studies and dem- the enzyme LTA4 hydrolase (LTA4H). Alternatively, LTA4 onstrate the importance of unbiased study designs, such can be shunted into the cysteinyl branch and converted as whole genome association, to identify genes. Numerous to LTC 4. Although LTB 4 is predominantly involved in other genes have also been examined but the initial results monocyte chemotaxis and inflammatory signaling, the are very often not replicated in subsequent studies. These cysteinyl LTs promote bronchoconstriction and smooth discrepancies can be attributed to confounding factors muscle cell contraction. such as genetic heterogeneity, population stratification, To evaluate 5-LO as a positional candidate gene, the extent of linkage disequilibrium, and varying statisti- Mehrabian et al. [37] crossed 5-LO knockout mice onto cal methodologies. Such difficulties inherent in human a genetically hyperlipidemic background and observed studies underscore the need for appropriately designed a profound reduction in aortic lesion formation despite and sufficiently powered studies. cholesterol levels in excess of 500 mg/dL. These studies
  7. 7. 190 Genetics were among the first to demonstrate the importance of asthma previously led to the development of drugs targeting 5-LO in atherosclerosis. Other studies with 5-LO–defi- different parts of the pathway, such as zileuton, montelukast, cient mice have suggested the involvement of 5-LO in and zafirlukast. This raises the exciting possibility of admin- atherosclerosis-related phenotypes as well [38], and most istering existing drugs, or newly developed ones, to target recently, 5-LO has been implicated in metabolic traits, the 5-LO pathway for heart disease patients. Indeed, a recent such as adiposity and bone density [39]. Furthermore, report has demonstrated the efficacy of an ALOX5AP inhibi- deficiency of the LTB 4 receptors (BLTR-1 and BLTR-2) or tor for reducing inflammatory biomarkers associated with their pharmacologic antagonism reduces lipid accumula- atherosclerosis [47••]. Even though the therapeutic appli- tion and monocyte infiltration into the artery wall, and, cations of the 5-LO studies can be considered somewhat consequently, aortic lesion formation [40,41]. serendipitous, these studies illustrate how identification By extending these findings to genetic studies in of nonconventional genetic factors for CVD can lead to humans, an important role for 5-LO pathway genes in potentially new strategies for pharmacologic intervention. CVD has also been observed (Table 2). For example, in a cohort study of healthy individuals, Dwyer et al. [42••] reported that certain alleles of an ALOX5 promoter Conclusions polymorphism, consisting of a variable number of Sp1 For the more common forms of CVD, a large number of transcription factor binding sites, were associated with association studies with candidate genes have been per- significantly increased carotid IMT. This study also dem- formed but few have been convincingly confirmed. Those onstrated a gene-dietary interaction where the effect of that have been confirmed explain only a very small fraction the proatherogenic alleles was exacerbated in individuals of genetic susceptibility in humans. Over the past few years, who consumed high levels of arachidonic acid, the sub- new evidence has emerged suggesting that genes that would strate for 5-LO. However this effect was blunted in those otherwise not be considered good candidates based on their individuals who consumed high levels of omega-3 fatty roles in pathways unrelated to known risk factors can play acids, substrates that competitively bind to 5-LO and prevent an important role in the development of CVD as well. Given the binding of arachidonic acid. the difficulty of dissecting complex traits in humans, mouse In a subsequent study, Helgadottir et al. [43••] per- models can also become increasingly important in the iden- formed a genome-wide linkage analysis of 296 Icelandic tification of novel genes, as exemplified by identification of families and reported linkage of MI to chromosome 13q12- the 5-LO pathway for CVD. Given the genetic complexity 13. Using fine mapping to narrow the locus, a four-SNP of CVD, it is likely that there are important gene-gene inter- haplotype of the 5-LO activating protein gene (ALOX5AP) actions, particularly between genes in the same pathway, was found to occur at significantly higher frequency in which have yet to be uncovered. The ability to explore such MI and stroke cases compared with control subjects. hypotheses will require large sample sizes and more sophis- Stimulated neutrophils from male MI cases carrying this ticated analytic methodology. Newly developed genomic, “Icelandic” haplotype also produced higher levels of LTB4, statistical, and bioinformatics tools, such as the sequences consistent with a proatherogenic role for this at-risk allele. of the mouse and human genomes, microarrays, and high- In contrast, a different haplotype was associated with MI throughput genotyping technologies, should facilitate the in patients from the United Kingdom, suggesting the exis- search for CVD genes and may lead to more effective diag- tence of multiple susceptibility alleles in the population. nosis, risk assessment, and treatments. Importantly, association of the ALOX5AP gene and CVD has been observed in other cohorts as well, replicating the initial results and lending further support for the role of Acknowledgments the 5-LO pathway in CVD [44••,45••]. This work is supported by NIH grant HL079353 (H.A. and Very recently, SNP variants of LTA4H have also been M.M), American Heart Association grant 0435223N (H.A.), associated with MI in case-control cohorts from Iceland the Robert E. and Mary R. Wright Foundation (H.A.), and and the United States [46••]. Interestingly, the increased the James H. Zumberge Innovation Fund (H.A.). risk of MI in blacks was threefold higher than that in whites, suggesting the existence of interactions with other genes in individuals of African ancestry. Although this References and Recommended Reading latter study implicates the LTB 4 branch of the pathway Papers of particular interest, published recently, in atherogenesis, it is still not clear whether the cysteinyl have been highlighted as: LTs are also involved. Thus, additional studies with the • Of importance other 5-LO pathway genes will be required to genetically •• Of major importance dissect the mechanism by which LTs contribute to CVD. 1. Lusis AJ: Atherosclerosis. Nature 2000, 407:233–241. From a clinical standpoint, these findings may have a 2. Lusis AJ, Weinreb A, Drake TA, Allayee H: Genetics of Athero- major impact on the development of novel treatments for sclerosis. In Textbook of Cardiovascular Disease. Edited by Topol CVD. Knowledge that 5-LO and leukotrienes are involved in EJ. Philadelphia: Lippincott-Raven; 2002:Chapter 98.
  8. 8. Nonconventional Genetic Risk Factors for CVD Tymchuk et al. 191 3.•• Wang Q, Rao S, Shen GQ, et al.: Premature myocardial 20.• Kiechl S, Lorenz E, Reindl M, et al.: Toll-like receptor 4 infarction novel susceptibility locus on chromosome polymorphisms and atherogenesis. N Engl J Med 2002, 1P34-36 identified by genomewide linkage analysis. 347:185–192. Am J Hum Genet 2004, 74:262–271. This study examined genetic variants of the TLR4 gene that had pre- This genome scan study identified a highly significant LOD score viously been shown to confer a diminished inflammatory response for MI in a large set of families with premature CAD. The impor- to bacterial endotoxin in carriers. These authors found that the same tance of this study was the exclusion of families with diabetes and SNPs were also protective against increased carotid atherosclerosis, hyperlipidemia to enrich for forms of CAD that are not associated supporting the role of inflammation in atherogenesis. with known risk factors. 21. Ameziane N, Beillat T, Verpillat P, et al.: Association of the 4. Yamada Y, Izawa H, Ichihara S, et al.: Prediction of the Toll-like receptor 4 gene Asp299Gly polymorphism with risk of myocardial infarction from polymorphisms in acute coronary events. Arterioscler Thromb Vasc Biol 2003, candidate genes. N Engl J Med 2002, 347:1916–1923. 23:e61–e64. 5. Hauser ER, Crossman DC, Granger CB, et al.: A genome- 22. Holloway JW, Yang IA, Ye S: Variation in the toll-like recep- wide scan for early-onset coronary artery disease in 438 tor 4 gene and susceptibility to myocardial infarction. families: the GENECARD Study. Am J Hum Genet 2004, Pharmacogenet Genom 2005, 15:15–21. 75:436–447. 23. Balistreri CR, Candore G, Colonna-Romano G, et al.: Role 6. Wang L, Fan C, Topol SE, et al.: Mutation of MEF2A in of Toll-like receptor 4 in acute myocardial infarction and an inherited disorder with features of coronary artery longevity. JAMA 2004, 292:2339–2340. disease. Science 2003, 302:1578–1581. 24. Topol EJ, McCarthy J, Gabriel S, et al.: Single nucleotide 7. Bhagavatula MR, Fan C, Shen GQ, et al.: Transcription polymorphisms in multiple novel thrombospondin genes factor MEF2A mutations in patients with coronary artery may be associated with familial premature myocardial disease. Hum Mol Genet 2004, 13:3181–3188. infarction. Circulation 2001, 104:2641–2644. 8. Weng L, Kavaslar N, Ustaszewska A, et al.: Lack of MEF2A 25. McCarthy JJ, Parker A, Salem R, et al.: Large scale association mutations in coronary artery disease. J Clin Invest 2005, analysis for identification of genes underlying premature 115:1016–1020. coronary heart disease: cumulative perspective from analy- 9. Fox CS, Cupples LA, Chazaro I, et al.: Genomewide link- sis of 111 candidate genes. J Med Genet 2004, 41:334–341. age analysis for internal carotid artery intimal medial 26. Wessel J, Topol EJ, Ji M, et al.: Replication of the association thickness: evidence for linkage to chromosome 12. between the thrombospondin-4 A387P polymorphism and Am J Hum Genet 2004, 74:253–261. myocardial infarction. Am Heart J 2004, 147:905–909. 10. Wang D, Yang H, Quinones MJ, et al.: A genome-wide scan 27. Yamada Y, Ichihara S, Izawa H, et al.: Genetic risk for coro- for carotid artery intima-media thickness: the Mexican- nary artery disease in individuals with or without type 2 American Coronary Artery Disease family study. Stroke diabetes. Mol Genet Metab 2004, 81:282–290. 2005, 36:540–545. 28. Listi F, Candore G, Lio D, et al.: Association between 11. Lange LA, Lange EM, Bielak LF, et al.: Autosomal genome- C1019T polymorphism of connexin37 and acute myocar- wide scan for coronary artery calcification loci in sibships dial infarction: a study in patients from Sicily. at high risk for hypertension. Arterioscler Thromb Vasc Biol Int J Cardiol 2005, 102:269–271. 2002, 22:418–423. 29. Newby AC, Johnson JL: Genetic strategies to elucidate the 12. Gretarsdottir S, Sveinbjornsdottir S, Jonsson HH, et al.: roles of matrix metalloproteinases in atherosclerotic plaque Localization of a susceptibility gene for common forms growth and stability. Circ Res 2005, 97:958–960. of stroke to 5q12. Am J Hum Genet 2002, 70:593–603. 30. Pollanen PJ, Lehtimaki T, Mikkelsson J, et al.: Matrix 13. Gretarsdottir S, Thorleifsson G, Reynisdottir ST, et al.: The metalloproteinase3 and 9 gene promoter polymorphisms: gene encoding phosphodiesterase 4D confers risk of joint action of two loci as a risk factor for coronary artery ischemic stroke. Nat Genet 2003, 35:131–138. complicated plaques. Atherosclerosis 2005, 180:73–78. 14.•• Ozaki K, Ohnishi Y, Iida A, et al.: Functional SNPs in 31. Pearce E, Tregouet DA, Samnegard A, et al.: Haplotype the lymphotoxin-alpha gene that are associated with effect of the matrix metalloproteinase-1 gene on risk of susceptibility to myocardial infarction. Nat Genet 2002, myocardial infarction. Circ Res 2005, 97:1070–1076. 32:650–654. 32. Shiffman D, Ellis SG, Rowland CM, et al.: Identification of This was one of the first whole-genome association studies carried four gene variants associated with myocardial infarction. out for complex diseases. The paper identifies functional SNPs in Am J Hum Genet 2005, 77:596–605. the lymphotoxin alpha (LTA) gene that increase the risk of MI. 33. Ohashi R, Mu H, Yao Q, Chen C: Cellular and molecular 15. Iwanaga Y, Ono K, Takagi S, et al.: Association analysis mechanisms of atherosclerosis with mouse models. between polymorphisms of the lymphotoxin-alpha gene Trends Cardiovasc Med 2004, 14:187–190. and myocardial infarction in a Japanese population. 34. Allayee H, Ghazalpour A, Lusis AJ: Using mice to dissect Atherosclerosis 2004, 172:197–198. genetic factors in atherosclerosis. Arterioscler Thromb Vasc Biol 16. Laxton R, Pearce E, Kyriakou T, Ye S: Association of the 2003, 23:1501–1509. lymphotoxin-alpha gene Thr26Asn polymorphism with 35. Wang X, Ishimori N, Korstanje R, et al.: Identifying novel severity of coronary atherosclerosis. Genes Immunol 2005, genes for atherosclerosis through mouse-human compara- 6:539–541. tive genetics. Am J Hum Genet 2005, 77:1–15. 17. Szolnoki Z, Havasi V, Talian G, et al.: Lymphotoxin-alpha 36. Mehrabian M, Allayee H: 5-lipoxygenase and athero- gene 252G allelic variant is a risk factor for large-ves- sclerosis. Curr Opin Lipidol 2003, 14:447–457. sel-associated ischemic stroke. J Mol Neurosci 2005, 37. Mehrabian M, Allayee H, Wong J, et al.: Identification of 5- 27:205–211. lipoxygenase as a major gene contributing to atherosclerosis 18. Yamada A, Ichihara S, Murase Y, et al.: Lack of association of susceptibility in mice. Circ Res 2002, 91:120–126. polymorphisms of the lymphotoxin alpha gene with myo- 38. Zhao L, Moos MP, Grabner R, et al.: The 5-lipoxygenase cardial infarction in Japanese. J Mol Med 2004, 82:477–483. pathway promotes pathogenesis of hyperlipidemia- 19.• Ozaki K, Inoue K, Sato H, et al.: Functional variation dependent aortic aneurysm. Nat Med 2004, 10:966–973. in LGALS2 confers risk of myocardial infarction and 39. Mehrabian M, Allayee H, Stockton J, et al.: Integrating geno- regulates lymphotoxin-alpha secretion in vitro. Nature typic and expression data in a segregating mouse population 2004, 429:72–75. to identify 5-lipoxygenase as a susceptibility gene for obesity This paper identifies galectin-2 as a protein that interacts with lym- and bone traits. Nat Genet 2005, 37:1224–1233. photoxin alpha and regulates its secretion. A SNP in the galectin-2 40. Aiello RJ, Bourassa PA, Lindsey S, et al.: Leukotriene B4 gene (LGALS2) was also found to be associated with MI. receptor antagonism reduces monocytic foam cells in mice. Arterioscler Thromb Vasc Biol 2002, 22:443–449.
  9. 9. 192 Genetics 41. Subbarao K, Jala VR, Mathis S, et al.: Role of leukotriene B4 46.•• Helgadottir A, Manolescu A, Helgason A, et al.: A variant leceptors in the development of atherosclerosis: potential of the gene encoding leukotriene A4 hydrolase confers mechanisms. Arterioscler Thromb Vasc Biol 2004, 24:369–375. ethnicity-specific risk of myocardial infarction. Nat 42.•• Dwyer JH, Allayee H, Dwyer KM, et al.: Arachidonate Genet 2005, In press. 5-lipoxygenase promoter genotype, dietary arachidonic This study builds on earlier mouse studies and identifies polymor- acid, and atherosclerosis. N Engl J Med 2004, 350:29–37. phisms in three 5-lipoxygenase pathway genes that are associated This study builds on earlier mouse studies and identifies polymor- with various CVD traits. A clinical trial further demonstrated that phisms in three 5-lipoxygenase pathway genes that are associated a 5-LO pathway inhibitor decreases biomarkers of inflammation with various CVD traits. A clinical trial further demonstrated that a that are associated with CVD. 5-LO pathway inhibitor decreases biomarkers of inflammation that 47.•• Hakonarson H, Thorvaldsson S, Helgadottir A, et al.: are associated with CVD. Effects of a 5-lipoxygenase-activating protein inhibitor 43.•• Helgadottir A, Manolescu A, Thorleifsson G, et al.: The on biomarkers associated with risk of myocardial infarc- gene encoding 5-lipoxygenase activating protein confers tion: a randomized trial. JAMA 2005, 293:2245–2256. risk of myocardial infarction and stroke. Nat Genet 2004, This study builds on earlier mouse studies and identifies polymor- 36:233–239. phisms in three 5-lipoxygenase pathway genes that are associated This study builds on earlier mouse studies and identifies polymor- with various CVD traits. A clinical trial further demonstrated that phisms in three 5-lipoxygenase pathway genes that are associated a 5-LO pathway inhibitor decreases biomarkers of inflammation with various CVD traits. A clinical trial further demonstrated that a that are associated with CVD. 5-LO pathway inhibitor decreases biomarkers of inflammation that 48. Pajukanta P, Cargill M, Viitanen L, et al.: Two loci on are associated with CVD. chromosomes 2 and X for premature coronary heart 44.•• Lohmussaar E, Gschwendtner A, Mueller JC, et al.: ALOX5AP disease identified in early- and late-settlement popula- gene and the PDE4D gene in a central European popula- tions of Finland. Am J Hum Genet 2000, 67:1481–1493. tion of stroke patients. Stroke 2005, 36:731–736. 49. Broeckel U, Hengstenberg C, Mayer B, et al.: A comprehen- This study builds on earlier mouse studies and identifies polymor- sive linkage analysis for myocardial infarction and its phisms in three 5-lipoxygenase pathway genes that are associated related risk factors. Nat Genet 2002, 30:210–214. with various CVD traits. A clinical trial further demonstrated that a 50. Harrap SB, Zammit KS, Wong ZY, et al.: Genome-wide 5-LO pathway inhibitor decreases biomarkers of inflammation that linkage analysis of the acute coronary syndrome suggests are associated with CVD. a locus on chromosome 2. Arterioscler Thromb Vasc Biol 45.•• Helgadottir A, Gretarsdottir S, St Clair D, et al.: Association 2002, 22:874–878. between the gene encoding 5-lipoxygenase-activating protein and stroke replicated in a Scottish population. Am J Hum Genet 2005, 76:505–509. This study builds on earlier mouse studies and identifies polymor- phisms in three 5-lipoxygenase pathway genes that are associated with various CVD traits. A clinical trial further demonstrated that a 5-LO pathway inhibitor decreases biomarkers of inflammation that are associated with CVD.