The genomics revolution, including the complete sequence of the human genome, has greatly advanced our understanding of the molecular basis of Mendelian disorders, which are largely the result of necessary and sufficient mutations that are individually rare. As fruitful as genome-targeted efforts have been in the field of single-gene disorders, progress in understanding the genetic architecture of complex traits has been slower and more tenuous. These traits are governed by the interplay between genes, epigenetic factors, and the environment. In this scenario, complex traits are likely to be modulated by multiple genes (and possibly by multiple genetic variants within a gene), which are individually neither necessary nor sufficient to determine a trait. In the field of cardiovascular genetics, the multitude of different outcomes assessed, often using slightly different definitions of the phenotype in question, add to the complexity. Thus, reproducibly identifying genetic variants that impact these traits was a daunting task until recently, when the popularization of microarray technology made it possible to survey large cohorts at a large number of loci in order to scan the genome for variants associated with disease. Genetic studies of cardiovascular disease (CVD) in particular are largely dichotomized into studies of genes that influence CVD risk factors, such as lipids and blood pressure, and studies that focus on clinical outcomes. In this chapter, we summarize the progress made in the search for genes affecting cardiovascular disease risk both by candidate-gene studies and genome-wide association studies (GWAS), while focusing on three specific CVD events (myocardial infarction, stroke, and sudden cardiac death), with the idea that genetic variants that directly affect clinical outcomes would not necessarily be identified only through studies  of CVD risk factors, and often require a direct interrogation of the clinical outcome of interest, allowing for more accurate risk stratification. This is particularly important in sudden cardiac death, where two-thirds of victims do not have CVD risk profiles that would warrant intervention under current guidelines. The results of this search for genetic variants of interest are summarized in Table 21.1.


*QRS interval is included as a subclinical phenotype for SCD, due to the limited direct studies of SCD


Myocardial infarction (MI) is an often fatal manifestation of coronary artery disease (CAD), with an annual incidence of approximately 935,000 cases in the United States, 172,000 of whom will die from the disease (Roger et al., 2012). The proximal cause of MI is believed to be a thrombosis event triggered by an atherosclerotic plaque rupture, which occludes a coronary artery and leads to necrosis of the myocardium. In a long-term follow-up study of ~21,000 monozygous (MZ) and dizygous (DZ) twins, heritability of fatal coronary events was 57% and 38%, respectively, for men and women (Zdravkovic et al., 2002), underlining the importance of genetic factors in susceptibility to MI. Several clinically relevant risk factors have been identified, many of which display significant heritability and thus are, at least in part, under genetic influence (Table 21.2). Indeed, rare Mendelian mutations that affect these risk factors can lead to premature coronary artery disease (Table 21.3) and have been signposts of important pathways that have directly led to successful drug therapies, including the cholesterol metabolism pathway that emerged from studies of familial hypercholsterolemia (FH).


Risk Factors with a Significant Genetic Component (Heritability) Total cholesterol (40–60%)

HDL-cholesterol (45–75%)

Total triglycerides (40–80%) Body mass index (25–60%) Blood pressure (50–70%)

Lp(a) levels (90%)

Homocysteine levels (45%)

Type 2 diabetes (40-80%)

Fibrinogen (20–50%)

C-reactive protein (20–50%) Gender


Environmental risk factors: Smoking

Diet Exercise Infection

*Adapted from Lusis et al., 2004.


Familial hypercholesterolemia LDLR Defective binding of LDL by receptor
Familial defective APOB APOB Reduced binding affinity of APOB to LDLR
Sitosterolemia ABCG5, ABCG8 Increased absorption of plant  sterols
Autosomal recessive hypercholesterolemia ARH             Defective endocytosis of  LDLR
APOA1 deficiency APOA1 Deletion or loss-of-function mutations that lead to very low HDL
Tangier disease ABCA1 Impaired cholesterol efflux in macrophages
Homocystinuria CBS Leads to increased thrombotic tendency

*Adapted from Watkins et al.,  2006.

The majority of research efforts prior to 2007 were focused on identifying genes that modulate common variation in traditional cardiovascular risk factors. A major breakthrough was made with the popularization of GWAS and the ability to genotype a large patient population for a million single- nucleotide polymorphisms (SNPs) or more. The efforts to identify common genetic variants associated with CAD and MI have produced a list of over 30 loci showing robust association that has been replicated across multiple studies and cohorts (Kathiresan et al., 2009; Samani et al., 2007; Schunkert et al., 2011).

We highlight the progress made in the search for genes associated with MI by both the candidate gene approach and the genome-wide association approach, by discussing four specific examples: apolipoprotein E (APOE), coagulation proteins, 9p21, and SORT1, in detail.


Apolipoprotein E, a low-density lipoprotein (LDL) receptor ligand, is an important player in the metabolism of cholesterol and triglycerides, where it mediates the clearance of chylomicron and very low-density lipoprotein (VLDL) from plasma. Utermann and colleagues (Utermann et al., 1977) first described the effects of three common allelic variants of APOE (termed E2, E3, and E4, and with frequencies of 8%, 77%, and 15%, respectively, in Caucasian populations) on type III familial hyperlipoproteinemia, in which more than 95% of affected individuals were homozygous for the rare allele. The common genotype, E3/E3, is used as the reference group in most studies, and individuals who carry the E2 allele have ~14 mg/dl lower LDL levels, and E4 carriers have ~7 mg/dl higher LDL levels (Motulsky and Brunzell, 2002).

Numerous studies have examined the association between the E2/E3/E4 variants and coronary heart disease (CHD), including a large-scale meta- analysis incorporating 121 studies with 37,850 cases and 82,727 controls (Bennet et al., 2007). The meta-analysis, which was stratified by the number of participants in an individual study at a cutoff of at least 1,000 healthy controls and 500 cases, demonstrated a moderate increased risk for E4 carriers (odds ratio [OR] 1.06, 95% confidence interval [CI], 0.99–1.13), and a significant decreased risk for E2 carriers (OR 0.80, 95% CI 0.70–0.90) in the group with larger study size. In addition, the study also established a nearly linear relationship between APOE status and both LDL cholesterol levels and risk for CVD, putting forward the possibility that the differential risk associated with the different APOE isoforms may be mediated by LDL cholesterol, a well-established risk factor for CAD. Indeed, while most of the studies in the meta-analysis did not report odds ratios adjusted for lipids, and thus the meta-analysis did not determine whether the APOE variants influence risk for CHD independently of lipids, several studies that have examined the data for an association of the E4 allele after adjusting for lipid levels report conflicting results (Humphries et al., 2001; Lahoz et al., 2001; Volcik et al., 2006; Ward et al., 2009).


The most common pathogenetic pathway of acute myocardial infarction is through thrombosis, generally triggered by atherosclerotic plaque rupture. Thus, a great number of candidate gene studies have involved the examination of genetic variants in genes involved in coagulation and fibrinolytic pathways. Ye and colleagues (Ye et al., 2006) performed a meta- analysis of 191 studies to determine the relationship between CAD and variants in seven genes involved in the thrombotic process: factor V Leiden, factor VII G10976A, prothrombin G20210A, plasminogen activator inhibitor-1 (PAI-1) [-675] 4G/5G, and three platelet glycoprotein (GP) variants (GPIa, C807T, GPIba T[-5]C, GPIIIa C1565T). In contrast to an earlier study (Boekholdt et al., 2001), which examined the association of several of the same variants with MI and found that associations for these genetic variants were either weak (PAI-1, fibrinogen) or absent (factor V, prothrombin), Ye and colleagues reported a mild association with factor V Leiden (OR 1.12, 95% CI 0.91–1.36) and prothrombin (OR 1.91, 95% CI 0.91–1.55). Despite this weak and conflicting association with MI, stronger, consistent associations are seen with stroke, and these variants are discussed in greater detail later in the chapter in that context.

Given the difficulty in identifying genetic variants that play a significant role in susceptibility to MI/IHD through a candidate gene approach, a problem observed for most complex diseases, family-based strategies have also been implemented. While many regions have been implicated through linkage analysis, these studies often result in large candidate regions, often comprising hundreds of genes. Therefore, their utility in identifying specific gene variants associated with disease is limited, though some successes have been reported, for instance in the association of genes ALOX5AP and PDE4D. with stroke (details in next section). With both a rapid reduction in genotyping costs and a vast increase in throughput, the focus has turned to genome-wide association studies.

Cardiovascular disease has proven to be an excellent example to illustrate the power of genome-wide association studies to find loci that contribute to common complex disorders. The high incidence of CVD, combined with the ready availability of large cohorts with detailed data on traditional cardiovascular risk factors, has led to several large association studies that have brought the list of loci associated with either CVD or MI to more than

  1. A majority of these loci come from three large multi-cohort Samani and colleagues used data from a combined 2801 cases and 4582 controls, replicated the association of the 9p21 region, and identified six additional loci associated with coronary artery disease (Samani et al., 2007). Four of these loci were also shown to be associated with myocardial infarction in a large-scale four-stage study by Kathiresan and colleagues (Kathiresan et al., 2009), who also reported three additional novel loci. In the third and largest association study for CAD, Schunkert and colleagues (Schunkert et al., 2011) carried out a meta-analysis of 14 studies to obtain a sample size of 22,233 cases and 64,762 controls of European descent. In addition to confirming the 10 previously associated loci, the study also identified 13 novel loci associated with CAD, only three of which showed any significant association with traditional CAD risk factors. From this long list of loci (see Table 21.4), we will focus on 9p21 and SORT1 as specific examples of GWAS discoveries resulting in interesting insights about biology and disease.



*Adapted from Kathiresan and Srivastava,  2012.


One of the most strongly associated signals from GWAS has been in the noncoding region on chromosome 9p21.3. After its initial discovery in a cohort of early-onset MI patients from the Ottawa Heart Study and deCode project (Helgadottir et al., 2007; McPherson et al., 2007), the region was fine- mapped to a 58kb locus with multiple tagged SNPs in tight linkage disequilibrium (LD), and it has shown consistent association with disease independent of traditional CAD risk factors, in multiple cohorts of various ethnic backgrounds (Cheng et al., 2011; Gori et al., 2010; IBC 50K CAD Consortium, 2011; Shen et al., 2008; Takeuchi et al., 2012; Xie et al., 2011). In a meta-analysis of 47 studies comprising 35,872 cases and 95,837 controls of either European or Asian descent, Palomaki and colleagues surveyed data from one of three common lead SNPs in the region (rs1333049, rs10757274, rs2382207) that are in tight LD, to confirm that the region had a small but significant effect on risk for MI (OR 1.25, 95% CI 1.21–1.29) (Palomaki et al., 2010). It is important to mention that while these results have been replicated in a number of European and Asian cohorts, the results from studies including African-Americans have been more conflicting (Beckie et al., 2011; Kral et al., 2011; Patel et al., 2011; Yamagishi et al., 2009). Although the risk locus is devoid of a protein-coding gene, it lies within a well-described noncoding RNA in the INK locus (ANRIL) (Pasmant et al., 2007) and overlaps with upstream cyclin-dependent kinase inhibitor genes CDKN2B and CDKN2A, which have been studied for their role as cell cycle regulators and tumor suppressors. While the genes lie in the same LD block, they are also approximately 100 kb upstream of the implicated risk region, and the direct evidence for their putative role in the modulation of cardiovascular disease did not emerge until Axel and colleagues developed a mouse model with a homozygous deletion of the orthologous region on the mouse chromosome 4, which showed reduced levels of cardiac expression of both genes (Visel et al., 2010). There have been numerous studies that have looked at the direct effect of expression of the CDKN2A, CDKN2B, and ANRIL (Cunnington et al., 2010; Folkersen et al., 2009; Harismendy et al., 2011; Jarinova et al., 2009; Liu et al., 2009) with conflicting results on the direction and magnitude of the effect of region in the development of CAD.

To add to the confusion, 9p21 has turned out to be somewhat of a hot  spot for GWAS hits for a number of conditions, including type 2 diabetes (T2D) (Diabetes Genetics Initiative of Broad Institute of Harvard and MIT, Lund University, and Novartis Institutes of BioMedical Research et al., 2007; Scott et al., 2007; Zeggini et al., 2007; Zeggini et al., 2008), intracranial aneurysm and abdominal aortic aneurysms (AAA) (Helgadottir et al., 2008), and a number of different cancers (Amos et al., 2011; Antoniou et al., 2012; Enciso-Mora et al., 2012; Rajaraman et al., 2012; Sherborne et al., 2010; Shete et al., 2009; Yang et al., 2010). While the link to cancer can be explained by the presence of CDKN2A/B, two well-established tumor suppressor genes, the presence of a strong association to T2D, a traditional CAD risk factor, paved the ground for the tempting possibility of a shared biological function of the region that would affect both conditions. In an elegant study, Helgadottir and colleagues showed that the region was in fact that tagged by two separate lead SNPs (rs10757278 for CAD,  and rs10811661 for T2D) which were in adjoining LD blocks (Helgadottir et al., 2008). While the SNP associated with CAD also showed association with five other arterial diseases, with the strongest association being AAA (OR = 1.31, 95% CI 1.22–1.42) and intracranial aneurysms (OR = 1.29, 95% CI 1.16–1.43), rs10811661, the T2D SNP, showed no  significant association with either CAD or the other arterial diseases. This result has since been replicated in several studies (Biros et al., 2010; Bown et al., 2008; Gori et al., 2010) and highlights the complexity of the genomic region and makes the process of going from an associated locus to a causal gene even more challenging. While the mechanism by which the risk “region” affects atherosclerotic processes remains to be determined, the presence of a consistent association in multiple cohorts makes 9p21 a prominent area of research efforts.


The elucidation of the role of Sortilin1, a multi-ligand sorting receptor in cholesterol metabolism, highlights the power of GWAS findings when put in the context of appropriate functional studies.

Samani and colleagues first implicated the 1p31 region in a GWAS for CAD in 2007 (OR 1.29, 95% CI 1.10–1.21) (Samani et al., 2007), which has since been replicated in a number of large-scale GWAS for both MI and CAD (Kathiresan et al., 2009; Schunkert et al., 2011). The SNPs tagged in these studies lie in a noncoding region, between genes PSRC1 and CELSR1, and in the same LD block as SORT1, making identification of the causal variant and the gene mediating the association challenging.

SORT1 was first implicated as the putative gene of interest by Linsel- Nitschke and colleagues (Linsel-Nitschke et al., 2010). This work identified an expression quantitative trait locus (eQTL), uncovering an association between one of the GWAS SNPs, rs599839, and SORT1 mRNA levels. In addition, they demonstrated a significant increase in LDL cholesterol uptake in HEK293 cells with over-expression of SORT1 and laid the foundation for the hypothesis that increased SORT1 expression could potentially have a protective role in CAD. The role of SORT1 in LDL cholesterol metabolism was further investigated by Musunuru and colleagues in a series of experiments that showed the direct effect of Sort1 knockdown and over- expression on plasma LDL cholesterol and lipoprotein levels in murine models of atherosclerosis. They also presented evidence to show that a common polymorphism, rs12740374, previously shown to be associated with CAD, creates a novel CCAAT-enhancer-binding protein  (C/EBP) binding site and hence mediates an increased LDL cholesterol level via a hepatic secretory pathway. However, in direct contrast, Kjolby et al. showed that in a Sort1, Ldlr double knockout mouse model, plasma LDL cholesterol levels, and thus the atherosclerotic burden, are reduced when compared to a LDLR- null mouse (Kjolby et al., 2010). Considering the difference in mouse manipulations and timing of over-expression, the differences are not altogether surprising, but they definitely raise questions about the role of SORT1 in the biology of cholesterol metabolism and its subsequent effect on disease processes.

The success of GWAS is demonstrated by the fact that, in addition to identifying genes that are implicated as putative candidates because of their biological relevance in the disease process, we are now also able to identify a long list of genes that show a strong, definite association with CVD through yet-unknown mechanisms. Indeed, in a recent review, Kathiresan and Srivastava (Kathiresan and Srivastava, 2012) divided the list of loci mapped by GWAS into those known to be associated with an established risk factor like LDL-cholesterol or blood pressure, and those where the association is established but the mechanism is unknown.

Herein lies the challenge before us: to improve our ability to identify the causal variant, and thereby the underlying mechanism of action. Over the next couple of years, we will need to make a strong effort to determine the complete list of associated loci, and more importantly, to use this list to direct research efforts to better understand the biology of normal cardiovascular processes and myocardial infarction. Aside from identifying novel molecular pathways, the importance of these genetic variants from a clinical standpoint is often hard to interpret, given the small effect size of most GWAS hits and the ability to easily measure traditional cardiovascular risk factors. The argument in favor of using variants that have shown repeated association in several studies for either risk prediction has been shaky at best, with different groups reporting conflicting levels of success in using genetic data for risk prediction (Drenos et al., 2007; Kathiresan et al., 2008; Kathiresan et al., 2009; Paynter et al., 2010). In a large literature-survey-based test of  a “genetic score” combining the effects of 101 SNPs with traditional cardiovascular risk factors, Paynter and colleagues showed no significant improvement in risk determination or classification in a cohort of 19,213 women (Paynter et al., 2010). However, it is important to mention that while these data suggest that genetic variants, at this point in time, have little value in cross-sectional measures of risk, there is strong evidence to suggest that genetic data might provide a better measure of lifetime risk than individual cross-sectional measures of risk factors. This has been successfully shown by Cohen and colleagues, who observed significantly reduced levels of plasma LDL cholesterol (~15%) and incidence of cardiovascular disease in  carriers of the Arg47Leu allele of PCSK9 (hazard ratio 0.50, 95% CI 0.32–0.79) (Cohen et al., 2006). While the expected reduction of the CVD risk corresponding to the decrease in LDL-c levels that they observed is ~23%, analysis of the Atherosclerosis Risk in Communities (ARIC) prospective cohort showed a 47% decrease in R47L carriers, suggesting that there is an accumulated lifetime burden of reduced LDL-c levels that is not accurately captured by a single measure of lipid levels.


Stroke is one of the leading causes of death and disability in the developed world, with annual incidence of 795,000 (Roger et al., 2012). With limited treatment options available, focus has been on primary prevention, largely through modification of acquired risk factors (diabetes mellitus, smoking, high blood pressure, and atrial fibrillation) (Goldstein et al., 2001). However, as with many common diseases, rare monogenic conditions that cause stroke have been identified, and a great deal of progress has been made in identifying the underlying genetic defects. Indeed, the gene for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which has served as a model for inherited ischemic stroke, was identified as NOTCH3 in 1996 (Joutel et al., 1996). More recently, substantial progress has been made in cerebral cavernous malformations (CCM), with genes for all three types of CCM now identified. CCM1 is caused by mutations in KRIT (Laberge-le Couteulx et al., 1999); CCM2 is caused by mutations in MGC4607 (malcavernin) (Denier et al., 2004); and CCM3 is caused by Programmed cell-death protein 10 (Bergametti et al., 2005). While these genetic defects are rare in the general population, they have high penetrance; therefore, being able to identify carriers has a significant clinical impact. Whether any of these genes has a role in common forms of stroke remains to be determined, though a study by Dong and colleagues, in which they screened individuals with lacunar stroke for coding mutations in NOTCH3, did not find any association (Dong et al., 2003). This does not, however, rule out the involvement of common variants, more likely to be found in noncoding conserved regions and involved in gene regulation.

While rare, cerebral venous thrombosis (CVT), which accounts for less than 1% of all strokes, is a complex disease, with numerous etiological risk factors (for review, see Agostoni et al., 2009), including genetic factors. Marjot and colleagues (Marjot et al., 2011) conducted a comprehensive meta- analysis of candidate gene studies for CVT, identifying 26 case-control studies covering six polymorphisms in six genes. With a sample size of 1183 CVT cases and 5189 controls, they demonstrated significant associations for three genes (discussed below), including factor V Leiden/G1691A (OR 2.40, 95% CI 1.75–3.30), prothrombin/G20210A (OR 5.48, 95% CI 3.88–7.74), and MTHFR/C677T (OR 2.30, 95% CI 1.20–4.42).


Bertina and colleagues (Bertina et al., 1994) identified an arginine-to-glycine (R506Q) mutation (termed “Leiden allele”) in factor V in a family with activated protein C (APC) resistance and prone to thrombosis. APC limits clot  formation  by  inactivation  of  factors  Va  and  VIIIa,  and  the   Leiden mutation is predicted to alter the amino acid at the APC cleavage site in factor V, causing factor V to be less efficiently degraded. Thus, individuals who carry factor V Leiden have increased thrombin generation and a hypercoaguable state, which could explain the increased risk for stroke associated with this allele (Dahlback, 1995).


Poort and colleagues (Poort et al., 1996) first identified the G20210A single- base-pair substitution in the 3′ untranslated region of the parent gene, coagulation factor II (F2), and demonstrated its association with elevated plasma prothrombin levels and increased risk for venous thrombosis. Subsequent studies have demonstrated that prothrombin levels were probably due to increased thrombin generation (Franco et al., 1999). More recently, the mechanism by which G20210A alters prothrombin levels has been established. Ceelie and colleagues (Ceelie et al., 2004) have demonstrated that the G20210A mutation results in a more effective poly (A) site (a poly [A] tail is required for mRNA to be efficiently exported from the nucleus and translated into protein in the cytoplasm), leading to elevated mRNA levels, resulting in increased prothrombin production and thrombin formation. Thus, like factor V Leiden, the G20210A mutation probably leads to a pro- coagulant state, thereby increasing risk of stroke.


MTHFR catalyzes the conversion of 5,10- methylenetetrahydrofolate to 5- methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine. Frosst and colleagues (Frosst et al., 1995) identified a cytosine- to-thymine single-base-pair substitution at position 677 (C677T)  that converts an alanine to a valine residue, and produces a thermolabile form of the protein. They demonstrated that this variant was associated with reduced enzyme activity and increased levels of homocysteine. Elevated levels of homocysteine are associated with increased risk for stroke (Wald et  al., 2002), thus, the C677T variant is likely to contribute to increased risk of stroke directly due to its reduced ability to metabolize homocysteine.

More common forms of stroke can be divided into two major varieties: ischemic and hemorrhagic. The majority of strokes are ischemic    (80–90%), and can be further subdivided into: 1) large-vessel occlusive disease, usually due to atherosclerosis and plaque formation; 2) small-vessel occlusive disease, which have involvement of small, perforating end-arteries in the brain; and 3) cardiogenic stroke, which is secondary to blood clots from a diseased heart. Traditionally, occlusive disease has been considered to be due to atherosclerosis, and cardiogenic stroke due to atrial fibrillation secondary to mitral-valve stenosis; however, arguments have been put forward that all three forms probably have a significant atherosclerotic component (Gulcher et al., 2005). Given the heterogeneous nature of the stroke phenotype, assumptions about the underlying etiology of disease can have a significant impact on the ability to identify genetic determinants of stroke susceptibility.

The leap from the existence of genes for monogenic and rare forms of stroke to the likely existence of genes contributing to risk for common forms of stroke is bolstered by numerous studies that have shown that a genetic component to susceptibility to common forms of stroke probably exists. A comprehensive analysis of these studies, in which all genetic epidemiology studies of ischemic stroke from 1966 to 2003 were systematically reviewed, was conducted by Flossmann and colleagues (Flossmann et al., 2004). Based on twin (OR 1.65, 95% CI 1.2–2.3), case-control (OR 1.76, 95% CI 1.7–1.9), and cohort (OR 1.30, 95% CI 1.2–1.5) studies, they concluded that there is a modest but significant genetic component to the risk for ischemic stroke in the general population. Most genetic studies have focused on candidate genes under case-control designs, and Casas and colleagues (Casas et al., 2004) performed a meta-analysis of these studies on ischemic stroke, incorporating 32 genes studies across ~18,000 cases and ~58,000 controls. They identified significant associations for factor V Leiden (OR 1.33, 95% CI 1.12–1.58), methylenetetrahydrofolatereductase (MTHFR) C677T (OR = 1.24, 95% CI 1.08–1.42),   prothrombin   G20210A   (OR   =   1.44,   95%   CI   1.11–1.86) (discussed above, in the context of CVT), as well angiotensin-converting enzyme (ACE) insertion/deletion (OR 1.21, 95% CI 1.08–1.35). A more recent meta-analysis conducted by Hamzi et al. (Hamzi et al., 2011) reviewed 300 manuscripts for five candidate genes among 152,797 individuals (45,433 cases and 107,634 controls) and confirmed the associations for prothrombin (OR 1.57, 95% CI 1.23–2.89) and ACE (OR 1.11, 95% CI 1.02–1.25), but did not find significant results for factor V Leiden or MTHFR for ischemic stroke. Therefore, they concluded that there are common variants in several genes involved in common forms of stroke, each with a modest effect.  Meta-analyses of the ACE insertion/deletion variant (see below) in non–European descent individuals also revealed a significant association with ischemic stroke, indicating the importance of this variant across different ethnic groups (Ariyaratnam et al., 2007; Wang et al., 2012).


ACE plays an important role in blood pressure regulation and electrolyte balance, and ACE inhibitors have been at the forefront of therapy for treating hypertension and reducing risk for CVD. Indeed, ACE is an important regulator of the renin-angiotensin-aldosterone system through both its ability to hydrolyze angiotensin I into angiotensin II, a potent vasopressor, and its ability to inactivate bradykinin, a potent vasodilator that may stimulate nitric oxide production (Kim and Iwao, 2000). ACE is found on the surface of vascular endothelial cells and in circulating plasma, and animal studies have shown the importance of ACE in regulating blood pressure (Esther et al., 1997; Krege et al., 1995). In 1990, Rigat and colleagues (Rigat et al., 1990) identified an insertion/deletion polymorphism (I/D) that was responsible for up to 50% of the variation in circulating levels of ACE. While the molecular basis of how the I/D polymorphism affects circulating ACE levels is not entirely clear, a study using nearby polymorphisms to measure specific expression of the I and D alleles indicates that the D allele leads to higher expression of ACE mRNA (Suehiro et al., 2004).

Given the widespread use of ACE inhibitors in clinical treatment, and the high frequency of the I/D polymorphism in the general population (~30% in Caucasian populations), the ACE I/D polymorphism provides a prime target for testing the potential impact of a genetic variant on choice of drug therapy (pharmacogenetics). Arnett and colleagues (Arnett et al., 2005) have reported the results of a double-blind, active-controlled randomized trial of antihypertensive treatment in which they examined the impact of the ACE I/D polymorphism on response to four different medications (chlorthalidone, amlodipine, lisinopril, and doxazosin). The study included  37,939 participants ≥55 years of age with ≥1 risk factor for CVD. These individuals were followed up for four to eight years, with primary outcomes including fatal coronary heart disease (CHD) and/or nonfatal MI, and secondary outcomes including stroke, all-cause mortality, combined CHD, and combined cardiovascular disease. ACE I/D genotype was not predictive for CHD (though the risk for stroke was consistent with the meta-analysis of Casas et al., 2004), nor did it modify the response to treatment with the different antihypertensive medications. These results were surprising, as one would predict that those with the D/D genotype, and therefore higher ACE levels, would be more responsive to the ACE inhibitor therapy (lisinopril). Indeed, these results provide a warning for making the leap between genetics and treatment. Despite a functional variant in a gene whose product is a direct target of one of the therapies, the choice of therapy did not affect the outcome. There were however, some differences in outcome according to gender and diabetes status, but given the number of hypotheses tested, further follow-up is needed to verify any of these observations.

In addition to candidate-gene studies that led to the revelation of the association of the above-discussed genes with stroke, traditional genetic approaches have also focused on using large pedigrees in family-based linkage studies. While these studies have been at the forefront of identifying genes for Mendelian diseases, they are often difficult to implement for phenotypes that occur late in life, such as stroke, due to the difficulty in obtaining informative pedigrees. However, deCODE Genetics has leveraged the combination of extensive genealogical records and medical records in Iceland to be able to perform these types of studies.


Gretarsdottir and colleagues (Gretarsdottir et al., 2002) initially performed a genome-wide linkage scan in 476 patients with stroke within 179 extended pedigrees from Iceland, and identified a locus on 5q12 (Log of odds [LOD] score = 4.40), which they designated as “STRK1.” They employed a broad definition of stroke, including individuals with either ischemic or hemorrhagic stroke, as well as TIA (transient ischemic attack), which they considered an ischemic event, arguing that the same pathophysiological mechanisms are responsible for both. In a subsequent study, the same group fine-mapped this locus using a population-based case-control  study composed of 864 Icelandic affected individuals and 908 controls, implicating PDE4D, a regulator of intracellular levels of cyclic  adenosine monophosphate (cAMP) (Gretarsdottir et al., 2003). PDE4D mRNA is expressed in cardiac myocytes and may be involved in excitation–contraction coupling (Lehnart  et  al.,  2005).  However,  this  association  was  limited to ischemic stroke, and specifically to the combined cardiogenic and carotid forms (using Trial of Org 10172 in Acute Stroke Treatment [TOAST] subcategories). Subsequent studies have yielded ambiguous replication results, and two large meta-analyses, with the most recent containing >10,000 cases and >10,000 controls, have not demonstrated an association with ischemic stroke (Bevan et al., 2008; Lovkvist et al., 2012).


Helgadottir and colleagues (Helgadottir et al., 2004) reported a finding of linkage and association with ALOX5AP and both stroke and MI in an Icelandic population. They identified a specific haplotype (HapA) that is relatively common and carried in 27% of patients with stroke (Relative risk [RR] 1.7, P <0.0001). The association with both MI and stroke was particularly intriguing, and in the same paper Helgadottir and colleagues identified another haploytpe (HapB) that was associated with MI in an independent British cohort. They went on to demonstrate that the synthesis of leukotriene B4 (LTB4) in ionomycin-stimulated neutrophils from patients with a history of MI is greater than from controls without MI and that this difference is largely accounted for by carriers of the HapA haplotype. LTB4, which is a biolipid inflammatory and vasoactive mediator, is produced from LTA4, which is produced from arachidonic acid by ALOX5AP. Thus, the association study is backed by functional evidence that points to a specific mechanism by which variants in ALOX5AP may increase risk for both stroke and MI: elevated levels of LTB4 might contribute to increased inflammation, a known risk factor for CVD events through the development and atherosclerosis and/or plaque instability. As with PDE4D, replication studies have yielded ambiguous results, and the largest meta-analysis to date, with>5,000 cases and >4,500 controls, does not show a significant association of the HapA haplotype and ischemic stroke (Zintzaras et al., 2009).


As noted above for MI, the advent of GWAS has begun to change the landscape of stroke genetics (see Table 21.5). The first successful GWAS for ischemic stroke (Gretarsdottir et al., 2008) identified a signal near PITX2 (paired-like homeodomain transcription factor 2), which had previously been associated with atrial fibrillation (AF) (Gudbjartsson et al., 2007). AF, one of the most common forms of electrical instability, is characterized by chaotic electrical activity of the atria, and plays a major role in cardioembolic stroke (Lip and Tse, 2007). While the association was initially also reported for non- cardiogenic stroke (Gretarsdottir et al., 2008), subsequent studies have validated the association with cardiogenic stroke (International Stroke Genetics Consortium [ISGC] et al., 2012), but not overall ischemic stroke (Carty et al., 2012). The utility of using AF as an endophenotype to identify cardiogenic stroke–associated variants is further validated by the observation of an association between stroke and SNPs at the ZFHX3 locus (Gudbjartsson et al., 2007; ISGC et al., 2012), which had previously been associated with AF (Benjamin et al., 2009; ISGC et al., 2012).



ACE 17 . . .
ALOX5AP 13 rs17216473
Angiopoietin-1 8 rs2507800
APOA (LPA) 6 . . .
APOE 19 . . .
CRP 1 rs2794521
CYP4AII 1 . . .
CYP4F2 19 rs2108622
CYPIIB2 8 rs1799998
DDAH1 1 . . .
eNOS (NOS3) 7 rs1799983
Factor V Leiden 1 . . .
Fibrinogen 4 . . .
GP1BA 17 . . .
GPIIIa 17 . . .
IL-6 7 . . .
LTC4S2 5 rs730012
MTHFR 1 rs2274976
NPY 7 rs16147
PAI-1 (Serpine) 7 . . .
Paroxonase-1 7 rs662
PDE4D 5 rs12188950
Prothrombin 11 . . .
SGK1 6 rs1057293
TNF-Alpha 10 . . .
VKORC1 16 rs9923231


CDKN2A/B 9 rs4977574
NINJ2 12 rs11833579
PCSK9 1 rs11206510
PITX2 4 rs1906591
PRKCH 14 rs2230500
ZFHX3 16 rs7193343

*Adapted from Bevan et al., 2012.


In the first study of prospectively identified stroke in the general population, Ikram and colleagues identified a single locus, NINJ2 (Ikram et al., 2009). NINJ2 encodes ninjurin2, an adhesion molecule that is upregulated in response to nerve injury. However, this locus was not  validated by subsequent studies (ISGC et al., 2012; ISGC and Wellcome Trust Case- Control Consortium 2, 2010). A more recent GWAS for ischemic stroke, conducted in >9,000 cases and >11,000 controls, replicated associations for cardioembolic stroke near PITX2 and ZFHX3, as well for the 9p21 locus (previously implicated in MI; see above) with large-vessel disease (Gschwendtner et al., 2009). They also reported a novel finding with the HDAC9 locus and large vessel stroke (OR 1.42, 95% CI 1.28–1.57). HDAC9 encodes histone deacetylase 9, an enzyme involved in regulating chromatin structure and gene transcription (Haberland et al., 2009). Using a Bayesian statistical   framework   to   formally   test   whether   different   variants were associated with all subtypes of ischemic stroke or specific subtypes, they clearly demonstrate the importance of subtype classification and provide strong evidence of heterogeneity of genetic effects across stroke subtypes. The PITX2 and ZFHX3 variants appear to only affect risk for cardioembolic stroke, whereas the 9p21 variants appear to broadly influence ischemic stroke, and HDAC9 is specific for large-vessel stroke. Based on this specificity, the authors postulate that a mechanism for association of HDAC9 with stroke through accelerated atherosclerosis is possible, but note that this hypothesis is highly speculative.

In an example of population-specific findings, a gene-based association study using ~50,000 tag SNPs was conducted in ~1100 Japanese cerebral infarct cases, and identified a nonsynonymous SNP in PRKCH (V374I), which encodes protein kinase C eta (Kubo et al., 2007). The authors go on to show that the identified variant appears to be functional, resulting in higher autophosphorylation and kinase activity, which activates its downstream signaling pathway. The variant has a frequency of about 20% in Asian populations, but less than 1% in European ancestry individuals, and is not observed in the Yoruba from Ibadan (International HapMap Consortium, 2005). This finding has been validated in a Chinese population (Wu et al., 2009), as well as in a meta-analysis of >3,600 cases of ischemic stroke and>4,500 controls drawn from Chinese and Japanese populations (Li et al., 2012). PRKCH is a serine-theronine kinase that is mainly expressed in vascular endothelial cells and foamy macrophages (which play an important role in atherosclerosis), and is involved in regulation of cell differentiation, proliferation, and apoptosis. Indeed, increased expression of PRKCH was correlated with progression of coronary atherosclerotic lesion type, providing strong evidence for a mechanism by which altered PRKCH levels influence risk of stroke (Kubo et al., 2007).


A series of GWAS have focused on intracranial aneurysm, a major cause of hemorrhagic stroke, and identified six loci using discovery and replication cohorts from Europe and Japan comprising >5,800 cases and >14,000 controls (Bilguvar et al., 2008; Yasuno et al., 2010; Yasuno et al., 2011). These loci include the 9p21 locus, which is implicated in CAD and large- vessel ischemic stroke (see above). Additional loci include SOX17,  CNNM2, KL/STARD13, RBBP8, and EDNRA. The author note that a common pathway tying these genes together is cell cycle progression, and these genes may affect proliferation and senescence of progenitor-cell populations (Yasuno et al., 2010). Indeed, SOX17 is required for endothelial formation and maintenance, and Sox17-/- mice show vascular abnormalities (Sakamoto et al., 2007). EDNRA, which encodes a G protein-coupled receptor for endothelin, is a particularly intriguing candidate, as it mediates the vasoconstriction and mitogenic effects of EDN1 (Alberts et al., 1994; Suzuki et al., 1999). Yasuno and colleagues (Yasuno et al., 2011) note  that the effects of EDNRA variants on IA risk may occur in two distinct ways, depending on whether the variant increases or decreases EDNRA-mediated signaling. An increase in signal might promote development of atherosclerosis, whereas a decreased signal might lead to an inability to adequately repair the vasculature after vascular injury. Understanding the specific nature of the risk variant may have important pharmacogenetic implications, as selective EDNRA antagonists are in clinical trials for treatment of subarachnoid hemorrhage (clazosentan) (Kramer and Fletcher, 2009; Macdonald et al., 2008; Macdonald et al., 2013; Vergouwen et al., 2012).


Sudden cardiac death continues to be one of the leading causes of death in the United States. According to the U.S. Centers for Disease Control and Prevention, about 462,000 of the 2,400,000 (19.3%) U.S. deaths in 1999 were classified as “sudden cardiac deaths” (SCDs) using their definition of SCD as including all deaths “due to cardiac disease that occurred out of hospital (~341,000) or in an emergency department, or one in which the decedent was reported ‘dead on arrival’” (Zheng et al., 2002). From the standpoint of preventive care, SCD poses a huge burden, since fewer than 10% of SCD victims survive, and approximately one-third of all victims manifest SCD as their first clinical event. Approximately two-thirds of SCD victims do not have clinical symptoms that would warrant preventive intervention. Therefore, the ability to identify individuals who are at high risk for SCD is crucial, and advances in genetics may fill this gap.

As for stroke and MI, a great deal of progress has been made identifying the genes involved in Mendelian forms of disease that contribute to increased risk of SCD. Mutations in coding sequences in at least seven cardiac sarcolemmal, sodium, potassium, and calcium ion channels subunit genes (i.e., KVLQT1 [KCNQ1], HERG [KCNH2], SCN5A, minK  [KCNE1], RYR2, MiRP1 [KCNE2], and Kir2.1 [KCNJ2]) result in increased susceptibility to SCD (Priori and Napolitano, 2004). Electrophysiological dysfunction, manifested as delayed myocardial cell depolarization and repolarization, is caused by mutations in the proteins encoded by these genes, as was originally discovered by Keating, Schwartz, Moss, Priori, and others during the 1990s, and it is now known to underlie a whole family of related pro-arrhythmic conditions, exemplified by the Long QT syndrome (LQTS) and Brugada’s syndrome (Keating and Sanguinetti, 2001; Splawski et al., 2000). Mutations in these same genes also result in converse disorders, such as “short QT syndrome,” which also enhances SCD risk (Brugada et al., 2004; Gaita et al., 2003).

Multiple etiologies probably contribute to increased SCD risk, including susceptibilities that arise from genetic variations in sarcomeric proteins, such as beta-myosin heavy chain (MyHC), cardiac troponin T (cTnT), and myosin binding protein-C (MyBP-C), which underlie SCDs that occur in patients with inherited hypertrophic cardiomyopathies (Marian and Roberts, 2001). Another important factor is genetic changes that impact early patterning events during embryogenesis, and subsequently cause disturbances in cardiac electrical function from development through maturation. Chien and collaborators were among the first to report that genetic alterations affecting early transcription-factor expression may lead to enhanced arrhythmia susceptibility (Nguyen-Tran et al., 2000). Similar alterations  in developmental factors were also implicated in rare familial vascular defects that appear to result in enhanced susceptibility to myocardial infarction (Wang et al., 2003).

The existence of rare inherited monogenic disorders, such as the LQTS and Brugada syndromes, demonstrate that mutations in ion-channel genes, structural proteins, and calcium-handling genes can increase susceptibility to lethal arrhythmias (Keating and Sanguinetti, 2001; Splawski et al., 2000). It is thus a small leap to propose that subtler variations in these same genes may predispose to more common forms of SCD. Two population-based studies have demonstrated that a family history of SCD increases the risk for SCD approximately 1.8-fold, independent of other traditional cardiovascular disease risk factors (Friedlander et al., 1998; Jouven et al., 1999). The first study, conducted in the United States by Friedlander and colleagues (Friedlander et al., 1998), analyzed associations with “primary cardiac events” in a cohort of men and women attended by first responders in King County, Washington (235 cases, 374 controls). The second, done in Paris by Jouven and colleagues (Jouven et al., 1999), analyzed deaths in a cohort of 7,746 asymptomatic middle-aged males, using retrospective autopsy and clinical data analyses to ascribe cardiac deaths to either SCD or MI. Multifactorial statistical analyses indicated that the occurrence of SCD in a parent results in a 1.6–1.8-fold increase in SCD susceptibility, despite controlling for conventional risk factors indicative of coronary disease (e.g., cholesterol sub-fractions, blood pressure, obesity, tobacco use, etc.). In a very limited number of cases in the Parisian study, where there was a history of both maternal and paternal SCD events (n = 19), the relative risk in offspring was ~9 (P = 0.01), indicating an additive genetic model. Elevated  incidence of SCD in the Paris study segregated independently of elevated familial incidence of myocardial infarction, suggesting genetic  factors that specifically associate with risk for SCD, rather than factors that may underlie overall CVD risk (e.g., atherosclerosis).


The idea that ion-channel sequence variations that alter cardiac de- or re- polarizing currents in patients with rare inherited syndromes, like LQTS, may also contribute to enhanced SCD susceptibility seen in more common forms of cardiac disease represents an attractive hypothesis. Splawski and colleagues (Splawski et al., 2002) have given some insight into  this paradigm. In their study, a single-nucleotide sequence variant in the SCN5A Na channel gene found in African Americans, S1102Y, was associated with a modest enhancement of arrhythmia risk. The aberrant allele was estimated to be present in up to 4.6 million African Americans: a level of prevalence far beyond all previously established SCD susceptibility alleles combined, and it was not identified in other ethnic populations sampled. In in vitro transfection experiments, the Y1102 allele accelerated channel activation, providing a plausible mechanism by which this variant may increase the likelihood of altered cardiac repolarization and arrhythmia. This finding was followed  up in a series of 289 sudden deaths in blacks by Burke and colleagues (Burke et al., 2005). Individuals were classified into four categories: 1) controls, 2) cardiac deaths with clear anatomical substrate, 3) cardiac deaths with no anatomical substrate except mild to moderate cardiac hypertrophy, and 4) unexplained cardiac arrhythmias. The frequency of the Y1102 allele was significantly higher in those with SCD in the absence of a  clear morphological abnormality (categories 3 and 4, combined n = 65). These findings strongly suggest that this allele is a risk factor for SCD in African Americans, but require confirmation in a larger cohort.

SCN5A variants have also been studied in non–African ancestry populations, yielding mixed results. An examination of all coding exons in 67 SCD cases with known coronary artery disease and 91 CAD controls in the Oregon Sudden Unexpected Death Study (primarily of European descent) found no association between coding variants and SCD risk (Stecker et al., 2006). In a Han Chinese population, the A1673G variant in SCN5A has been observed to modify risk for SCD (Chen et al., 2004; Fang et  al., 2008), though this SNP has not been found to be associated in other populations with SCD (Doolan et al., 2008; Stecker et al., 2006). Finally, in a sequencing study of SCN5A and four potassium-channel genes (KCNQ1, KCNH2, KCNE1, and KCNE2), Albert and colleagues (Albert et al., 2008) identified a significantly higher proportion of variants in SCN5A in women who died suddenly. An examination of common variants in these same genes in a nested case-control analysis of 516 cases and 1,522 matched controls of European ancestry demonstrated two intronic SNPs significantly associated with SCD, one in SCN5A and one in KCNQ1 (Albert et al., 2010). While clearly requiring additional replication, together these results  suggest that both common and rare variants in SCN5A may contribute to altered risk of SCD.

Thus, while several marginal associations have been reported for ion- channel genes, none of these results has been convincingly replicated in independent studies, and therefore they remain unproven. Indeed, SCD is likely to be the result of multiple pathways that contribute to increased susceptibility to arrhythmias, including atherosclerosis and thrombosis, electrogenesis and propagation, and initiating influences and triggers (Figure 21.1) (Spooner et al., 2001a; Spooner et al., 2001b). These pathways are likely to involve different genes, and thus extensive phenotyping of samples becomes important. In the absence of being able to distinguish SCD due to different underlying etiologies (e.g., structural defects vs. ion-channel defects), all samples will fall under the rubric of “SCD,” and the power to find genetic determinants is greatly reduced (Arking et al., 2004). Several studies are attempting to address this issue, including the Oregon Sudden Unexpected Death Study, which recruits samples through the emergency medical system and attempts to get electrocardiogram data on all SCDs and autopsy data when available (Chugh et al., 2003). There is also the need to obtain prospective data in order to assess attributable risk for SCD- susceptibility variants, and that effort is ongoing in the ARIC and CHS cohorts (ARIC-Investigators, 1989; Fried et al., 1991). In the  absence of large, well-phenotyped SCD cohorts, a great deal of focus has been on subclinical phenotypes, including the QT interval.

Figure 21.1 Potential genetic contributors to sudden cardiac death (SCD). Potential and documented elements of susceptibility are suggested in three broad pathways: 1) those that lead to progressive atherosclerosis and frank coronary disease and the likelihood of an occlusive infarction and ischemic arrhythmias; 2) those involved in electrogenesis and intromyocardial conduction pathways; and 3) those that may influence the initiation of aberrant triggering events and the perpetuation of an arrhythmia. Adapted from Spooner et al., 2001, and Arking et al., 2004.


The QT interval is a measure of cardiac repolarization and is subject to the joint control of the depolarizing Na+ (INa) currents, Ca2+ (ICa) currents,  and the repolarizing slow (IKs) and rapid (IKr) K+ currents. QT interval (corrected for heart rate) is a moderately heritable trait (25% to 52% heritability) (Busjahn et al., 1999; Carter et al., 2000; Newton-Cheh et al., 2005), and extremes of QT interval have been associated with increased risk for SCD in both Mendelian forms (LQTS and short QT syndrome [SQTS]), as well as in population-based settings (de Bruyne et al., 1999; Dekker et al.,  1994; Dekker et al., 2004; Elming et al., 1998; Okin et al., 2000; Schouten et al., 1991; Sharp et al., 1998). Taken together, these observations suggest that QT interval is likely to have a genetic component (moderate heritability), and that genetic variants that modify QT interval may also modify risk of SCD.


Testing the utility of endophenotypes to identify disease-related genes was a major motivation behind one of the first successful GWAS, in which the investigators identified a common variant in the 5′ region of the NOS1AP gene associated with a 2–3 ms increase in QT interval per minor allele. NOS1AP encodes an adapter protein that physically bridges neuronal nitric oxide synthase with its targets and modulator proteins. Specifically how NOS1AP variants modulate QT interval is currently unknown, but over- expression of NOS1AP in guinea pig ventricular myocytes results in shortening of the cardiac action potential, a decrease in the L-type Ca2+ (ICa) current, and a smaller increase in the rapid delayed rectifier K+ current (IKr), with a resultant shortening of the QT interval (Chang et al., 2008). The genetic association has been extensively replicated (Arking et al., 2009; Eijgelsheim et al., 2009; Lehtinen et al., 2008; Post et al., 2007; Raitakari et al., 2008; Tobin et al., 2008). In a follow-up study of NOS1AP with SCD in the combined Atherosclerosis Risk in Communities  Study and Cardiovascular Health Study cohorts (498 cases, 19,295 controls), Kao and colleagues (Kao et al., 2009) demonstrated that the NOS1AP SNP most strongly associated with QT interval, rs16847548, was associated with risk for SCD in white American adults, with the QT-prolonging variant associated with increased SCD risk (p = 0.002). Individuals homozygous for the risk allele were approximately 72% more likely to die of SCD than individuals homozygous for the non-risk genotype, even after adjusting for age, sex, and heart rate. It is important to note the risk allele is common, with 39% of the general white American population carrying one copy and 5% carrying two copies. These findings have been confirmed by a second independent study (Eijgelsheim et al., 2009).


In addition to QT interval, QRS (cardiac ventricular conduction) and RR (inverse heart rate) intervals have also been associated with cardiovascular mortality and SCD (Desai et al., 2006; Jouven et al., 2001). The  publication of GWAS identifying numerous variants associated with these traits (Eijgelsheim et al., 2010; Sotoodehnia et al., 2010) has allowed a more comprehensive assessment of the role of electrocardiogram (ECG)-associated SNPs in SCD risk. In one such study, using data generated from an SCD GWAS composed of 1,283 SCD cases and >20,000 controls, Arking and colleagues (Arking et al., 2011) examined 49 SNPs associated (p < 5 × 10–8) with QRS, QT, and RR intervals. In a test looking at direction of the genetic effect, the ECG-trait-prolonging allele was significantly more  often associated with increased risk of SCD (31 of 49, p = 0.03), with this effect almost entirely due to the QRS/QT-associated SNPs (28 of 40, p = 0.006). Three loci, including PLN, KCNQ1, and NOS1AP, showed nominal association with SCD, while a fourth locus, TKT/CACNA1D/PRKCD, was significant even after Bonferroni correction to account for the number of loci tested. The TKT/CACNA1D/PRKCD association is particularly intriguing due to the observation that the QRS-prolonging allele was protective for risk of SCD, which is counter to the effect observed with the measured trait (longer QRS duration is associated with increased risk of SCD). This result raises the possibility that the effect of the SNP variant on risk of SCD may not be mediated through its effect on QRS interval. A similar result was also seen in NOS1AP, where one of the alleles that were associated with SCD had no effect on QT interval (Kao et al., 2009).


While focusing on candidate genes and endophenotypes has yielded several compelling candidates, there is no doubt that additional genes play a role in SCD, and they are not likely to be identified through these approaches. Several GWAS with SCD as the phenotype of interest have been published, two of which have reported genome-wide significant findings. The AGNES (Arrhythmia Genetics in the Netherlands) cohort, which is composed of individuals with a first myocardial infarction and ventricular fibrillation (VF) who survived to hospital admission (n = 515) compared with individuals with myocardial infarction alone (n = 457), reported a SNP, rs2824292 in the 21q21 locus, with an OR of 1.78 (95% CI 1.47–2.13, p = 3.36 × 10–10),  and an OR of 1.49 (95% CI 1.14–1.95, p = 0.004) in a replication sample of 146 out-of-hospital SCD cases and 391 controls (Bezzina et al., 2010). This SNP is a common variant (allele frequency of 47%) located in an  intergenic region. The nearest gene, CXADR (~100 kb away), encodes a viral receptor implicated in viral myocarditis (Bowles et al., 1986), but it is not directly implicated by this study.


A second GWAS, comprising a total of 4,400 SCD cases and >30,000 controls, all of European ancestry, reported a significant signal at the 2q24.2 locus, with the strongest SNP, rs4665058 (p = 1.8 × 10–10), mapped to an intron of the BAZ2B gene. This locus contains three genes expressed in the heart but not previously known to play a role in cardiac biology (BAZ2B, WDSUB1, and TANC1). The risk allele had a study-size-weighted frequency of 1.4% and increased the risk for SCD by 1.92-fold per allele (95% CI 1.57– 2.34). Based on non-human primate sequence data, the risk allele is ancestral; thus its low frequency in European-ancestry populations suggests strong negative selection, as fewer than 0.8% of ancestral alleles have reached a frequency of 1.4% or lower. A search for missense/splice mutations correlated with rs4665058 (r2 > 0.8) using pilot 1 data from the 1000 Genomes Project (November 2010 release) (Durbin et al., 2010) was unsuccessful, indicating that the functional variant is probably regulatory in nature. The authors note that the meta-analysis consisted of both population- based and case-control studies, with some of the case-control studies using CAD controls as opposed to population-based controls. They thus suggest that the consistent results in studies with both CAD controls and population- based controls (see supplementary figure 2 from Arking et al., 2011) provide evidence that the risk associated with rs4665058 may be specific to SCD rather than a generic CAD risk factor. Somewhat surprisingly, this study did not replicate the 21q21 association seen in the AGNES cohort, despite adequate power. This lack of replication may reflect an association specific to the underlying population from which the AGNES cohort is drawn, or may be limited to the highly specific AGNES phenotype of individuals with a first myocardial infarction who survived a VF event, as opposed to a more broadly defined class of SCD observed in the general population.



One approach to identifying genetic determinants of complex traits has been to identify families that exhibit monogenic forms of the phenotype of interest, with the idea that any genes identified are likely to be involved in complex forms of the phenotype as well. This approach has several merits, including less phenotypical heterogeneity, since presumably all affected individuals in the pedigree are manifesting the same phenotype. Additionally, one can use traditional linkage analysis, which does not require recruiting a control population: however, if not done carefully, this can lead to both false positives and false negative results. This approach was adopted by Wang and colleagues (Wang et al., 2003), who studied a large family that displayed an autosomal dominant form of CAD. They performed genome-wide linkage analysis, and identified a significant association on chromosome 15q26. With 93 genes in the associated region, they focused on the transcription factor MEF2A, largely due to the overall expression pattern; MEF2A was expressed in blood vessels during mouse early embryogenesis (Edmondson et  al., 1994), and expression was similar to vascular endothelial growth factor receptor 2 (VEGFR2) and the Von Willebrand factor (Subramanian and Nadal-Ginard, 1996). These data led Wang and colleagues to speculate that MEF2A can be an early marker for vasculogenesis. They sequenced the gene in the affected individuals, and identified a 21-bp deletion in exon 11 (termed “Δ7aa”) that resulted in the loss of seven conserved amino acids and was present in all affected members in the family. They further went on to demonstrate altered cellular trafficking for the mutant protein, and that its ability to activate atrial natriuretic factor is reduced. In a subsequent study, the same group examined 207 CAD/MI patients for mutations in MEF2A, identifying three novel mutations in exon 7 in four patients, and no mutations in 191 controls (Bhagavatula et al., 2004). They demonstrated that these mutations significantly reduce the transcriptional activity of MEF2A, and suggest that “a significant percent of the CAD/MI population may carry mutations in MEF2A.”

The combination of both family- and population-based evidence would seem to present strong evidence for the involvement of MEF2A with CAD/MI. However, a subsequent study by Weng and  colleagues (Weng et al., 2005) raised some doubts about the strength of the effect of MEF2A variants on susceptibility to CAD/MI. They sequenced MEF2A exons in 300 white individuals with documented CAD with onset before age 55 years (men) or 65 years (women), and in 300 elderly controls (men >60 yrs, women >70 yrs) who did not have signs or symptoms of CAD. Of five missense mutations identified, one was unique to the CAD individuals, one was unique to the controls, and three were common to both groups. They  further observed the 21-bp deletion in three unrelated, unaffected individuals, and demonstrated that the deletion does not segregate with early CAD. They conclude that “these studies support that MEF2A mutations are not a common cause of CAD in white people and [we] argue strongly against a role for the MEF2A 21-bp deletion in autosomal dominant CAD.” A similar negative association was reported by Kajimoto and colleagues in a  Japanese population (Kajimoto et al., 2005) and by Gonzalez and colleagues (Gonzalez et al., 2006) in a Spanish cohort for the Δ7aa allele, though Gonzalez and colleagues reported a positive association for a rare Pro279Leu mutation (OR 3.06, 95% CI 1.17–8.06).

In an accompanying commentary to the Weng and colleagues paper, Altshuler and Hirschhorn (Altshuler and Hirschhorn, 2005) concluded that the role of MEF2A variants in CAD has not been established, and used the MEF2A studies to illustrate a number of criteria that should be imposed when performing similar studies. They note that in a complex disease like CAD, it may not be particularly uncommon to find large pedigrees that appear to display monogenic forms of the disease. Thus, they propose looking for an unusual phenotype shared by affected individuals, such as early-onset or a syndromic form of disease. The phenotype also needs to be consistent across family members, which can be difficult with CAD, in which some individuals are “affected” by virtue of having had prior coronary events, while others may have angiographically defined disease in the absence of events. Given these concerns, linkage across multiple families with identical ascertainment criteria should be observed.

In the event that a linkage signal in a region is detected, similar concerns arise when trying to identify the underlying functional variant. The observation of rare, potentially functional variants segregating within  a family is not uncommon, and any such variation observed in the linkage region will, by virtue of being in a disease-linked region, segregate with the disease. Thus, Altshuler and Hirschhorn propose that for a specific gene in a linked region to be associated with disease, it must meet one or more of the following criteria:

Multiple different mutations exist, each of which co-segregates with disease; There is confirmatory evidence for a particular allele in a case-control study; There are multiple rare variants that have been well ascertained in controls;

There is observation of a de novo mutation in an affected child (but not in his parent);

There is strong evidence of the effects of a human mutation in a model organism that recapitulates the human disease phenotype.

With the cost of genotyping decreasing and sample-collection increasing, the number of such studies being performed is on the rise; therefore, adopting rigorous criteria, as outlined above, for deeming a gene to be involved in complex disease is warranted.


Our understanding of the genetic landscape of cardiovascular disease has significantly changed in the last five years. We now have a long list of associated loci that have been robustly replicated in a number of different cohorts. However, the daunting challenge before us now is to uncover the underlying causal variant. There are many challenges that impede our ability to do so.

First, common genetic variants are of modest effect (OR <2), which has been a significant factor in the failure to identify and reproducibly demonstrate an effect for a given variant. Most studies have simply been underpowered, greatly contributing to the confusion in the literature. Indeed, as large studies with participant size soaring to over 200,000 become the norm, the list of reproducibly associated loci climbs steadily. Second, our ability to pick out genes involved in disease based on our limited understanding of biology is insufficient, especially given that half the  genes in the genome are of largely unknown function. Third, most genes that have been studied have not been studied in a comprehensive way, limiting our ability to compare across studies and to truly exclude genes (as opposed to a specific variant) from association with disease. Fourth, functional variants are likely to be influenced by sex and/or environment (e.g., smoking, diet); thus, comprehensively collecting data on study populations is essential. With genotyping no longer an impediment to progress, the focus going forward is likely to shift from collecting and assessing larger populations to identify additional genetic variants, to finding ways to interrogate the function of the linked loci, identify the underlying causal genes, and use this information to better predict general population risk for cardiovascular disease.


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