Despite the clinical heterogeneity of CAD and MI as described above, the phenomenon of familial clustering of CAD and collections of large pedigrees with multiple members in multiple generations provided an opportunity to perform linkage analysis and gene discovery. In recent years, three potential causal genes and their responsible mutations for pedigrees with apparent Mendelian autosomal dominant (AD) CAD and MI have been identified (below and Table 2). The common feature of these mutations is co-segregation with the phenotype in the index kindred and a presence in unrelated cohorts. Functional analyses of these genes also supported their potential involvement in the pathogenesis of CAD and MI. However, the potential roles of these familial CAD causal genes and mutations in general population are unknown. Further validation with additional pedigrees and experimental models is warranted.

MEF2A: MEF2A is a transcription factor that belongs to the monocyte enhancer factor (MEF) family. MEF2A is expressed in blood vessels during embryogenesis as an early marker of vasculogenesis and interacts with a variety of molecules that are known to be involved in cardiovascular pathogenesis. Wang et al[34] performed a genome-wide linkage analysis of a large kindred group consisting of 13 individuals and designated the first AD CAD gene (adCAD1) at chromosomal location 15q26 within a region containing approximately 93 genes including MEF2A. Considering the relevance of MEF2A in vasculogenesis, sequencing of the MEF2A gene revealed a 21-base pair deletion in exon 11 of the MEF2A gene that was present in all 10 affected living members but not in unaffected individuals[34]. This D7aa MEF2A mutation co-segregated with the CAD phenotype in an AD manner in the index pedigree. Interestingly, the same exon 11 deletion in MEF2A was reported in other CAD individuals from different ethnic backgrounds[35-37]. Missense mutations in exon 7 of MEF2A, resulting in a loss-of-function of MEF2A, were also found in premature CAD individuals, but not in age-, ethnicity- and BMI-matched controls lacking angiographic evidence of CAD[38,39]. Further studies have demonstrated that the deletion of 7 amino acid leads to defective trafficking of MEF2A in a dominant-negative manner. Consequently, MEF2A entry into the nucleus is blocked, which is crucial for the ability of MEF2A to regulate gene expression. It is plausible that functional abnormalities in D7aa MEF2A lead to cellular abnormalities in endothelial cells and vascular smooth muscle cells, which in turn participate in processes associated with atherogenesis. The discovery of the mutation in MEF2A in CAD resulted in significant hope of genetic testing for CAD. However, conflicting reports demonstrating the lack of an association between MEF2A gene mutations and CAD in other cohorts has raised doubts concerning a causal role of MEF2A in CAD[40-42]. In particular, two individuals carrying the D7aa MEF2A mutation did not appear to have CAD before the reported age of CAD development, while members of the same family who developed CAD carried the normal MEF2A gene[41]. However, potential genetic or environmental modifiers may reduce the phenotypic penetration. Polymorphisms that do not change MEF2A transcriptional activation are not associated with an increased risk of CAD[43], and the actual prevalence of functional MEF2A mutations in the general population is not yet known.

Low-density lipoprotein receptor-related protein 6: Mani et al[44] studied a group of extreme outlier kindred with an extraordinary prevalence of premature CAD presenting an almost uniform presence of hypertension, hypercholesterolemia, type II DM, obesity and an absence of cigarette smoking in affected individuals. The extreme familial clustering and segregation of phenotypes (both premature CAD and risk factors) was transmitted as a highly penetrant AD trait[44]. Genome-wide linkage analysis revealed strong linkage between the familial trait and markers of chromosome 12p, which spans 750 kb and contains only six annotated genes [ETV6, BCL2L14, low-density lipoprotein receptor-related protein 6 (LRP6), MANSC1, LOH12CR1 and DUSP16]. Sequencing of the candidate gene LRP6 revealed the causal variant, a missense substitution of R611C in a conserved EGF-like domain of LRP6. LPR6 functions as a co-receptor with Frizzle proteins for Wnt ligands. Functional studies of the LRP6_R611C mutation revealed a dominant-negative decrease in Wnt signaling. Although there was complete linkage of LRP6_R611C and hypertension, high low-density lipoprotein (LDL), TG and a prevalence of DM2 in the index kindred, the frequency of LRP6_R611C was very rare in general population. Recently, a polymorphism in LRP6 intron 2 was found to be associated with the presence and severity of angiographic CAD in a Chinese case-control study[45]. A common variant of LRP6, rs2302685 (a non-synonymous coding sequence SNP in exon 14, T>C, p Ile1062Val), was initially found to be associated with late-onset Alzheimer’s disease in Caucasians[46] followed by carotid artery atherosclerosis[47] and, very recently, CAD[48]. Ilr1062Val variant reduces Wnt/β-catenin signaling and anti-apoptotic activities in cultured cells and arterial walls. Additional missense mutations in the LRP6 gene that correlate to its extracellular domain have been identified by the sequencing of exons and the promoter of LRP6 in premature CAD patients and controls in different Chinese cohorts. All of these missense mutations cause loss of function (LoF) via reduced Wnt signaling activity and attenuated human umbilical vein endothelial cell proliferation in vitro[48].

CYP27A1: A large pedigree with well-defined familial traits together with massive parallel sequencing technology and bioinformatics computational and statistical tools has dramatically accelerated the pace of the discovery of disease-causing genes[49]. The identification of potential causative mutations in the CYP27A1 gene that co-segregated with a familial AD CAD phenotype is one recent example. Inanloorahatloo et al[50] performed whole-exome sequencing of two affected members in a large group of AD kindred with premature CAD. An in silico un-biased algorithm identified two candidate variants, c. G674A (p. Arg225His) in CYP27A1 and c. A241T (p.Ile81Phe) in NTRK2, which were further sequenced in all of the available members of the kindred. The variant c.G674A (p. Arg225His) in CYP27A1 co-segregated with the CAD status. The CYP27A1 gene encodes the sterol 27-hydroxylase involved in cholesterol and 25-hydroxy vitamin D3 synthesis. The amino acid p. Arg 225 that was affected by the identified variant is highly conserved in paralogous and orthologous proteins, suggesting its functional importance. This variant was not observed in 500 ethnically matched controls without a history of cardiac disease. Furthermore, an additional four disease-specific variants in the CYP27A1 gene were discovered by sequencing the CYP27A1 exons in 7 out of 100 unrelated CAD patients. Although disease-causing variants of the CYP27A1 gene were considered relatively rare, potential disease-causing variants reached up to 7% in Iranian patients with CAD. To date, potential CAD-causing CYP27A1 variants have not been reported in other populations, and the prevalence of these variants in the general population is unknown. The mechanism underlying the possible causal role of mutant CYP27A1 in atherosclerotic CAD remains uncharacterized. Recently, CYP27A1-deficient mice in an ApoE-deficient background exhibited a 10-fold reduction of aortic atherosclerosis after challenge with a high-fat diet associated with a 2-fold reduction of total plasma cholesterol, LDL, and very low-density lipoprotein (VLDL) as well as a 2-fold elevation of high-density lipoprotein (HDL). These results suggested that CYP27A1 regulates cholesterol homeostasis, and alterations of its activities may subsequently lead to atherosclerosis[51].

ST6GALNAC5: The ST6GALNAC5 gene is the newest addition to the group of causal genes for familial CAD. InanlooRahatloo et al[52] studied a highly inbred Iranian pedigree of AD premature CAD. Unbiased GWAS combined with whole-exome sequencing of two affected members identified a polymorphism, G295A, in the ST6GALNAC5 gene that resulted in a p. Val99Met mutation. Targeted sequencing of all of the available members confirmed the co-segregation between this variant and the CAD phenotype. A search of ST6GALNAC5 mutations in other Iranians with confirmed CAD revealed a p.*337Qext*20 mutation in two unrelated patients with CAD (2 out of 160). Interestingly, one of the patients who carried this p.*337Qext*20 stop-loss mutation had one sibling with CAD and two unaffected siblings; a genetic analysis of the family again showed co-segregation of the mutation with disease status. ST6GALNAC5 encodes sialyltransferase 7e, a member of the sialyltransferase family. Sialyltransferases add sialic acids (acetylated derivatives of neuraminic acid) to the termini of carbohydrate chains in glycoproteins and glycolipids. Elevated sialyltransferase activity in blood cells and serum sialic acid levels[53] are associated with atherosclerosis and CAD. In vitro functional studies of proteins encoded by these two mutated ST6GALNAC5 genes revealed a two-fold increase in sialyltransferase 7e enzymatic activity[52]. Given that: (1) an un-biased approach was applied in the identification of the ST6GALNAC5 mutation in a large CAD pedigree; (2) convincing evidence demonstrates the co-segregation of the ST6GALNAC5 mutation with the familial CAD/MI phenotype; (3) additional functional mutations have been identified in the ST6GALNAC5 gene in unrelated CAD/MI patients and families; (4) no functional variations were identified in affected family members and unrelated controls; and (5) the available evidence supports the notion that sialic acid and sialyltransferase activity are involved in the pathogenesis of atherosclerotic arterial disease, it is reasonable to conclude that gain-of-function mutations in ST6GALNAC5, such as p. Val99Met and p.*337Qext*20, are monogenic causal genes for CAD. The prevalence of functional ST6GALNAC5 gene mutations in the general population and in patients with CAD is unknown. The mechanism by which mutant ST6GALNAC5 causes atherosclerosis and CAD and its potential role in targeted therapy or in the prevention of CAD remain unknown.

Serum lipid levels, particularly elevated LDL cholesterol and HDL, are important risk factors for the development of atherosclerotic CAD. Mutations in genes involved in lipid metabolism have been identified and demonstrated to be causal for dyslipidemia and related atherosclerotic CAD and MI with variable penetration. These genes and their mutations in association with CAD and MI have been extensively reviewed in the literature[54] and are presented in Table 2. Depending on the primary abnormality in lipid metabolism and its effects on CAD, monogenic lipid disorders can be categorized into primary elevated LDL (LDL receptor, ApoB-100, PCSK9, and LDLRAp or ARH), a primary reduction of HDL (ApoA1 in primary hypoalphalipoproteinemia, ABCA1 in Tangier disease, and the lecithin:cholesterol acyltransferase (LCAT) gene in Norum disease and Fish-Eye disease), and primary elevated triglycerides (TGs) (LPL, ApoC-II in type Ib hyperlipoproteinemia and the ABCG5/8 gene in Sitosterolemia).

Genes and variants that are the primary cause of high LDL cholesterol: Mutations in genes encoding the LDL receptor, apolipoprotein B-100 (an LDL receptor ligand) and the pro-protein convertase subtilisin kexin type 9 (PCSK9) cause AD familial hypercholesterolemia (FH). Patients who harbor homozygous [low-density lipoprotein receptor (LDLR)] mutations (1 in 1 million) display a 6- to 10-fold increase in plasma LDL-C from birth and experience CAD/MI in early childhood. The early atherosclerosis observed in children who are homozygous for FH is not associated with any other risk factors that suggest that elevated LDL alone can produce atherosclerosis in humans. Carriers of heterozygous LDLR mutations, demonstrating a frequency of 1/500 in the general population, display a 2-fold increase in low-density lipoprotein cholesterol (LDL-C) levels from birth and are at risk to suffer CAD and MI at 30s years of age. Approximately 5% patients with CAD and MI under the age of 60 years carry heterozygous LDLR mutations. A total of 1741 sequence variants (1122 unique variants) have been recorded in the British Heart Foundation LDLR database[55] (http://www.ucl.ac.uk/ldlr/Current/summary.php?select_db=LDLR&show=sum). These mutations are present in the form of exonic substitutions, small exonic rearrangements, large rearrangements, promoter variants, intronic variants, variant sin the 3’ untranslated sequence, point mutations, splice site mutations, and large deletions. These mutations are equally distributed throughout the gene[56,57]. Genetic testing of all known LDLR variants is available. This test is often considered as the first step in a stepwise genetic analysis for FH followed by tests to assess the ApoB-100 and PCSK9 genes[58].

Apo B-100 is a unique protein component in lipoproteins originating from the liver (VLDL, IDL, and LDL). Apo B-100 is also required for the synthesis, assembly, and secretion of hepatic TG-rich lipoproteins, and it binds to heparin and various proteoglycans found in arterial walls. The most important function of Apo B-100 is to bind the LDLR via its LDLR-binding domain to mediate the clearance of LDL from plasma. Two mutations in the Apo B-100 gene, C10580G (p. Arg3527Gln)[59] and C10800T (p. Arg3531Cys), result in the alteration of LDLR binding affinity. In addition, these mutations cause familial ligand-defective hypercholesterolemia (OMIM 144010) and are associated with early atherosclerotic arterial disease[60]. The frequency in the unselected general population of the Arg3527Gln and Arg3531Cys mutations is approximately 1 in 500 and 1 in 3000, respectively. Most recently, by taking advantage of whole-exome sequencing and linkage analysis of an AD hypercholesterolemia pedigree, a third mutation (p. Arg50Trp) was identified[61].

Linkage analysis of two large French ADH pedigrees resulted in the identification of two mutations in the PCSK9 gene (1p34.1-1p32), which encodes a protein that is also known as a neural apoptosis-regulated convertase 1 (NARC-1)[62]. A total of 9 gain-of-function mutations in PCSK9 genes in families with ADH have been reported[63]. These mutations cause a decreased number of LDLR, elevated levels of serum total and LDL cholesterol, and phenotypes of tendon xanthomas, premature CAD, MI and stroke. SNP rs11206510 (risk allele T) located in the PCSK9 gene is also associated with an increased risk of CAD and MI in an unbiased GWAS study[64]. However, loss-of-function mutations in PCSK9 identified by exome sequencing of individuals with extremely low LDL levels in the Atherosclerosis Risk in Communities study (ARIC) and Dallas Heart Study cohorts revealed that these mutations led to hypocholesterolemia and were protective against CAD and MI[65]. Secreted PCSK9 protein functions as an LDLR chaperone, binds to the EGF-A domain of the LDLR, decreases receptor recycling to the cell surface and promotes lysosomal degradation. Although the contribution of the PCSK9 gain-of-function mutation in ADH is rather small (< 3%), elucidation of the PCSK9 gain-of-function in ADH has shed light on the potential for the development of cholesterol-lowering agents by reducing the circulatory level of PCSK9 (PCSK9 inhibitors). These discoveries have resulted in the development of PCSK9 inhibitors as novel cholesterol-lowering agents.

Approximately 50 individuals of Mediterranean or Middle Eastern origin carry homozygous mutations in the autosomal recessive hypercholesterolemia (ARH) gene. ARH (1p34-1p35) was subsequently cloned and named LDL receptor adaptor protein 1 (LDLRAP1), which encodes a phosphotyrosine-binding domain protein and is required for LDLR internalization in hepatocytes. Two mutations, 432insA in exon 4 causing FS170Stop (ARH1) and the nonsense mutation G65A in exon 1 (p. trp22ter), account for most of the known cases of ARH in Sardinia[66]. The third mutant was a result of an ancient recombination between ARH1 and ARH2. In addition, 4 Italian ARH individuals from the mainland carried homozygous ARH1. Overall, ARH mutations are rare[67].

Genes and variants that are primary causes of low HDL cholesterol: Approximately 40% of patients with CAD have a low level of high-density lipoprotein cholesterol (HDL-C; < 40 mg/dL per current guidelines or age and sex-adjusted plasma HDL-C levels below the 10^th percentile). Prospective cohort studies also suggest that low HDL-C is a significant, independent risk factor for CAD. An estimated 50% to 70% of the variations in HDL-C in the human populations are due to genetic factors, and the majority remain undefined[68].

Apolipoprotein A1 (Apo AI) is the major apolipoprotein in HDL-C and is a key determinant of the levels and metabolism of HDL-C. Apo AI functions as a cofactor for LCAT, which is responsible for the formation of most cholesterol esters in the plasma. Apo AI also promotes the efflux of cholesterol from cells. ApoAI mutations cause AD familial hypoalphalipoproteinemia. The homozygous loss of ApoAI leads to a complete absence of Apo AI and HDL-C levels < 5 mg/dL with normal LDL-C and TG levels. Heterozygous LoF Apo AI carriers have HDL-C levels that are approximately 50% less than normal HDL-C levels. ApoAI gene polymorphisms are associated with decreased HDL and an increased risk of premature CAD[69]. Yamakawa-Kobayashi et al[70] analyzed sequence variations in the ApoAI gene in Japanese children with low levels of HDL (below the first percentile in the general population) and found 3 frameshift and 1 splice site mutation with possible deleterious effects. They estimated the frequency of hypoalphalipoproteinemia due to ApoAI mutations to be 6% in subjects with low HDL cholesterol and 0.3% in the general Japanese population[70]. The A164S variant of the ApoAI gene identified by sequencing of the ApoAI gene in 190 Copenhagen City Heart Study participants predicts an increased risk of IHD [hazard ratio (HR) 3.2, 95%CI: 1.6-6.5], MI (5.5, 95%CI: 2.6-11.7) and overall mortality (2.5, 95% CI: 1.3-4.8). Despite comparable levels of plasma lipids and lipoprotein, including HDL-C and ApoAI, in A164S heterozygotes, heterozygous A164S carriers exhibit a decrease in survival by more than 10 years (P < 0.0001) compared with non-carrier controls[71,72]. In addition, two ApoAI variants (ApoAI_Paris and ApoAI_Milano) were associated with a reduced risk of CAD, suggesting the occurrence of cardioprotective effects[73].

The ATP-binding cassette transporter (ABCA1) is involved in the initial phase of reverse cholesterol transport and the egress of free intracellular cholesterol and phospholipids from extrahepatic cells. Homozygous LoF ABCA1 variants are causal factors for the rare Tangier disease, which results in extremely low HDL-C levels, a 40% reduction of LDL-C compared with the general population, and an increased risk of early CAD[74,75]. A total of 200 LoF mutations in the ABCA1 gene have been reported (http://www.hgmd.cf.ac.uk/ac/gene.php?gene=ABCA1, last accessed on August 6, 2014). Heterozygous carriers of the ABCA1 mutation exhibit an approximately 50% reduction in HDL-C without alterations in the levels of LDL-C and an increased risk of premature CAD[76]. The frequency of heterozygous carriers of ABCA1 mutations is estimated as approximately 3:1000 in the general population[77]. The R219K polymorphism in the ABCA1 gene is associated with a reduced risk of CAD, suggesting that this polymorphism provides protective effects against the disease[78].

LCAT catalyzes the esterification of free cholesterol with acyl groups derived from lecithin as an essential step in the maturation of HDL-C. Homozygous LoF in the LCAT gene causes rare autosomal recessive Norum disease with very low HDL (< 5^th percentile), elevated TGs and decreased LDL-C. Greater than 80 genetic variants in the LCAT gene have been identified and reported to be associated with 29% of the individuals with low levels of HDL-C in the Netherlands[79]. However, the association between low levels of HDL-C caused by LCAT deficiency and an increased risk of CAD is not as certain as the associated risk of ApoAI[80]. This phenomenon may be explained by the observation that LCAT deficiency mainly causes decreased levels of ApoAII. HDL particles containing ApoAI, but not ApoAII, possess “anti-atherogenic” effects.

Genes and variants that are primary causes of elevated TGs: Plasma TGs predominantly occur in the form of intestinally synthesized chylomicrons (CMs), remnants in the postprandial state and hepatically synthesized VLDL in the fasted state. Plasma TG levels are a polygenic trait and is influenced by environmental factors and lifestyles, i.e., diet, physical activities and tobacco use. Large epidemiological studies have demonstrated that plasma TG concentrations are a strong independent risk factor for CAD[81].

Lipoprotein lipase (LPL) is the rate-limiting enzyme in converting VLDL to LDL. Homozygous LoF mutations in LDL genes cause LPL deficiency in rare (approximately 1 in a million) AR type I hyperlipoproteinemia characterized by marked hypertriglyceridemia with a decrease in HDL and LDL, eruptive xanthoma, hepatosplenomegaly, recurrent pancreatitis, and in some cases, premature atherosclerotic arterial disease. Greater than 100 LoF variants have been identified[82]. Sequencing data have suggested that rare LPL variants are actually common in patients with elevated TG levels[83]. Variants of LPL genes that are negatively associated with plasma levels of TG, positively associated with HDL and inversely associated with a risk of CAD have also reported, suggesting potential protective genetic variants against CAD[84]. ApoC-II is an activator of LPL. A homozygous LoF ApoC-II deficiency results in rare AD type Ib hyperlipoproteinemia with extremely elevated TG and chylomicron levels in the plasma, causing recurrent pancreatitis and, in some cases, ApoCII_{St. Michael} (Gln70Pro)[85] and other conditions[86], leading to premature ischemic vascular disease. The significance of these mutations in general population remains to be explored.

Sitosterolemia is characterized by hyperabsorption and the retention of dietary cholesterol and sterols, including plant and shellfish sterols, leading to high levels of plant sterols in the plasma, the development of tendon and tuberous xanthomas, accelerated atherosclerosis, and premature CAD. LoF mutations in ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2) cause sitosterolemia. All of the probands identified in the sitosterolemia pedigree have homozygous mutations in either ABCG5 or ABCG8[87]. The prevalence of ABCG5 and ABCG8 heterozygous carriers and their effects on cholesterol metabolism and atherosclerotic disease in the general population remain unclear.

Monogenic traits and their causal genetics only explain a small proportion of the genetics of CAD and MI. Prior to the completion of human genome sequencing, CAD and MI gene discovery largely employed pedigree-based linkage analysis and positional cloning with the limited availability of genomic markers. The Human Genome Project, HapMap project, and 1000 Genomes Project provided a reference of 3.2 billion nucleotide base pairs of the human genome and 3 million single nucleotide polymorphisms (SNPs) distributed throughout the genome. These SNPs serve as high-density genomic markers for the entire genome. The developments of high-density microchips containing millions of SNPs, high-throughput analytic technology, and powerful biostatistics data mining tools have permitted genome-wide association studies (GWAS). GWAS is a non-hypothesis-driven, unbiased analysis of the potential associations between traits of interest (disease, phenotypes, etc.) and genomic markers (SNPs) consisting of tens of thousands of cases and controls[88]. In 2007, SNPs located in 9p21 were identified as strongly associated with CAD and MI based on the results of four nearly simultaneous publications. Numerous GWAS studies have been subsequently conducted, involving tens of thousands of CAD and MI cases and controls inclusive of a large spectrum of demographic, geographic and ethnic backgrounds. The largest meta-analysis of GWAS data reported by the CARDIoGRAMplusC4D Consortium included a total of 63746 CAD cases and 130681 control subjects and confirmed/identified 46 CAD susceptibility loci[84]. Together with a 6q21 locus that was identified in the Chinese Han population by Wang et al[89] and an additional 3 CAD susceptibility variants identified by IBC 50K[90], which were not confirmed in the CARDIoGRAMplusC4D Consortium study, a total of 50 GWAS-identified CAD susceptibility genomic loci were reported. Recent reviews have focused on GWAS discovery of CAD and MI susceptibility loci[91-95].

The journey to understand genetic/genomic architecture of CAD and MI continues with steady progress. Figure 2 provides a complete map of the genes and loci that are reported in literature to be causal for, susceptible to, or associated with the risk of CAD and MI identified by either linkage analysis and/or association analyses with either candidate-gene or genome-wide approaches. The genes and mutations that are potentially causal for monogenic CAD and MI families are also presented in Table 2.

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Figure 2 Chromosome map of genes reported to be causal for, susceptible to and associated with coronary artery disease and myocardial infarction in the literature. Genes in red are causal genes for monogenic familial CAD and MI. CAD: Coronary artery disease; MI: Myocardial infarction.

The ultimate goals of the study of the genetics of CAD and MI are to reveal genes and their products involved in the development of CAD and MI, understand the molecular and cellular pathophysiology and subsequently establish risk stratification strategies to direct prevention and also develop effective therapeutic approaches. Despite the total of 147 genes (Figure 2) with variants that are causative for or associated with CAD and MI, these genes only explain less than 20% of the heritability of CAD and MI. Furthermore, the biological functions and pathophysiological roles of most of the gene variants and genomic loci are not fully understood. Early attempts to use genetic risk markers of CAD to predict long-term outcomes were not successful, and many challenges remain before the ultimate goal of understanding the genetics of CAD can be realized.

Searching for unexplained heritability: GWAS is based on the hypothesis of a “common disease, common variant”. The sensitivity of GWAS in detecting a significant association between genetic variants and traits is limited to high frequency variants (5%)[96]. Regarding CAD and MI, most of the GWAS-identified variants individually or in combination confer relatively small increments in risk (1.1- to 1.5-fold) and explain only a small proportion of heritability. The well-recognized sources for the missing heritability of complex traits[97-99] include: (1) larger numbers of variants with a smaller effect that remains to be identified; (2) a low minor allele frequency (0.5%-5%) or rare variants (< 0.5%) with a larger frequency that are not detectable using the available arrays; (3) structural variants (i.e., copy number of variants due to insertion or deletions, inversions, or translocation) are poorly captured by SNPs; (4) a low power to detect gene-gene interactions; and (5) an inability to detect gene-environment interactions using the current GWAS methodology. Strategies have been suggested and explored to overcome these pitfalls, including: (1) analyzing phenotypically well-defined cases and controls[100], increasing the numbers of participants[84], and utilizing extreme phenotype groups[54,101]; (2) developing powerful biostatistics tools to enrich the signal and detect sensitivities and to capture the additive effects of variants, gene-gene interactions and rare variants[102,103]; (3) integrating available functional information, i.e., eQTLs, protein structure/function predictions, and known pathways and networks related to the traits, to prioritize GWAS signals[104] and performing integrative analyses[105]; (4) customizing fine mapping SNPs or use next-generation sequencing regions of interest to capture rare variants and structural variants; and (5) considering the epigenomic regulation of gene expression.

Many current phenotypes are subjectively measured and may represent numerous underlying biological processes. Misclassifying a phenotype can reduce power in GWAS relative to expectations based on power calculations of idealized homogeneous populations. Strong genotypic effects that are important in a small homogeneous subgroup could have a small or even negligible effect within an entire population[106].

Functional annotation of known genes and variants: Numerous genes and variants associated with CAD and MI have been explored in the last two decades. Candidate gene approaches (positional cloning, linkage analysis, and candidate-gene association analysis) provide a direct link between the pathogenesis of CAD/MI and candidate genes. Variants or chromosomal loci identified by GWAS however do not associate with specific genes or pathways. Only one-third of the 45 CAD loci reported in the largest CAD and MI GWAS study contain a known functionally relevant candidate gene[84]. The 9p21 locus is the CAD locus discovered by GWAS and remains the strongest association with CAD in the human genome. However, the SNPs defining the 9p21 association with CAD are all located in intergenic locations rather than in coding or regulatory regions. Functional annotation of the 9p21 locus in association with CAD has focused on the two closest protein-coding genes, CDKN2A and CDKN2B, and an additional CDKN2B antisense noncoding RNA (ANRIL). The systematic functional annotation of these CAD and MI gene variants and loci will provide insight regarding the pathophysiology of the disease. These functional studies will require a combination of tissue, cell and animal model systems as well as assessments at the levels of gene expression, protein modification and metabolism[107,108].

Protective genetic factors against CAD and MI: Atherosclerosis is a multi-decade pathological process. A simplified pathologic process (Figure 3A) includes: (1) endothelial injury; (2) lipid particle deposition (fatty streak formation); (3) local cellular and inflammatory responses (early atheroma formation). The process is followed by (4) atheroma progression with the formation and expansion of the necrotic core, fibrous cap, matrix accumulation and various degrees of plaque instability; and (5) intertwined with various degrees of thrombosis formation[109]. The standard human genetic approach to CAD involves searching for genetic risk factors or susceptibility genes for the disease. Could genetic factors be protective against atherosclerosis and CAD and MI? Protective genetic factor against CAD and MI may antagonize the effects of genetic risk factors and potentially explain a portion of the missing heritability. The presence of protective genetic factors will alter the predictive value for CAD and MI using risk variants. The identification of protective genetic factors may shed light on the mechanisms of CAD and MI and further guide the development of strategies or therapy.

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Figure 3 The balance between athero-protective (A) and pro-atherogenic (B) factors involved in the development of atherosclerosis. Obstructive atherosclerotic arterial disease results from the loss of balance between these two factors. A: A list of factors and processes that provide protective effects against atherogenesis; B: A list of factors and processes that promote atherogenesis. HDL: High-density lipoprotein; LDL: Low-density lipoprotein.

Clinical practice often encounters individuals of an advanced age with multiple TRF for CAD and MI without demonstrated angiographic evidence of CAD. The offspring of centenarians have a significantly reduced rate of cardiovascular complications[110]. The current understanding of the pathophysiology of atherosclerosis and CAD and MI supports the notion that the development and progression of atherosclerosis is an accumulating effect of the imbalance between athero-protective and pro-atherogenic processes (Figure 3B). Approximately 500 genes reported in the literature have been tested for their effects on atherogenic processes in atherosclerosis-prone mouse models (ApoE deficiency or LDL receptor deficiency with a high-fat diet) with transgenic (gain-of-function), knockout (LoF) or both genetic modifications. LoF mutations in approximately half of these genes accelerate atherosclerosis in a mouse model, whereas gain-of-function mutations significantly reduced atherosclerosis. It is plausible to suggest that these genes normally function as protective factors against atherosclerosis. In contrast, the remaining half of these reported genes exert pro-atherogenic effects that are normal or consistent with their gain-of-function mutations[111] (a complete list of these genes is available from the authors upon request).

Most genomic association studies have been designed to identify CAD/MI susceptibility genes or polymorphisms. The largest genetic study to assess the impact of common genomic variation on the risk of CAD reported a total of 45 CAD susceptibility loci and an additional 104 likely independent SNPs that were associated with an increased risk of CAD and MI, explaining approximately 10.6% of the heritability. No protective variants were reported[84]. Candidate gene-based association studies identified polymorphisms that are significantly associated with a reduced risk of CAD and MI (Table 3). However, the results obtained for many of these potentially protective genetic loci against CAD are conflicting.

HDL provides protection against atherosclerotic CAD partially through its anti-oxidative effects. Serum paraoxonase is responsible for most of the antioxidant properties of HDL. Human paraoxonase is encoded by the family of PON1, PON2 and PON3 genes. Low serum PON1 activity is associated with an increased risk of CAD and its severity[112,113]. Many candidate gene association studies have revealed that PON1 (Leu55Met, Gln193Arg) and PON2 (Ser311Cys) polymorphisms are associated with the risk of CAD[114] and its angiographic severity[115]. For example, an association study in a single center of consecutive patients who underwent coronary angiography revealed a significant dose-dependent association of the PON1 genotypes (192 Q/R) and serum PON1 (QQ192 > QR192 > RR192) as well as an inverse association with systemic indices of oxidative stress. In addition, 192 Q (QQ and QR) was associated with a decreased risk of cardiovascular and all-cause mortality[116]. The PON1/PON2 haplotype comprising M55, Q1192 in PON1 and Cys 311 in PON2 is associated with a significant protective effect against the risk of MI[117]. PON1-deficient mice in an ApoE^-/- background fed a high-fat diet exhibit significantly exaggerated atherosclerosis compared with ApoE^-/- mice carrying the wild type PON1 gene[118,119]. Germline transgenic or transient adenoviral vector-mediated overexpression of atheroprotective PON1 (55L/192Q) in ApoE^-/- mice revealed protective effects against atherosclerosis with ApoE^-/- without transgenic PON1[120,121]. Multiple layers of evidence suggest that genetic polymorphisms in PON1 and PON2 lead to an increase in serum paraoxonase activity that may provide protective effects against CAD. However, the frequency of these variants in the general population remains to be determined.