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Identification of genetic roots of coronary artery disease by combining stepwise genome wide association studies with transcriptomic and functional genomic investigation of relevant genetic variants

Final Report Summary - CARDIOGENICS (Identification of genetic roots of coronary artery disease by combining stepwise genome wide association studies with genomic investigation …)

The heart is the organ that works non-stop to keep the blood circulation going. Its own oxygen needs are supplied by blood vessels that form a crown at the 'top' of the heart, hence the name Coronary artery disease (CAD) for constrictions in these arteries. Most often, these constrictions are caused by atherosclerosis, a condition when plaques of cholesterol and cells obstruct the blood flow. In the European Union (EU), CAD is the leading cause of disability and death. Despite significant advances in the management of CAD and its major complication, heart attack, the burden of disease continues to increase, particularly in the Eastern European countries. Worldwide, CAD is predicted to be the leading cause of morbidity and mortality by 2020. The CARDIOGENICS project aimed at discovering genetic variations leading to CAD and Myocardial infarction (MI), thereby uncovering the underlying disease mechanisms and helping develop new treatments. However, without the support of numerous heart patients and blood donors, whose samples were used by researchers to perform tests, these goals would remain unattainable.

Atherosclerosis is a complex disease, caused by multiple genetic and environmental factors. Likewise, a multifactorial etiology applies to many of the underlying cardiovascular risk factors including hypercholesterolemia, hypertension, diabetes mellitus, and smoking addiction. Thus, endogenous (genetic) and exogenous (nutrition, physical activity, therapy etc.) mechanisms may affect the manifestation of atherosclerotic lesions either directly or indirectly via modulation of traditional risk factors. On a cellular level atherosclerosis is also complex characterised by endothelial dysfunction, lipid and matrix accumulation, Smooth muscle cell (SMC) proliferation and migration, calcification, inflammation, and, finally, thrombus formation. In this scenario, the potential involvement of genetically modulated mechanisms is indicated.

For long, evaluation of the family history served as a guide to approach a patient's genetic risk for coronary events. Beyond the information conferred by a positive family history, identification of the underlying gene defects is thought to improve risk prediction and the knowledge of pathogenetic mechanisms.

Consequently, over the past three decades, a great deal of research has focused on defining such genetic components of myocardial infarction, atherosclerosis and its risk factors. The hope for the future is that knowledge of the genes and gene variants will lead to improvements in the diagnosis and treatment of coronary disease. Indeed, emerging data suggest that some of these gene variants identified over the last years allow improve genetic risk prediction with sufficient reproducibility for the clinical setting. Initially this research focused on candidate genes that hypothetically might affect known traits involved in the atherosclerotic process including the reninangiotensin system, lipoprotein metabolism, inflammation or coagulation. Many of these attempts failed replication in consecutive studies. Another difficulty in this research was that, unlike Mendelian traits, genetic studies on complex cardiovascular disorders are complicated by variable cosegregation between the risk allele and the disease. In fact, many genetic variants associated with the disorders were found to be relatively common in the overall population and thus - albeit to a variable degree - prevalent in both healthy and affected individuals.

Subsequently, genome wide linkage analysis searched without a priori hypothesis for chromosomal regions shared in family members with myocardial infarction. While this approach allowed identification of several chromosomal regions harbouring myocardial infarction genes, it proved to be difficult to precisely define these.

However, success came with Genome wide association (GWA) studies that most recently identified multiple gene variants reproducibly associated with coronary heart disease, hypercholesterolemia, or diabetes mellitus. Surprisingly, most of the genes identified thus far were not expected to play a role in the development of atherosclerosis. Thus, an important task for the immediate future is to understand the fundamental pathophysiological mechanisms affected by these genes in the development of atherosclerosis. Accordingly, functional information on these genetic factors and related gene expression as well as protein expression patterns is very much in need. Subsequently, genetic research may enhance diagnostic testing and development of new treatment targets.

In 2007, just at the beginning of CARDIOGENICS, three GWA studies successfully identified variants on chromosome 9p21.3 to be strongly associated with risk of coronary artery disease and myocardial infarction. Since then, similar studies have firmly associated about 32 chromosomal loci all of which increase the susceptibility to coronary disease.

This 'gold rush of genomic discovery' was preceded by identification of the full human DNA sequence, followed by the cataloguing of common Single nucleotide polymorphisms (SNPs) at these bases by SNP consortium, and development of arrays that allow simultaneous typing of millions such SNPs at relatively low cost. Based on these assets, GWA studies compared the allele frequency of SNPs across the entire genome, with dense coverage of all chromosomes, between cases and controls. For high resolution and an unbiased view on the entire genome thousands of individuals had to be studied in this fashion. In order to meet this goal the research community formed international consortia and shared information for subsequent meta-analyses. Such joint effort ultimately resulted in successful identification of multiple genetic variants that are associated with an increased susceptibility to coronary disease (so-called risk alleles).

Genome-wide arrays preferentially contain SNPs that are frequently found in a population as those offers the highest statistical power to detect association. Accordingly, almost all currently identified risk alleles for coronary disease are common. For example, in an individual of European descent the probability to carry one or two risk alleles at the chromosome 9p21.3 locus is 51 and 27 %, respectively. Thus, only 22 % of Europeans are free of this specific genetic risk factor for myocardial infarction. Given the large number and the high frequency of risk alleles that have been identified thus far, virtually every person in our population carries multiple genetic variants that increase susceptibility to coronary disease.

Each risk allele increases the probability of coronary disease only by a relatively small margin, i.e. 5-15 relative percent per allele. There are two exemptions. One is a rare allele on chromosome 6q25.3 that markedly increases lipoprotein (a) levels and coronary disease risk by 54 %. The other one is the relatively frequent one on chromosome 9p21.3 which increases risk by 29 % per allele.

SNPs that are associated with a disease in a GWA study are not necessarily causal. Rather, they mark a small region on a chromosome (a linkage disequilibrium block), in which such causal variant is likely to be located. Thus, a challenge that immediately follows the discovery of association between a SNP and a disease is the search for the affected gene and the causal variant(s) at the chromosomal locus. Studies that link SNPs with expression levels of genes (eQTL analysis) had been instrumental in this respect. The findings from these studies (conducted within CARDIOGENICS) suggested that at least some of the risk alleles affect expression of nearby genes in a quantitative and tissue-specific fashion and thereby modify the risk of coronary disease.

The rapidly growing list of chromosomal regions and genes associated with the risk of coronary disease is surprising in many aspects. Firstly, some of the strongest genetic effects were found in regions without known protein-coding genes. Secondly, only a minority of genetic variants showing clear association with coronary disease were also found to be associated with one of the traditional risk factors. Thirdly, in regions where annotation of the association signal to a specific gene was possible, only a few of these genes had previously been implied in the pathogenesis of coronary disease (ADAMTS7, COL4A1/A2, etc.). Finally, some of the loci did not only affect coronary disease risk but also multiple other, seemingly unrelated phenotypes (pleiotropy).