| There is ample interest in the genetic epidemiology of coronary heart disease (CHD). Completely ignored, however, is its epigenetic epidemiology despite the fact that epigenetic mechanisms control the expression of the human genome. We hypothesise that epigenetic variation significantly contributes to the risk of CHD both directly by causing aberrant gene expression and indirectly through masking or exposing DNA sequence variation. To powerfully test the role of epigenetics in CHD, we have devised a novel framework that is effective, feasible and well-founded in recent literature. Our main interest is DNA methylation, which currently is one of the best-understood epigenetic mechanisms and reflects other epigenetic layers. Crucial is our focus on epigenetic changes induced in utero. These changes arise well before the onset of (sub-clinical) CHD allowing strong causal inferences. Moreover, epigenetic changes are heritable and, if induced early in development, they will be propagated throughout the adult body. Hence, readily available lymphocytes effectively mark epigenetic states of loci in other non-accessible tissues that directly influence disease aetiology. This principle has now been proven for multiple loci and enables large-scale applications in human subjects. We designed a 3-phased framework exploiting this principle to for the first time test the effect of epigenetic variation on CHD risk. In a discovery phase, we will identify epigenetic variation induced early in development either by genetic factors or by prenatal malnutrition using two tailored epidemiological studies. The former will be identified using twin studies; we already successfully identified the IGF2 locus. The latter will be detected by comparing subjects who were prenatally exposed to famine with their unexposed siblings (Dutch Hunger Winter Study). We assembled a first set of candidate loci implicated in CHD risk and suggested to have an epigenetically labile state. These will be scrutinised using a novel mass spectrometry-based method, fully operational in our lab, enabling high-throughput quantitative measurements of the DNA methylation of single CpGs. In addition, we will employ a genomic approach using CpG island arrays enabling hypothesis-free discoveries. In the next phase, epigenetic variation identified in phase 1 should pass two tests. First, the variation should be stable during ageing and consistent over tissues so that lymphocytes are indeed valuable markers. This will be tested in unique material: within the twin study, participants donated blood and buccal cells over a 10-20 years. Second, the epigenetic variation should be functional. To this end, Taqman gene expression studies will be performed. Epigenetic variation passing both tests from phase 2 will be studied in phase 3 for clinical relevance to CHD in a case-control (500/500) study nested in PROSPER. To optimally extract predictive patterns, an integrated assessment will be made of both epigenetic and genetic variation at the loci using a battery of data-mining tools. If successful, our study will pave the way for large-scale, integrated assessments of epigenetic and genetic variation, which is not being made in current epidemiological studies, despite the fact that both can be measured using the genomic DNA available. Importantly, epigenetic variation, in contrast to DNA sequence variation, is amendable to therapeutic treatment, opening the prospect of new preventative strategies. Objectives We will test the hypothesis that epigenetic variation induced early in development contributes to CHD risk in three phases: 1. Discovery - Loci whose epigenetic state is vulnerable to undergo a change in utero are thought to be common in the human genome. Our first goal is to establish a set of such loci using differential exposure to prenatal malnutrition and genetic influences as read-outs. Clearly, no Internet resource does exist yet for epigenetic variation as it does for e.g. SNPs. 2. Depth Loci identified in phase 1 should meet three criteria to guarantee that studies aimed at testing their relevance for CHD in humans are feasible. Epigenetic variation should be (i) stable over time, (ii) correlated across tissues and (iii) influence gene expression. This ensures that DNA from lymphocytes reliably marks epigenetic variation as well as biological relevance. 3. Clinical relevance Loci meeting the criteria of phase 2, will be tested in a large, prospective epidemiological study for their association with CHD. Of note, establishing clinical relevance of epigenetic variation is our primary objective and will be our priority throughout the project. As soon as an individual promising locus is identified in the preceding phases, it will immediately be tested in phase 3. Relevance for cardiovascular diseases Establishing risk profiles is a major theme in cardiovascular research into preventative strategies. Currently, such profiles predominantly encompass genetic risk factors and plasma markers. Completely ignored, however, is epigenetic variation, which is indicated to be a major contributor to inter-individual differences in gene expression. Risk-profiles will gain significantly in predictive power if this level of biological information is also included. This is particularly expected since the effect of widely studied SNPs may very well be masked or exposed by the superimposed epigenetic state of the locus. In our proposed study we will develop such risk-profiles focussing on CHD endpoints using a large epidemiological study. Apart from better prediction and increased understanding of the biological processes underlying the development of CHD, epigenetic epidemiology spurs on new preventative strategies. Epigenetic variation, in contrast to DNA sequence variation, is amendable therapeutic intervention. Several drugs targeting DNA methylation are under development and the first one treating a rare disease is on the market (Azacitidine). In addition, a simple measure as methyl (e.g. folate) supplementation is indicated to influence epigenetic patterns and, in animal studies, supplementation was shown to even reverse deleterious epigenetic changes induced early in development. Keywords |
