This resulted in a set of 796 genes, with at leas one significant CpG site, that where entered into the enrichment analysis. muscle cell differentiation. This study show that GDF-15 levels are associated with differences in DNA methylation in blood cells, and a subset from the loci are also differentially methylated in participants with MI. However , there might be interactions between GDF-15 levels and methylation in other tissues not addressed in this study. These results provide novel links between GDF-15 and cardiovascular disease. == Introduction == Growth-differential factor-15 (GDF-15) is a member of the transforming growth factor- (TGF-) family members (1). Under physiologic PD 334581 conditions, GDF-15 is expressed in low to moderate levels in most healthy tissues. GDF-15 levels may increase in response to pathological stress associated with inflammation or tissue damage and an overexpression continues to be found in several malignancies including breast, melanoma, colorectal, pancreatic and prostate cancer (24). Growing evidence indicate that the GDF-15 level in plasma may be a new biomarker intended for risk stratification and therapeutic decision making in cardiovascular disease (5, 6). GDF-15 is expressed in the myocardium of patients with acute myocardial infarction (MI)(7) and has been shown to be increased in blood from patients with acute MI (8). Raised GDF-15 levels has also been associated with risk of mortality in patients with acute coronary syndrome (ACS) or chronic heart failure (9). GDF-15 levels are also higher in healthy individuals older than 65 years than in more youthful individuals (10). Epigenetics is generally used to denote the regulation of genes that cannot be attributed to the DNA sequence. It includes a number of different mechanisms of which PD 334581 DNA methylation and histones modifications being the most commonly analyzed. DNA methylation is the heritable and reversible attachment of a methyl group to a nucleotide. In mammals, DNA methylation appears to be specific PD 334581 to cytosine, predominantly to CpG (cytosine-phosphate-guanine) dinucleotide. In promoter regions, CpG-sites in many cases are found because clusters called CpG islands. Methylation of promoter CpGs can expose stable changes in gene expression, which might lead to silencing of that gene (11, 12). In this study, we performed a genome-wide DNA methylation study to determine the relationship between GDF-15 levels and DNA methylation. DNA methylation status in blood cells has been decided at more than 475, 000 sites distributed throughout the genome in two independent populace based study cohorts. == Results == == Genome-wide DNA methylation study == DNA methylation levels of 485, 512 CpG sites were measured in blood in 743 participants from NSPHS, using the Illumina 450K chip. A total of 472, 477 sites (99. 89%) exceeded the QC. Of these, 470, 789 CpG -sites were autosomal and have been included in downstream analyses. 11, 232 CpG-sites were located on the X chromosome and analysed in males and females separately and 416 CpG-site on the Y chromosome was analysed only in males. None from the CpG-sites located on the sex-chromosomes exceeded PD 334581 the False discovery price (FDR)q-values < 0. 05 threshold and was not further investigated. All individuals passed QC for the NSPHS populace. After eliminating technical regulates, 717 samples with DNA methylation and GDF-15 data available remained for the epigenetic analyses. In NSPHS, GDF-15 levels increased with age (p=6. 98 x 10-41) and with fraction of granulocytes (p=1. 60 x 10-7), but there was no significant difference between sexes, nor with variation in fractions of other cell types ITGAV (p> 0. 05). A total of 31 DNA autosomal methylation sites were significantly associated (False discovery rate PD 334581 (FDR)q-values < 0. 05) with GDF-15 levels in NSPHS (Figure 1, Table 2, Supplementary Table 2). The 31 significant sites mapped to 23 genes. Of these, 16 CpG-sites, corresponding to 11 genes, were replicated in PIVUS (FDR q < 0. 05) with all the same direction of effect (Table 2, Supplementary Table 2). Some of the probes used for measuring DNA methylation overlaps with known SNPs that might influence the probe specificity. Including SNPs located within the probe positions (Seesupplementary methodsfor more information) did not influence the relationship between the CpG methylation and the GDF-15 levels for any from the replicating sites (Supplementary Table 1). == Figure 1 . == Manhattan plot intended for the genome-wide DNA methylation study in NSPHS. Significance threshold is marked in with a grey range (FDRq-value 0. 05) == Table 2 . Genome-wide DNA methylation study summary statistics for Discovery (NSPHS), Replication cohort (PIVUS) and meta-analysis (NSPHS+PIVUS). == Among the replicated sites, four.