Identification of differentially methylated BRCA1 and CRISP2 DNA regions as blood surrogate markers for cardiovascular disease

Abstract

Genome-wide Illumina InfiniumMethylation 450 K DNA methylation analysis was performed on blood samples from clinical atherosclerosis patients (n = 8) and healthy donors (n = 8) in the LVAD study (NCT02174133, NCT01799005). Multiple differentially methylated regions (DMR) could be identified in atherosclerosis patients, related to epigenetic control of cell adhesion, chemotaxis, cytoskeletal reorganisations, cell proliferation, cell death, estrogen receptor pathways and phagocytic immune responses. Furthermore, a subset of 34 DMRs related to impaired oxidative stress, DNA repair, and inflammatory pathways could be replicated in an independent cohort study of donor-matched healthy and atherosclerotic human aorta tissue (n = 15) and human carotid plaque samples (n = 19). Upon integrated network analysis, BRCA1 and CRISP2 DMRs were identified as most central disease-associated DNA methylation biomarkers. Differentially methylated BRCA1 and CRISP2 regions were verified by MassARRAY Epityper and pyrosequencing assays and could be further replicated in blood, aorta tissue and carotid plaque material of atherosclerosis patients. Moreover, methylation changes at BRCA1 and CRISP2 specific CpG sites were consistently associated with subclinical atherosclerosis measures (coronary calcium score and carotid intima media thickness) in an independent sample cohort of middle-aged men with subclinical cardiovascular disease in the Aragon Workers' Health Study (n = 24). Altogether, BRCA1 and CRISP2 DMRs hold promise as novel blood surrogate markers for early risk stratification and CVD prevention.

Figure 1

Flowchart of DMP and DMR selection.

Figure 2

Differentially methylated CpG probes between healthy and atherosclerotic individuals. Sig-DMPs were selected based on FDR < 0.15 and Δ beta >5%. (A) Volcano plot showing 465 hypo- and 247 hypermethylated probes meeting the selection criteria. (B) PCA plot demonstrating the separation of atherosclerosis patients for CVD (grey) and healthy individuals (black) based on the DNA methylation values of the 712 DMPs.

Figure 3

Genomic distribution of sig-DMPs based on (A) gene regions, (B) CpG-island regions and (C) chromatin segmentation states (based on GM12878 cell type data). Significant enrichment or depletion of sig-DMPs (P-value < 0.05), determined by the Fisher’s exact test, are marked by an asterisk.

Figure 4

Metacore based pathway enrichment analysis of gene associated DMRs in atherosclerosis reveals a significant (P-value < 0.05; −log(P-value) < 1.3) enrichment of CVD related pathways related to leukocyte chemotaxis and adhesion.

Figure 5

Deconvolution of immune cell type blood composition. The approach described by Houseman et al. was applied to determine the relative immune cell type fraction (Y-axis) in healthy and atherosclerosis blood samples. Statistical significant differences in cell type contribution between healthy and atherosclerosis blood samples were calculated by a student t-test.

Figure 6

GeneMANIA network of the 34 sig-DMRs not affected by age or cell type composition and consistently differentially methylated in both blood and plaque tissue.

Figure 7

MassARRAY EpiTYPER validation of BRCA1 and CRISP2. The mean methylation values of each measured region are represented in boxplots. The student t-test was used to calculate the significance of the methylation difference between healthy and atherosclerotic blood samples.

Figure 8

DMRs associated with BRCA1 and CRISP2 in an independent publicly available cohort (Zaina et al.). The Zaina et al. cohort comprises of 30 donor-matched atherosclerotic plaque tissue samples and 19 carotid plaque samples.