Methylome of circulating cell free DNA as a novel biomarker tool: from acute coronary syndrome to broader risk stratification of cardiovascular disease

Abstract Background Cell death during tissue injury leads to release of chromosomal DNA which is promptly degraded. However, short DNA fragments wrapped around nucleosomes survive longer in the bloodstream and can be isolated as circulating cell-free DNA (ccfDNA). Oncology field pioneered the use of cfDNA as clinical biomarker by identifying sequence modifications induced in cancer cells. Importantly, ccfDNA also retains the same epigenetic information as the tissue of origin. Despite major advances for disease diagnosis and therapy, coronary artery diseases (CAD) and associated conditions such acute myocardial infarction (AMI) or heart failure (HF) are responsible for leading number of deaths worldwide. Preventive and surveillance strategies with non-invasive testing are needed. Purpose Evaluation of ccfDNA methylation profiles as prognostic non-invasive biomarker for risk stratification of acute coronary syndromes and their potential application in preventive and surveillance strategies in other cardiovascular disorders. Methods CcfDNA isolated from citrate-plasma collected from our discovery cohort, comprising healthy controls, STEMI, NSTEMI and unstable angina (UA) patients, was analyzed applying Whole Genome Bisulfite Sequencing (WGBS) using a low-input BS-seq (PBAT) protocol. Computational analysis generated differentially methylated regions (DMRs) by tiling CpG areas and setting a threshold of minimal 25% methylation difference compared to the healthy group. To assess the validity of our findings, a selection of disease-specific DMRs were tested using targeted methylation sequencing protocol in a new set of patients. This procedure consisted in enzymatic conversion of ccfDNA methylated sites followed by probe-specific enrichment libraries and sequencing at a Novaseq 6000 platform. Results The study identified a total of 1941 DMRs and from those 486 were STEMI, 223 NSTEMI and 684 UA specific. From the identified DMRs, 74.13% from STEMI, 72.68% from NSTEMI and 61.62% from UA annotated with heart-related diseases using DisGeNET gene-disease associations. Interestingly, most of identified DMRs located within intronic regions (~60%) and also annotated as enhancers and lncRNAs. The independent validation with targeted methylation sequencing identified 566, 306 and 248 DMRs, respectively, and reduced the number of disease-specific DMRs to 171 in STEMI, 115 in NSTEMI and 82 in UA. Conclusion and Future Approaches Methylation profiles of ccfDNA showed potent