Cell-free DNA Methylomics Identify Tissue Injury Patterns in Pediatric ARDS

Cell-free DNA (cfDNA) is released into the circulation from dead and dying cells, and is elevated in critical illness syndromes including sepsis and acute respiratory distress syndrome (ARDS). Plasma cfDNA has been implicated as a damage-associated molecular pattern (DAMP) that propagates ongoing inflammation. However, the tissue source of circulating cfDNA from different pathological processes has not been fully elucidated, and identification of the cellular origin of cfDNA may provide insight into the mechanisms underlying the early inflammatory response. As cfDNA retains the unique methylation pattern of its tissue of origin, we can leverage cfDNA methylome to map the presumed tissue of origin of these DAMPs. We conducted a pilot study to assess the cfDNA methylation pattern in pediatric ARDS. This was a secondary analysis of a cohort of children with Berlin-defined ARDS from the Children’s Hospital of Philadelphia (CHOP) enrolled between 2014 and 2019, with plasma collected within 24 hours of ARDS onset after obtaining prospective consent from caregivers. In this study, we aimed to include subjects with higher levels of plasma cfDNA to assess the feasibility of our methodology. We randomly selected 24 children with cfDNA levels above the median value and 6 intubated non-ARDS controls. We isolated and quantified cfDNA from plasma, and performed methylation analysis using the Infinium MethylationEPIC and MethylationEPIC v2.0 BeadChip. Processing IDAT files generated from Infinium BeadChip-based data was performed using the openSesame workflow within the SeSAMe package. This workflow effectively addresses critical processing challenges, including signal background correction, dye bias adjustment, inference of color channel swaps, masking of suboptimal signals, and precise extraction of DNA modification levels (beta values).