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The use of cardiopulmonary bypass (CPB) is thought to cause delayed cardiac damage. DNA methylation-based liquid biopsies are novel biomarkers for monitoring acute cardiac cell death. We assessed cell-free DNA molecules as markers for cardiac damage after open-heart surgery. Novel cardiomyocyte-specific DNA methylation markers were applied to measure cardiac cfDNA in the plasma of 42 infants who underwent open-heart surgery. Cardiac cfDNA was elevated following surgery, reflecting direct surgery-related tissue damage, and declined thereafter in most patients. The concentration of cardiac cfDNA post-surgery correlated with the duration of CPB and aortic cross clamping. Strikingly, cardiac cfDNA at 6 h predicted duration of mechanical ventilation and maximal vasoactive-inotropic score better than did maximal troponin levels. Cardiac cfDNA reveals heart damage associated with CPB, and can be used to monitor cardiac cell death, to predict clinical outcome of surgery and to assess performance of cardioprotective interventions.

DNA methylation is a fundamental epigenetic mark that governs chromatin organization, cell identity, and gene expression. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 207 healthy tissue samples.Replicates of the same cell-type are >99.5% identical, demonstrating robustness of cell identity programs to genetic variation and environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny, and identifies methylation patterns retained since gastrulation. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hyper-methylated loci are rare and are enriched for CpG islands, polycomb targets, and CTCF binding sites, suggesting a novel role in shaping cell type-specific chromatin looping. The atlas provides an essential resource for interpretation of disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.Summary paragraphDNA methylation, a fundamental epigenetic mark, governs chromatin organization and gene expression1, thus defining the molecular identity of cells and providing a window into developmental processes with wide-ranging physiologic and clinical ramifications. Current DNA methylation datasets have limitations, typically including only a fraction of methylation sites, many from cell lines that underwent massive changes in culture or from tissues containing unspecified mixtures of cells2–6.We present a human methylome atlas based on deep whole-genome bisulfite sequencing of 39 sorted, primary cell types and use this dataset to address fundamental questions in developmental biology, physiology and pathology. Biological replicates are >99.5% identical, demonstrating unappreciated robustness to genetic variation and environmental perturbations. Clustering recapitulates key elements of tissue ontogeny, identifying methylation patterns retained since gastrulation. Loci uniquely unmethylated in individual cell types identify novel transcriptional enhancers and are enriched for tissue-specific transcription factors binding motifs. In contrast, loci uniquely hyper-methylated in specific cell types are rare, enriched for CpG islands and polycomb targets, and overlap CTCF binding sites, suggesting a novel role in shaping cell-type-specific chromatin looping. Finally, the atlas facilitates fragment-level deconvolution of tissue and plasma methylomes across thousands of cell-type specific regions to quantify their individual components at unprecedented resolution.The human cell-type-specific methylation atlas provides an essential resource for studying gene regulation by defining cell-type-specific distal enhancers and regulators of 3D organization, for identifying pathological changes in DNA methylation, and for the interpretation of methylation-based liquid biopsies.A deep methylation atlas of 39 human cell types, sorted from healthy samplesMethylomes record developmental history of cellsThousands of novel cell type-specific methylation markersHypo-methylation uncovers cell type-specific regulatory map of distal enhancersHyper-methylation across CTCF sitesCell type-specific biomarkers facilitate fragment-level deconvolution of tissues and cfDNA

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2–5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

Much remains unknown regarding the response of the immune system to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination.We employed circulating cell-free DNA (cfDNA) to assess the turnover of specific immune cell types following administration of the Pfizer/BioNTech vaccine.The levels of B cell cfDNA after the primary dose correlated with development of neutralizing antibodies and memory B cells after the booster, revealing a link between early B cell turnover-potentially reflecting affinity maturation-and later development of effective humoral response. We also observed co-elevation of B cell, T cell, and monocyte cfDNA after the booster, underscoring the involvement of innate immune cell turnover in the development of humoral and cellular adaptive immunity. Actual cell counts remained largely stable following vaccination, other than a previously demonstrated temporary reduction in neutrophil and lymphocyte counts.Immune cfDNA dynamics reveal the crucial role of the primary SARS-CoV-2 vaccine in shaping responses of the immune system following the booster vaccine.This work was supported by a generous gift from Shlomo Kramer. Supported by grants from Human Islet Research Network (HIRN UC4DK116274 and UC4DK104216 to R.S. and Y.D.), Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Alex U Soyka Pancreatic Cancer Fund, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation, the Helmsley Charitable Trust, Grail, and the DON Foundation (to Y.D.). Y.D. holds the Walter and Greta Stiel Chair and Research Grant in Heart Studies. I.F.-F. received a fellowship from the Glassman Hebrew University Diabetes Center.

BackgroundCirculating biomarkers for lung damage are lacking. Lung epithelium-specific DNA methylation patterns can potentially report the presence of lung-derived cell-free DNA (cfDNA) in blood, as an indication of lung cell death.MethodsWe sorted human lung alveolar and bronchial epithelial cells from surgical specimens, and obtained their methylomes using whole-genome bisulfite sequencing. We developed a PCR sequencing assay determining the methylation status of 17 loci with lung-specific methylation patterns, and used it to assess lung-derived cfDNA in the plasma of healthy volunteers and patients with lung disease.ResultsLoci that are uniquely unmethylated in alveolar or bronchial epithelial cells are enriched for enhancers controlling lung-specific genes. Methylation markers extracted from these methylomes revealed that normal lung cell turnover probably releases cfDNA into the air spaces, rather than to blood. People with advanced lung cancer show a massive elevation of lung cfDNA concentration in blood. Among individuals undergoing bronchoscopy, lung-derived cfDNA is observed in the plasma of those later diagnosed with lung cancer, and to a lesser extent in those diagnosed with other lung diseases. Lung cfDNA is also elevated in patients with acute exacerbation of COPD compared with patients with stable disease, and is associated with future exacerbation and mortality in these patients.ConclusionsUniversal cfDNA methylation markers of normal lung epithelium allow for mutation-independent, sensitive and specific detection of lung-derived cfDNA, reporting on ongoing lung injury. Such markers can find broad utility in the study of normal and pathologic human lung dynamics.