Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease

Joshua Moss ^1,2 Judith Magenheim ¹ Daniel Neiman ¹ Hai Zemmour ¹ Netanel Loyfer ² Amit Korach ³ Yaacov Samet ⁴ Myriam Maoz ⁵ Henrik Druid ⁶ Peter Arner ⁷ Keng-Yeh Fu ⁸ Endre Kiss ⁸ Kirsty L. Spalding ^7,8 Giora Landesberg ⁹ Aviad Zick ⁵ Albert Grinshpun ⁵ A.M. James Shapiro ¹⁰ Markus Grompe ¹¹ Avigail Dreazan Wittenberg ¹ Benjamin Glaser ¹² Ruth Shemer ¹ Tommy Kaplan ² Yuval Dor ¹

¹Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel
²School of Computer Science and Engineering, The Hebrew University, Jerusalem, Israel
³Department of Cardio-Thoracic Surgery, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
⁴Department of Vascular Surgery, Hadassah-Hebrew University Medical Center, Jerusalem
⁵Department of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem
⁶Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden
⁷Department of Medicine, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden
⁸Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
⁹Dept of Anesthesiology and Critical Care Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
¹⁰Department of Surgery and the Clinical Islet Transplant Program, University of Alberta, Edmonton, Alberta, Canada
¹¹Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, Oregon, USA
¹²Dept of Endocrinology and Metabolism Service, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

Methylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated using in silico simulations as well as in vitro mixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), red blood cell/platelet precursors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. Furthermore, the observation that healthy cfDNA reflects dynamics of cells residing in the bone marrow such as platelet precursors (megakaryocytes) makes cfDNA a potential tool for the detection and monitoring of patients with hematological disturbances. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics.