Absolute measurement of the tissue origins of cell-free DNA in the healthy state and following paracetamol overdose

Danny Laurent,Fiona Semple,Jennifer Coe,Stuart J Forbes,James W Dear,Philip J Starkey Lewis,Mark J Arends,Holly A Black,Elaine Rose,Timothy J Aitman

doi:10.1186/s12920-020-0705-2

Abstract

BackgroundDespite the emergence of cell-free DNA (cfDNA) as a clinical biomarker in cancer, the tissue origins of cfDNA in healthy individuals have to date been inferred only by indirect and relative measurement methods, such as tissue-specific methylation and nucleosomal profiling.MethodsWe performed the first direct, absolute measurement of the tissue origins of cfDNA, using tissue-specific knockout mouse strains, in both healthy mice and following paracetamol (APAP) overdose. We then investigated the utility of total cfDNA and the percentage of liver-specific cfDNA as clinical biomarkers in patients presenting with APAP overdose.ResultsAnalysis of cfDNA from healthy tissue-specific knockout mice showed that cfDNA originates predominantly from white and red blood cell lineages, with minor contribution from hepatocytes, and no detectable contribution from skeletal and cardiac muscle. Following APAP overdose in mice, total plasma cfDNA and the percentage fraction originating from hepatocytes increased by ~ 100 and ~ 19-fold respectively. Total cfDNA increased by an average of more than 236-fold in clinical samples from APAP overdose patients with biochemical evidence of liver injury, and 18-fold in patients without biochemically apparent liver injury. Measurement of liver-specific cfDNA, using droplet digital PCR and methylation analysis, revealed that the contribution of liver to cfDNA was increased by an average of 175-fold in APAP overdose patients with biochemically apparent liver injury compared to healthy subjects, but was not increased in overdose patients with normal liver function tests.ConclusionsWe present a novel method for measurement of the tissue origins of cfDNA in healthy and disease states and demonstrate the potential of cfDNA as a clinical biomarker in APAP overdose.

Highlights

Despite the emergence of cell-free DNA as a clinical biomarker in cancer, the tissue origins of cfDNA in healthy individuals have to date been inferred only by indirect and relative measurement methods, such as tissue-specific methylation and nucleosomal profiling
We demonstrate that hepatocytes are a minor contributor to the pool of circulating cfDNA in healthy mice, and show a substantial increase in hepatocyte-derived cfDNA in mice exposed to an overdose of paracetamol
Validation of droplet-digital PCR (ddPCR) assay for absolute quantification of cfDNA tissue origins We designed and validated a ddPCR assay to quantify recombination in the floxed membrane-localised tdTomato (mT)/Cell membrane-localised enhanced green fluorescent protein (mG) gene

Summary

Introduction

Despite the emergence of cell-free DNA (cfDNA) as a clinical biomarker in cancer, the tissue origins of cfDNA in healthy individuals have to date been inferred only by indirect and relative measurement methods, such as tissue-specific methylation and nucleosomal profiling. In 1948, Mandel and Metaìs described the presence of DNA in plasma [1], known as cell-free DNA (cfDNA) and known to be present in other bodily fluids, such as urine [2], saliva and cerebrospinal fluid [3]. Analysis of cfDNA is clinically useful for detecting genetic and epigenetic alterations in DNA by allowing repeated non-invasive or minimally invasive sampling from bodily fluids [8]. Analysis of foetal cfDNA in maternal plasma has been clinically implemented for noninvasive prenatal testing of chromosomal abnormalities and some monogenic disorders [9, 10]. Analysis of cfDNA in cancer genomics is evolving rapidly [11,12,13,14] and utility of cfDNA as a biomarker of internal tissue damage [15,16,17], in organ transplantation [18] and in autoimmune disease is being investigated [19, 20]

Methods

Results

Discussion

Conclusion