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. We 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. Analysis 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. We 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.

中文翻译：

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在健康状态下和扑热息痛过量后，绝对测量无细胞DNA的组织来源。

尽管无细胞DNA（cfDNA）作为癌症的临床生物标志物出现，但迄今为止，仅通过间接和相对测量方法（例如组织特异性甲基化和核小体分析）可以推断出健康个体中cfDNA的组织起源。我们在健康小鼠中和扑热息痛（APAP）过量后，使用组织特异性敲除小鼠品系对cfDNA的组织起源进行了首次直接，绝对的测量。然后，我们调查了APAP过量患者中总cfDNA的效用和肝脏特异性cfDNA作为临床生物标记物的百分比。对来自健康组织特异性基因敲除小鼠的cfDNA的分析表明，cfDNA主要来源于白血球和红血球谱系，而肝细胞的贡献很小，并且骨骼肌和心肌均无可检测的贡献。在小鼠中APAP用药过量后，总血浆cfDNA和源自肝细胞的百分比分数分别增加了约100倍和19倍。在有生化证据表明肝损伤的过量用药的APAP临床样品中，总cfDNA平均增加了236倍以上，而在没有生化表现明显肝损伤的患者中，总cfDNA平均增加了236倍。使用液滴数字PCR和甲基化分析对肝脏特异性cfDNA的测量显示，与健康受试者相比，在生化明显肝损伤的AP​​AP过量患者中，肝脏对cfDNA的贡献平均增加了175倍，但没有增加肝功能检查正常的药物过量患者。