Centre for Genomic and Experimental Medicine, Medical Research Council Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, UK.
Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
BMC Med Genomics. 2020 Apr 6;13(1):60. doi: 10.1186/s12920-020-0705-2.
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.
尽管无细胞游离 DNA（cfDNA）已成为癌症的一种临床生物标志物，但迄今为止，健康个体 cfDNA 的组织来源只能通过间接和相对的测量方法推断，例如组织特异性甲基化和核小体分析。
我们使用组织特异性敲除小鼠品系，对健康小鼠和扑热息痛（APAP）过量后的 cfDNA 进行了首次直接、绝对的组织起源测量。然后，我们研究了总 cfDNA 和肝特异性 cfDNA 作为 APAP 过量患者临床生物标志物的效用。
对来自健康组织特异性敲除小鼠的 cfDNA 进行分析表明，cfDNA 主要来源于白细胞和红细胞谱系，肝细胞贡献较小，骨骼肌和心肌无明显贡献。在小鼠 APAP 过量后，总血浆 cfDNA 和源自肝细胞的分数分别增加了约 100 倍和 19 倍。在具有生化肝损伤证据的 APAP 过量患者的临床样本中，总 cfDNA 平均增加了 236 倍以上，在没有生化明显肝损伤的患者中增加了 18 倍。使用液滴数字 PCR 和甲基化分析测量肝特异性 cfDNA 显示，与健康受试者相比，具有生化明显肝损伤的 APAP 过量患者中肝对 cfDNA 的贡献平均增加了 175 倍，但肝功能正常的过量患者中并未增加。
我们提出了一种测量健康和疾病状态下 cfDNA 组织来源的新方法，并证明了 cfDNA 在 APAP 过量中的临床生物标志物的潜力。
