Background Many studies have demonstrated the high efficacy of cell-free nuclear DNA in cancer diagnostics. Compared to nuclear DNA, mitochondrial DNA (mtDNA) exhibits distinct characteristics, including multiple copies per cell and higher mutation frequency. However, the potential applicability of cell-free mtDNA (cf-mtDNA) in plasma and urine remains poorly investigated. Methods Here, we comprehensively analyzed the fragmentomic and mutational characteristics of cf-mtDNA in urine and plasma samples from controls and cancer patients using next-generation sequencing. Results Compared to plasma cf-mtDNA, urine cf-mtDNA exhibited increased copy numbers and wider spread in fragment size distributions. Based on 2 independent animal models, urine cf-mtDNA originated predominantly from local shedding and transrenal excretion. Further analysis indicated an enhanced fragmentation of urine cf-mtDNA in renal cell carcinoma (RCC) and colorectal cancer (CRC) patients. Using the mtDNA sequence of peripheral blood mononuclear cells for reference, the mutant fragments were shorter than wild-type fragments in urine cf-mtDNA. Size selection of short urine cf-mtDNA fragments (<150 bp) significantly enhanced the somatic mutation detection. Our data revealed remarkably different base proportions of fragment ends between urine and plasma cf-mtDNA that also were associated with fragment size. Moreover, both RCC and CRC patients exhibited significantly higher T-end and lower A-end proportions in urine cf-mtDNA than controls. By integrating the fragmentomic and mutational features of urine cf-mtDNA, our nomogram model exhibited a robust efficacy for cancer diagnosis. Conclusions Our proof-of-concept findings revealed aberrant fragmentation and mutation profiles of urine cf-mtDNA in cancer patients that have diagnostic potential.

中文翻译：

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基于下一代测序的尿液游离 mtDNA 分析揭示癌症患者的异常片段化和突变谱

背景 许多研究已经证明无细胞核 DNA 在癌症诊断中的高效性。与核 DNA 相比，线粒体 DNA (mtDNA) 表现出不同的特征，包括每个细胞的多个拷贝和更高的突变频率。然而，血浆和尿液中无细胞 mtDNA (cf-mtDNA) 的潜在适用性仍然缺乏研究。方法 在这里，我们使用下一代测序全面分析了来自对照和癌症患者的尿液和血浆样本中 cf-mtDNA 的片段组和突变特征。结果 与血浆 cf-mtDNA 相比，尿液 cf-mtDNA 表现出增加的拷贝数和更广泛的片段大小分布分布。基于 2 个独立的动物模型，尿液 cf-mtDNA 主要来源于局部脱落和经肾排泄。进一步的分析表明，肾细胞癌 (RCC) 和结直肠癌 (CRC) 患者的尿液 cf-mtDNA 片段化增强。使用外周血单个核细胞的mtDNA序列作为参考，突变片段比尿液cf-mtDNA中的野生型片段短。短尿 cf-mtDNA 片段 (<150 bp) 的大小选择显着增强了体细胞突变检测。我们的数据揭示了尿液和血浆 cf-mtDNA 之间片段末端的碱基比例显着不同，这也与片段大小有关。此外，RCC 和 CRC 患者在尿液 cf-mtDNA 中的 T 端和 A 端比例均显着高于对照组。通过整合尿液 cf-mtDNA 的片段组和突变特征，我们的列线图模型显示出强大的癌症诊断功效。