VA San Diego Healthcare System, San Diego, California.
Division of Pulmonary and Critical Care and Sleep Medicine, University of California San Diego, San Diego, California.
Am J Physiol Lung Cell Mol Physiol. 2022 Jul 1;323(1):L84-L92. doi: 10.1152/ajplung.00128.2022. Epub 2022 Jun 14.
Increased plasma mitochondrial DNA concentrations are associated with poor outcomes in multiple critical illnesses, including COVID-19. However, current methods of cell-free mitochondrial DNA quantification in plasma are time-consuming and lack reproducibility. Here, we used next-generation sequencing to characterize the size and genome location of circulating mitochondrial DNA in critically ill subjects with COVID-19 to develop a facile and optimal method of quantification by droplet digital PCR. Sequencing revealed a large percentage of small mitochondrial DNA fragments in plasma with wide variability in coverage by genome location. We identified probes for the mitochondrial DNA genes, cytochrome B and NADH dehydrogenase 1, in regions of relatively high coverage that target small sequences potentially missed by other methods. Serial assessments of absolute mitochondrial DNA concentrations were then determined in plasma from 20 critically ill subjects with COVID-19 without a DNA isolation step. Mitochondrial DNA concentrations on the day of enrollment were increased significantly in patients with moderate or severe acute respiratory distress syndrome (ARDS) compared with those with no or mild ARDS. Comparisons of mitochondrial DNA concentrations over time between patients with no/mild ARDS who survived, patients with moderate/severe ARDS who survived, and nonsurvivors showed the highest concentrations in patients with more severe disease. Absolute mitochondrial DNA quantification by droplet digital PCR is time-efficient and reproducible; thus, we provide a valuable tool and rationale for future studies evaluating mitochondrial DNA as a real-time biomarker to guide clinical decision-making in critically ill subjects with COVID-19.
血浆中线粒体 DNA 浓度的增加与多种危重病的不良预后相关，包括 COVID-19。然而，目前血浆中无细胞线粒体 DNA 定量的方法既耗时又缺乏重现性。在这里，我们使用下一代测序技术来描述 COVID-19 危重症患者血浆中循环线粒体 DNA 的大小和基因组位置，以开发一种通过数字 PCR 进行定量的简便且优化的方法。测序显示，血浆中有很大比例的小线粒体 DNA 片段，其基因组位置的覆盖范围差异很大。我们在相对高覆盖区域中鉴定了线粒体 DNA 基因（细胞色素 B 和 NADH 脱氢酶 1）的探针，这些探针针对其他方法可能错过的小序列。然后，在没有 DNA 分离步骤的情况下，对 20 名 COVID-19 危重症患者的血浆进行了绝对线粒体 DNA 浓度的连续评估。与无或轻度急性呼吸窘迫综合征（ARDS）患者相比，中度或重度 ARDS 患者在入组当天的线粒体 DNA 浓度显著升高。对无/轻度 ARDS 存活患者、中/重度 ARDS 存活患者和非存活患者之间的线粒体 DNA 浓度随时间的比较表明，疾病更严重的患者浓度最高。通过数字 PCR 进行绝对线粒体 DNA 定量既省时又可重现；因此，我们为未来评估线粒体 DNA 作为 COVID-19 危重症患者实时生物标志物以指导临床决策的研究提供了有价值的工具和依据。
