New methods for analysing circulating cell‐free mitochondrial DNA

Abstract

Trauma, ischemia, inflammatory stress and environmental conditions can cause tissue injury and damage. This damage spurs the release of cellular components and debris into the extracellular environment, including damage associated molecular pattern (DAMP) molecules. These DAMPs are sensed by pattern recognition receptors that activate a cellular damage response to the pathological insult. Mitochondrial components have recently been recognised as potent DAMPs.1, 2 The mitochondrial genome shares features with bacterial genomes, due to their shared origin. Therefore, when mitochondrial DNA (mtDNA) is released into the extracellular space it is recognised as a foreign molecule and initiates an innate immune response.1, 2 Not only have DAMP molecules been of interest in studying the relationships between cellular damage and immune responses, but emerging evidence suggests that DAMPs may serve as ‘liquid biopsies’ to indicate tissue damage or disease status. For example, plasma levels of circulating cell-free mtDNA (ccf-mtDNA) can be isolated and quantified easily using quantitative real-time PCR (qPCR). The results of many studies suggest that there are higher levels of ccf-mtDNA after trauma, acute myocardial infarction, sepsis and in some inflammatory conditions.3 A recent systematic review examined published articles on utilising ccf-mtDNA as a predictor of mortality in critically ill patients.4 The authors noted that in their analysis there are inconsistencies in ccf-mtDNA isolation methods and quantification. For example, most often qPCR primers were designed against subunits of the NADH dehydrogenase (MT-ND, MT-ND1, MT-ND2 or MT-ND6).4 However, some studies use one primer set, others use multiple primer sets and some use primer sets recognising different genes in the mitochondrial genome. This may explain the discrepancies in the findings linking ccf-mtDNA to mortality in some studies but not in all. Therefore, there is a need for the development of technologies that improve upon these current methods to quantify ccf-mtDNA. Daly and colleagues recently developed a protocol that provides a new approach to analysing plasma ccf-mtDNA.5 In their approach, they utilised a target bait-capture kit consisting of biotinylated RNA probes complementary to the entire mitochondrial genome. First, the biotinylated probes bind to mitochondrial sequences and then are enriched, pooled, and sequenced on a standard Illumina instrument. Second, a Workflow Description Language-based computational pipeline aligns the reads to both the nuclear and mitochondrial genome and then custom programs call mitochondrial and nuclear coverage, insert fragment size and mtDNA variants. Nuclear genomes contain sequences of mitochondrial origin, called nuclear mitochondrial DNA segments (NUMTs). NUMT pseudogenes can also be enriched using these methods. Therefore, to exclude the possibility that reads are from the nuclear genome, this method excludes sequences that aligned to both nuclear and mitochondrial genomes. This approach may underestimate the mtDNA levels but increases classification accuracy. In fact, this protocol was shown to enrich for mean coverage of the mtDNA genome compared to whole genome sequencing. Therefore, this protocol allows for the simultaneous quantification of mtDNA levels across the entire mitochondrial genome, differentiation of fragment lengths, and the identification of mtDNA variants.5 The authors utilised their protocol to investigate ccf-mtDNA in comparison to clinical phenotypes of injury and severity scores in a cohort of 30 trauma patients.5 There were no significant differences in ccf-mtDNA abundance between patients with non-severe or severe injuries. The ccf-mtDNA fragment length was significantly shorter in patients with major trauma and in patients where acute lung injury occurred after admission compared to non-severe injury patients. There were also mtDNA variants that were discovered that were associated with various clinical scores of injuries and severity in this cohort. Therefore, the authors utilised samples from this trauma cohort to validate that their protocol can be implemented to examined mtDNA levels, fragment length, and variants.5 This technological advance has advantages over the standard qPCR methods, as the entire mitochondrial genome can be analysed in one protocol. However, additional experiments and validation studies should be performed to directly compare the sensitivity and accuracy of this protocol versus the standard qPCR methods. Nevertheless, these methods will give scientists an additional tool to examine ccf-mtDNA in relation to tissue damage or disease status. This work was funded by the Intramural Research Program of the National Institute on Aging, National Institutes of Health. The authors declare no conflict of interest.