Comparison of the structures and topologies of plasma extracted circulating nuclear and mitochondrial cell-free DNA.

Introduction: The function, origin and structural features of circulating nuclear DNA (cir-nDNA) and mitochondrial DNA (cir-mtDNA) are poorly known, even though they have been investigated in numerous clinical studies, and are involved in a number of routine clinical applications. Based on our previous report disproving the conventional plasma isolation used for cirDNA analysis, this work enables a direct topological comparison of the circulating structures associated with nuclear DNA and mitochondrial cell-free DNA. Materials and methods: We used a Q-PCR and low-pass whole genome sequencing (LP-WGS) combination approach of cir-nDNA and cir-mtDNA, extracted using a procedure that eliminates platelet activation during the plasma isolation process to prevent mitochondria release in the extracellular milieu. Various physical procedures, such as filtration and differential centrifugation, were employed to infer their circulating structures. Results: DSP-S cir-mtDNA mean size profiles distributed on a slightly shorter range than SSP-S. SSP-S detected 40-fold more low-sized cir-mtDNA fragments (<90 bp/nt) and three-fold less long-sized fragments (>200 bp/nt) than DSP-S. The ratio of the fragment number below 90 bp over the fragment number above 200 bp was very homogenous among both DSP-S and SSP-S profiles, being 134-fold lower with DSP-S than with SSP-S. Cir-mtDNA and cir-nDNA DSP-S and SSP-S mean size profiles of healthy individuals ranged in different intervals with periodic sub-peaks only detectable with cir-nDNA. The very low amount of cir-mtDNA fragments of short size observed suggested that most of the cir-mtDNA is poorly fragmented and appearing longer than ∼1,000 bp, the readout limit of this LP-WGS method. Data suggested that cir-nDNA is, among DNA extracted in plasma, associated with ∼8.6% of large structures (apoptotic bodies, large extracellular vesicles (EVs), cell debris…), ∼27.7% in chromatin and small EVs and ∼63.7% mainly in oligo- and mono-nucleosomes. By contrast, cir-mtDNA appeared to be preponderantly (75.7%) associated with extracellular mitochondria, either in its free form or with large EVs; to a lesser extent, it was also associated with other structures: small EVs (∼18.4%), and exosomes or protein complexes (∼5.9%). Conclusion: This is the first study to directly compare the structural features of cir-nDNA and cir-mtDNA. The significant differences revealed between both are due to the DNA topological structure contained in the nucleus (chromatin) and in the mitochondria (plasmid) that determine their biological stability in blood. Although cir-nDNA and cir-mtDNA are principally associated with mono-nucleosomes and cell-free mitochondria, our study highlights the diversity of the circulating structures associated with cell-free DNA. They consequently have different pharmacokinetics as well as physiological functions. Thus, any accurate evaluation of their biological or diagnostic individual properties must relies on appropriate pre-analytics, and optimally on the isolation or enrichment of one category of their cirDNA associated structures.

Pisareva E ，Roch B ，Sanchez C ，Pastor B ，Mirandola A ，Diab-Assaf M ，Mazard T ，Prévostel C ，Al Amir Dache Z ，Thierry AR ... -  《Frontiers in Genetics》

Impact of platelet activation on the release of cell-free mitochondria and circulating mitochondrial DNA.

Roch B ，Pisareva E ，Mirandola A ，Sanchez C ，Pastor B ，Tanos R ，Frayssinoux F ，Diab-Assaf M ，Anker P ，Al Amir Dache Z ，Thierry AR ... -  《-》

Association of the immediate perioperative dynamics of circulating DNA levels and neutrophil extracellular traps formation in cancer patients.

Elevated circulating DNA (cirDNA) concentrations were found to be associated with trauma or tissue damage which suggests involvement of inflammation or cell death in post-operative cirDNA release. We carried out the first prospective, multicenter study of the dynamics of cirDNA and neutrophil extracellular trap (NETs) markers during the perioperative period from 24 h before surgery up to 72 h after curative surgery in cancer patients. We examined the plasma levels of two NETs protein markers [myeloperoxidase (MPO) and neutrophil elastase (NE)], as well as levels of cirDNA of nuclear (cir-nDNA) and mitochondrial (cir-mtDNA) origin in 29 colon, prostate, and breast cancer patients and in 114 healthy individuals (HI). The synergistic analytical information provided by these markers revealed that: (i) NETs formation contributes to post-surgery conditions; (ii) post-surgery cir-nDNA levels were highly associated with NE and MPO in colon cancer [r = 0.60 (P < 0.001) and r = 0.53 (P < 0.01), respectively], but not in prostate and breast cancer; (iii) each tumor type shows a specific pattern of cir-nDNA and NETs marker dynamics, but overall the pre- and post-surgery median values of cir-nDNA, NE, and MPO were significantly higher in cancer patients than in HI. Taken as a whole, our work reveals the association of NETs formation with the elevated cir-nDNA release during a cancer patient's perioperative period, depending on surgical procedure or cancer type. By contrast, cir-mtDNA is poorly associated with NETs formation in the studied perioperative period, which would appear to indicate a different mechanism of release or suggest mitochondrial dysfunction.

Kudriavtsev A ，Pastor B ，Mirandola A ，Pisareva E ，Gricourt Y ，Capdevila X ，Thierry AR ，Cuvillon P ... -  《-》

An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma.

Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of ∼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.

Ware SA ，Desai N ，Lopez M ，Leach D ，Zhang Y ，Giordano L ，Nouraie M ，Picard M ，Kaufman BA ... -  《-》

Analysis of cf-mtDNA and cf-nDNA fragment size distribution using different isolation methods in BV-2 cell supernatant of starvation-induced autophagy.

Fragment size distribution, the important biological properties of cell-free DNA (cfDNA), provides useful information required for diagnostic assay development. However, besides methodological discrepancies, it varies due to the complicated origins and occurrences of in vivo cfDNA. In addition, limited data are available concerning the cfDNA associated with autophagy and distributional difference between cf-mitochondrial DNA (cf-mtDNA) and cf-nuclear DNA (cf-nDNA) fragments. Here we developed an in vitro model of mouse microglial cell (BV-2) with starvation-induced autophagy, in which cfDNA was isolated from the cell supernatant by ultrafiltration (UF) and column-based commercial kit (CC), respectively. Using Agilent 2100 Bioanalyzer, a DNA ladder pattern as the presence of peaks corresponding to mono-, di- and tri-nucleosomes was clearly visualized both in isolation products of UF and CC. However, we also detected shorter fragments than mono-nucleosome by UF. In comparing the UF and CC, we found that the former produced the higher recovery efficiency for spiked-in DNA of shorter fragments than mono-nucleosome in both water and medium, but the latter was superior for spiked-in DNA fragments which were longer than or equal to mono-nucleosome in medium. Combined with these two isolation methods, we have observed that autophagy-associated cf-mtDNA and cf-nDNA were both highly enriched in <mono-nucleosomes fragments more than 71%, and showed no significant differences in the relative percentages for these four fragment sizes. These results have improved our understanding of the fragment size distribution of autophagy-derived cf-mtDNA and cf-nDNA in vitro, and might further develop application of cfDNA as a diagnostic tool.

Gong Y ，Huang Q ，Deng Y ，Zhou L ，Yi X ，Zhu J ，Wu W ... -  《-》