Search

Background: Research on circulating mitochondrial DNA (cir-mtDNA) based diagnostic is insufficient, as to its function, origin, structural features, and particularly its standardization of isolation. To date, plasma preparation performed in previous studies do not take into consideration the potential bias resulting from the release of mitochondria by activated platelets., Methods: To tackle this, we compared the mtDNA amount determined by a standard plasma preparation method or a method optimally avoiding platelet activation. MtDNA extracted from the plasma of seven healthy individuals was quantified by Q-PCR in the course of the process of both methods submitted to filtration, freezing or differential centrifugation., Results: 98.7 to 99.4% of plasma mtDNA corresponded to extracellular mitochondria, either free or into large extracellular vesicles. Without platelet activation, the proportion of both types of entities remained preponderant (76-80%), but the amount of detected mtDNA decreased 67-fold., Conclusion: We show the high capacity of platelets to release free mitochondria in "in vitro" conditions. This represents a potent confounding factor when extracting mtDNA for cir-mtDNA investigation. Platelet activation during pre-analytical conditions should therefore be avoided when studying cir-mtDNA. Our findings lead to a profound revision of the assumptions previously made by most works in this field. Overall, our data suggest the need to characterize or isolate mtDNA associated various structural forms, as well as to standardize plasma preparation, to better circumscribe cir-mtDNA's diagnostic capacity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Chromosome stability is a key point in genome evolution, particularly that of the Y chromosome. The Y chromosome loss in blood and tumor cells is well established. Through processes that are common to other chromosomes too, the Y chromosome undergoes degradation and fragmentation in the blood stream before elimination. This process gives rise to circulating DNA (cirDNA) fragments, whose examination may provide potential insight into the role of DNA fragmentation in blood for the Y chromosome elimination. In this study, we employed shallow whole genome sequencing (sWGS) to comprehensively assess the total cirDNA and the individual chromosome fragment size profiles in the plasma of healthy male individuals. Here, we show that (i) the fragment size profiles of total circulating DNA (cirDNA) and DNA fragments originating from autosomes and the X chromosome in blood plasma are homogeneous, and have a remarkably low variability (mean CV = 7%) among healthy individuals, (ii) the Y chromosome has a distinct fragment size profile with the accumulation of the fragment < 145 bp and depletion of the dinucleosome-associated fragments (290-390 bp), and its fragment fraction in blood decreases with age. These results indicate a higher fragmentation of the Y chromosome compared to other chromosomes and this in turn might be due to its increased susceptibility to degradation. Our findings pave the way for an elucidation of the impact of chromosomal origin on DNA degradation and the Y chromosome biology., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: As circulating DNA (cirDNA) is mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood, apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA., Methods: We studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET-associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N = 28; systemic lupus erythematosus, N = 10; metastatic colorectal cancer, N = 10; and from healthy individuals, N = 114)., Results: Our studies reveal that gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24 h incubation in serum corresponded to mono-N DNA); "ex vivo" NET formation, as demonstrated by a concurrent 5-, 5-, and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2 and 41% of the remaining DNA after 2 h in serum corresponded to 1-10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8 h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO, and cirDNA was detected in plasma samples from lupus, COVID-19, and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI., Conclusions: Our work describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA., (© 2022. The Author(s).)

To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole-genome sequencing (WGS) from both double- and single-strand DNA library preparations (DSP and SSP, n = 7) and using quantitative PCR (Q-PCR, n = 116). The size profile in healthy individuals was remarkably homogenous when using DSP sequencing or SSP sequencing. CfDNA size profile had a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes, and chromatin of higher molecular size (>1000 bp) can be estimated as 67.5% to 80%, 9.4% to 11.5%, and 8.5% to 21.0%, respectively. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97%-98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between metastatic colorectal cancer patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content.

Circulating cell-free DNA (cfDNA) isolated from blood has been identified as a potential biomarker in numerous fields, and has been the object of intensive research over the past decade, although its original discovery dates back 60 years. While it is already used routinely in commercial and clinical practice in oncology and prenatal testing, other potential applications have emerged, including for diabetes, cardiovascular diseases, organ transplantation, autoimmune diseases, sepsis, trauma, and sport management. As with the discovery and development of any biomarker, preanalytical requirements and documentation are as important as analytical requirements. Except for the case of noninvasive prenatal testing and prenatal diagnosis, the implementation of cfDNA in a clinical setting remains limited because of the lack of standardization of cfDNA analysis. In particular, only a few attempts have been made to collect and pool scientific data on the relevant preanalytical factors, and no standard operating procedure has yet been set. For this report, we have performed a thorough and systematic search via MEDLINE ® for relevant preanalytical variables and patient factors. These form the basis of the guidelines we propose for analyzing nuclear cfDNA., (© 2019 American Association for Clinical Chemistry.)