Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jul 1;323(1):L84-L92.
doi: 10.1152/ajplung.00128.2022. Epub 2022 Jun 14.

Absolute quantification of plasma mitochondrial DNA by droplet digital PCR marks COVID-19 severity over time during intensive care unit admissions

Mark L Hepokoski  1   2 , Mazen Odish  2 , Michael T Lam  1   2   3 , Nicole G Coufal  4   5 , Mark L Rolfsen  6 , Gerald S Shadel  3 , Alexandra G Moyzis  3 , Alva G Sainz  3   7 , Puja G Takiar  6 , Sagar Patel  2 , Austin J Leonard  6 , Negin Samandari  1 , Emily Hansen  4 , Samantha Trescott  4 , Celina Nguyen  4 , Kristen Jepsen  8 , Gary Cutter  9 , Mark N Gillespie  10 , Roger G Spragg  2 , Roman Sasik  11 , Joachim H Ix  1   12
Affiliations

Absolute quantification of plasma mitochondrial DNA by droplet digital PCR marks COVID-19 severity over time during intensive care unit admissions

Mark L Hepokoski et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

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.

Keywords: ARDS; COVID-19; mitochondrial DNA.

PubMed Disclaimer

Conflict of interest statement

G. Cutter has the following conflicts of interest to disclose: Data and Safety Monitoring Boards: AI Therapeutics, AMO Pharma, Astra-Zeneca, Avexis Pharmaceuticals, Biolinerx, Brainstorm Cell Therapeutics, Bristol Meyers Squibb/Celgene, CSL Behring, Galmed Pharmaceuticals, Green Valley Pharma, Horizon Pharmaceuticals, Immunic, Mapi Pharmaceuticals LTD, Merck, Mitsubishi Tanabe Pharma Holdings, Opko Biologics, Prothena Biosciences, Novartis, Regeneron, Sanofi-Aventis, Reata Pharmaceuticals, NHLBI (Protocol Review Committee), University of Texas Southwestern, University of Pennsylvania, Visioneering Technologies, Inc. Consulting or Advisory Boards: Alexion, Antisense Therapeutics, Biogen, Clinical Trial Solutions LLC, Genzyme, Genentech, GW Pharmaceuticals, Immunic, Klein-Buendel Incorporated, Merck/Serono, Osmotica Pharmaceuticals, Perception Neurosciences, Protalix Biotherapeutics, Recursion/Cerexis Pharmaceuticals, Regeneron, Roche, SAB Biotherapeutics. G. Cutter is employed by the University of Alabama at Birmingham and is President of Pythagoras, Inc., a private consulting company located in Birmingham, Alabama. J. H. Ix leads an Investigator Initiated Research Grant supported by Baxter International, has served on Advisory Boards for Akebia, AstraZeneca, and Bayer, and serves as a member of a Data and Safety Monitoring Board for Sanifit International. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

Figures

Figure 1.
Figure 1.
Sequencing and mtDNA target sequence comparisons. A: average reads by mitochondrial genome location. B: size distribution of cell-free mtDNA reads. C: CYB vs. ND1 target sequence reads. D: CYB vs. ND1 copy number in plasma by droplet digital PCR. E: linear regression of CYB vs. ND1 copy number. For A, B, and C: n = 18 subjects; E: n = 111 samples. CYB, cytochrome B; mtDNA, mitochondrial DNA; ND1, NADH dehydrogenase 1.
Figure 2.
Figure 2.
Plasma mtDNA and disease severity. A: CYB copy number in no/mild vs. moderate/severe (mod/sev) ARDS on study day 1. B: ND1 copy number in no/mild vs. mod/sev ARDS on study day 1. C: CYB copy number on study day 1 and sequential organ failure assessment (SOFA) score. D: ND1 copy number on study day 1 and SOFA score. E: CYB copy number on study day 1 and modified Murray lung injury score. F: ND1 copy number on study day 1 and modified Murray lung injury score. **P < 0.01, n = 19 subjects. ARDS, acute respiratory distress syndrome; CYB, cytochrome B; mtDNA, mitochondrial DNA; ND1, NADH dehydrogenase 1.
Figure 3.
Figure 3.
Serial assessments of mtDNA and ARDS outcomes. A: serial CYB concentrations in patients with no/mild ARDS who survived vs. those with mod/severe ARDS who survived vs. nonsurvivors. B: serial ND1 concentrations in patients with no/mild ARDS who survived vs. those with mod/severe ARDS who survived vs. nonsurvivors. n = 6 subjects for no/mild ARDS survivors, n = 11 subjects for mod/severe ARDS survivors, and n = 3 subjects for nonsurvivors. ARDS, acute respiratory distress syndrome; CYB, cytochrome B; mtDNA, mitochondrial DNA; ND1, NADH dehydrogenase 1.
See this image and copyright information in PMC

References

    1. Tzotzos SJ, Fischer B, Fischer H, Zeitlinger M. Incidence of ARDS and outcomes in hospitalized patients with COVID-19: a global literature survey. Crit Care 24: 516, 2020. doi:10.1186/s13054-020-03240-7. - DOI - PMC - PubMed
    1. Guérin C, Reignier J, Richard JC, Beuret P, Gacouin A, Boulain T, Mercier E, Badet M, Mercat A, Baudin O, Clavel M, Chatellier D, Jaber S, Rosselli S, Mancebo J, Sirodot M, Hilbert G, Bengler C, Richecoeur J, Gainnier M, Bayle F, Bourdin G, Leray V, Girard R, Baboi L, Ayzac L. PROSEVA Study Group. Prone positioning in severe acute respiratory distress syndrome. N Engl J Med 368: 2159–2168, 2013. doi:10.1056/NEJMoa1214103. - DOI - PubMed
    1. West AP, Khoury-Hanold W, Staron M, Tal MC, Pineda CM, Lang SM, Bestwick M, Duguay BA, Raimundo N, MacDuff DA, Kaech SM, Smiley JR, Means RE, Iwasaki A, Shadel GS. Mitochondrial DNA stress primes the antiviral innate immune response. Nature 520: 553–557, 2015. doi:10.1038/nature14156. - DOI - PMC - PubMed
    1. Krychtiuk KA, Ruhittel S, Hohensinner PJ, Koller L, Kaun C, Lenz M, Bauer B, Wutzlhofer L, Draxler DF, Maurer G, Huber K, Wojta J, Heinz G, Niessner A, Speidl WS. Mitochondrial DNA and toll-like receptor-9 are associated with mortality in critically ill patients. Crit Care Med 43: 2633–2641, 2015. doi:10.1097/CCM.0000000000001311. - DOI - PubMed
    1. Wu G, Zhu Q, Zeng J, Gu X, Miao Y, Xu W, Lv T, Song Y. Extracellular mitochondrial DNA promote NLRP3 inflammasome activation and induce acute lung injury through TLR9 and NF-κB. J Thorac Dis 11: 4816–4828, 2019. doi:10.21037/jtd.2019.10.26. - DOI - PMC - PubMed

Publication types

Substances