Journal Article

Next-generation sequencing reveals mitogenome diversity in plasma extracellular vesicles from colorectal cancer patients

Stang, Espen, Fuglestad, Anniken Jørlo, Sanabria, Adriana M., Bjørnetrø, Tonje, Johansen, Christin, Kersten, Christian, Meltzer, Sebastian, Redalen, Kathrine Røe, Trøseid, Anne-Marie Siebke, Ree, Anne Hansen, Bousquet, Paula A., Lüders, Torben

2023

Overview

Background Recent reports have demonstrated that the entire mitochondrial genome can be secreted in extracellular vesicles (EVs), but the biological attributes of this cell-free mitochondrial DNA (mtDNA) remain insufficiently understood. We used next-generation sequencing to compare plasma EV-derived mtDNA to that of whole blood (WB), peripheral blood mononuclear cells (PBMCs), and formalin-fixed paraffin-embedded (FFPE) tumor tissue from eight rectal cancer patients and WB and fresh-frozen (FF) tumor tissue from eight colon cancer patients. Methods Total DNA was isolated before the mtDNA was enriched by PCR with either two primer sets generating two long products or multiple primer sets (for the FFPE tumors), prior to the sequencing. mtDNA diversity was assessed as the total variant number, level of heteroplasmy (mutant mtDNA copies mixed with wild-type copies), variant distribution within the protein-coding genes, and the predicted functional effect of the variants in the different sample types. Differences between groups were compared by paired Student’s t -test or ANOVA with Dunnett’s multiple comparison tests when comparing matched samples from patients. Mann–Whitney U test was used when comparing differences between the cancer types and patient groups. Pearson correlation analysis was performed. Results In both cancer types, EV mtDNA presented twice as many variants and had significantly more low-level heteroplasmy than WB mtDNA. The EV mtDNA variants were clustered in the coding regions, and the proportion of EV mtDNA variants that were missense mutations (i.e., estimated to moderately affect the mitochondrial protein function) was significantly higher than in WB and tumor tissues. Nonsense mutations (i.e., estimated to highly affect the mitochondrial protein function) were only observed in the tumor tissues and EVs. Conclusion Taken together, plasma EV mtDNA in CRC patients exhibits a high degree of diversity. Trial registration ClinicalTrials.gov: NCT01816607 . Registered 22 March 2013.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Analysis / Biomarkers / Biomedical and Life Sciences / Biomedicine / Biotechnology industry / Cancer / Cancer patients / Cancer Research / Care and treatment / Cell cycle / Colon cancer / Colorectal cancer / Correlation analysis / Diagnosis / DNA sequencing / Drug resistance / Extracellular vesicles / Extracellular vesicles in cancer progression / Formaldehyde / Genes / Genetic aspects / Genomes / Genomics / Health aspects / Health Promotion and Disease Prevention / Heteroplasmy / Leukocytes (mononuclear) / Medicine/Public Health / Metabolism / Microscopy / Missense mutation / Mitochondrial DNA / mtDNA / Mutation / Next-generation sequencing / Nucleotide sequencing / Oncology / Paraffin / Patients / Peripheral blood mononuclear cells / Phosphorylation / Plasma / Prevention / Proteins / Research Article / Scientific equipment and supplies industry / Surgical Oncology / Tumors