Topologic Analysis of Plasma Mitochondrial DNA Reveals the Coexistence of Both Linear and Circular Molecules

Background: Cellular mitochondrial DNA (mtDNA) is organized as a circular, covalently closed and double-stranded DNA. Studies have demonstrated the presence of short mtDNA fragments in plasma. It is not known whether circular mtDNA might concurrently exist with linear mtDNA in plasma. Methods: We elucidated the topology of plasma mtDNA using restriction enzyme Bfa I cleavage signatures on mtDNA fragment ends to differentiate linear and circular mtDNA. mtDNA fragments with both ends carrying Bfa I cleavage signatures were defined as circular-derived mtDNA, whereas those with no cleavage signature or with 1 cleavage signature were defined as linear-derived mtDNA. An independent assay using exonuclease V to remove linear DNA followed by restriction enzyme Msp I digestion was used for confirming the conclusions based on Bfa I cleavage analysis. We analyzed the presence of Bfa I cleavage signatures on plasma DNA ends in nonhematopoietically and hematopoietically derived DNA molecules by sequencing plasma DNA of patients with liver transplantation and bone marrow transplantation. Results: Both linear and circular mtDNA coexisted in plasma. In patients with liver transplantation, donor-derived (i.e., liver) mtDNA molecules were mainly linear (median fraction, 91%; range, 75%–97%), whereas recipient-derived (i.e., hematopoietic) mtDNA molecules were mainly circular (median fraction, 88%; range, 77%–93%). The proportion of linear mtDNA was well correlated with liver DNA contribution in the plasma DNA pool ( r = 0.83; P value = 0.0008). Consistent data were obtained from a bone marrow transplantation recipient in whom the donor-derived (i.e., hematopoietic) mtDNA molecules were predominantly circular. Conclusions: Linear and circular mtDNA molecules coexist in plasma and may have different tissue origins.