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

Abstract

BACKGROUND Cellular mitochondrial DNA (mtDNA) is organized as 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 BfaI cleavage signatures on mtDNA fragment ends to differentiate linear and circular mtDNA. mtDNA fragments with both ends carrying BfaI 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 MspI digestion was used for confirming the conclusions based on BfaI cleavage analysis. We analyzed the presence of BfaI 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.

Fetal mitochondrial DNA in maternal plasma in surrogate pregnancies: Detection and topology

“…As shown in Figure S1A, in contrast to plasma DNA of surrogate pregnant women without Bfa I treatment exhibiting a relatively smooth size distribution of mtDNA peaked at around 60 bp, plasma DNA with Bfa I digestion displayed an irregular distribution with a series of sporadic spikes up to 550 bp. The results were largely consistent with what was previously observed in plasma DNA of liver transplant patients 14 . However, we did not observe such differences in the size distribution of nuclear DNA molecules between before and after Bfa I digestion (Figure S1B).…”

“…Our data revealed that the linear and circular mtDNA coexisted in the plasma DNA of surrogate pregnant subjects. The fetal‐derived mtDNA in plasma was shown to be predominantly of a linear form (88%), which is reminiscent of the preponderance of linear liver‐derived mtDNA in the plasma of liver transplant recipients 14 . In contrast, half of surrogate maternal background mtDNA (mainly of hematopoietic and liver origin) appeared to be of circular origin.…”

“…The 4200 TapeStation (Agilent) and the Qubit 3.0 Fluorometer (Thermo Fisher Scientific) were used to evaluate the quality of the target‐captured DNA libraries. Targeted regions of interest included 1000 SNP (single nucleotide polymorphism) sites and human mtDNA regions for probe design and synthesis (Roche NimbleGen Inc.) as previously described 14 …”

“…The restriction enzyme Bfa I was used for differentiating linear and circular mtDNA as previously described 14 . This enzyme cleaves C^TAG sites and is expected to cut DNA once every 256 bp (1/4 4 ).…”

Identification and characterization of extrachromosomal circular DNA in maternal plasma

“…However, the above-mentioned studies predominantly focused on linear DNA fragments in plasma. We have recently demonstrated that there were different topologic forms (i.e., linear as well as circular) of plasma mitochondrial DNA (mtDNA) (13). Of interest, we observed that circular mtDNA molecules were predominantly of hematopoietic origin, whereas the liver-derived ones were predominantly linear.…”