Very Short Mitochondrial DNA Fragments and Heteroplasmy in Human Plasma

Abstract

Cell free DNA (cfDNA) has received increasing attention and has been studied in a broad range of clinical conditions. However, few studies have focused on mitochondrial DNA (mtDNA) in the cell free form. We optimized DNA isolation and sequencing library preparation protocols to better retain short DNA fragments from plasma, and applied these optimized methods to plasma samples from patients with sepsis. Our methods can retain substantially shorter DNA, resulting in an average of 11.5 fold increase in short DNA fragments yield (DNA <100bp). We report that cf-mtDNA in plasma is highly enriched in short-size cfDNA (30~60 bp). Motivated by this unique size distribution, we size-selected short cfDNA, which further increased the mtDNA recovery rate by an average of 10.4 fold. We then detected mtDNA heteroplasmy in plasma from 3 patients. In one patient who previously received bone marrow transplantation, different minor allele frequencies were observed between plasma and leukocytes at heteroplasmic sites, consistent with mixed-tissue origin for cfDNA. For the other two patients, the heteroplasmy pattern is also different between plasma and leukocyte. Our study shed new lights into the architecture of the cfDNA, and mtDNA heteroplasmy identified in plasma provides new potential for biomarker discovery.

Blood contains circulating cell‐free respiratory competent mitochondria

“…Whole Genome Sequencing of healthy plasma revealed a wide‐size ranging population of short mitochondrial DNA fragments mainly decreasing in abundance from 30 to 300 nucleotides. In contrast, NcfDNA had a more homogenous distribution, conventionally peaking at 167 bp, corresponding to mono‐nucleosomes (Figure D) . Paired‐end sequencing enables high‐resolution measurement of DNA size but has a practical upper limit of read‐out at ~1000 bp.…”

“…In contrast, NcfDNA had a more homogenous distribution, conventionally peaking at 167 bp, corresponding to mono-nucleosomes ( Figure 1D). 17,28 Paired-end sequencing enables high-resolution measurement of DNA size but has a practical upper limit of readout at ~1000 bp. Since no McfDNA was detected between 500 bp and 1000 bp, Whole Genome Sequencing only detected the minor fraction formerly revealed by gel electrophoresis ( Figure 1C).…”

The diverse origins of circulating cell‐free DNA in the human body: a critical re‐evaluation of the literature

“…Recent research by Zhang et al . (), on the other hand, identified circulating mtDNA fragments of 42 bp. It is possible that these differences in reported mtDNA sizes and forms may be due to different processing methods rather than being of physiological origin, as discussed in Section VI.…”

“…mtDNA can be released from mitochondria into the cytoplasm and extracellular environment during cellular clearance or repair processes, particularly autophagy, apoptosis, or necrosis (see Sections IV.2, III.3 and III.2; Zhang et al, ). The presence of circulating mtDNA was first reported by Zhong et al .…”

“…Circulating mtDNA is not always correlated with circulating nuclear DNA levels in certain pathological conditions, providing potentially unique pathophysiological information distinct from that of circulating nuclear DNA (Zhang et al, ). The lack of protection of mtDNA, due to the absence of histones, suggests that circulating mtDNA fragments should be shorter than circulating nuclear DNA.…”