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Very Short Mitochondrial DNA Fragments and Heteroplasmy in Human Plasma
Ruoyu Zhang1, Kiichi Nakahira2, Xiaoxian Guo1
1Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, USA.
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.
Insights
Researchers optimized methods to detect cell-free mitochondrial DNA (cf-mtDNA) in plasma, revealing its enrichment in short DNA fragments. This advance offers new potential for discovering biomarkers from cf-mtDNA heteroplasmy.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Cell-free DNA (cfDNA) is increasingly studied in various clinical conditions.
- Research on cell-free mitochondrial DNA (cf-mtDNA) remains limited.
- Understanding cfDNA architecture and its components like cf-mtDNA is crucial.
Purpose of the Study:
- To optimize protocols for isolating and sequencing short cfDNA fragments, particularly cf-mtDNA, from plasma.
- To characterize the size distribution of cf-mtDNA in plasma.
- To investigate cf-mtDNA heteroplasmy in sepsis patients.
Main Methods:
- Optimized DNA isolation and library preparation to retain short DNA fragments (<100bp).
- Applied optimized methods to plasma samples from sepsis patients.
- Utilized size selection to enrich for short cf-mtDNA fragments (30-60 bp).
- Detected and analyzed cf-mtDNA heteroplasmy by comparing plasma and leukocyte samples.
Main Results:
- Optimized methods increased short DNA fragment yield by 11.5-fold.
- cf-mtDNA was found to be highly enriched in short fragments (30-60 bp).
- Size selection further increased mtDNA recovery by 10.4-fold.
- Distinct cf-mtDNA heteroplasmy patterns were observed between plasma and leukocytes in sepsis patients, suggesting mixed tissue origins.
Conclusions:
- Optimized protocols enhance the recovery of short cfDNA, including cf-mtDNA.
- cf-mtDNA exhibits a unique size distribution in plasma.
- Plasma cf-mtDNA heteroplasmy analysis offers a promising avenue for biomarker discovery in clinical conditions like sepsis.
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