Plasma-derived cell-free mitochondrial DNA: A novel non-invasive methodology to identify mitochondrial DNA haplogroups in humans

Christopher Newell; Stacey Hume; Steven C Greenway; Lynn Podemski; Jane Shearer; Aneal Khan

doi:10.1016/j.ymgme.2018.10.002

Plasma-derived cell-free mitochondrial DNA: A novel non-invasive methodology to identify mitochondrial DNA haplogroups in humans

Mol Genet Metab. 2018 Dec;125(4):332-337. doi: 10.1016/j.ymgme.2018.10.002. Epub 2018 Oct 16.

Authors

Christopher Newell¹, Stacey Hume², Steven C Greenway³, Lynn Podemski², Jane Shearer⁴, Aneal Khan⁵

Affiliations

¹ Department of Medical Genetics, Cumming School of Medicine, University of Calgary, Alberta, Canada. Electronic address: cnewell@ucalgary.ca.
² Department of Medical Genetics, University of Alberta, Alberta, Canada.
³ Department of Cardiac Sciences, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada; Department of Pediatrics, Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Alberta, Canada.
⁴ Faculty of Kinesiology, University of Calgary, Alberta, Canada.
⁵ Department of Medical Genetics, Cumming School of Medicine, University of Calgary, Alberta, Canada; Department of Pediatrics, Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Alberta, Canada.

PMID: 30361042
DOI: 10.1016/j.ymgme.2018.10.002

Abstract

Background: Mitochondrial diseases are a clinically heterogeneous group of diseases caused by mutations in either nuclear or mitochondrial DNA (mtDNA). The diagnosis is challenging and has frequently required a tissue biopsy to obtain a sufficient quantity of mtDNA. Less-invasive sources mtDNA, such as peripheral blood leukocytes, urine sediment, or buccal swab, contain a lower quantity of mtDNA compared to tissue sources which may reduce sensitivity. Cellular apoptosis of tissues and hematopoetic cells releases fragments of DNA and mtDNA into the circulation and these molecules can be extracted from plasma as cell-free DNA (cfDNA). However, entire mtDNA has not been successfully identified from the cell free fraction previously. We hypothesized that the circular nature of mtDNA would prevent its degradation and a higher sensitivity method, such as next generation sequencing, could identify intact cf-mtDNA from human plasma.

Methods: Plasma was obtained from patients with mitochondrial disease diagnosed from skeletal muscle biopsy (n = 7) and healthy controls (n = 7) using a specially cfDNA collection tube (Streck Inc.; La Vista, NE). To demonstrate the presence of mtDNA within these samples, we amplified the isolated DNA using custom PCR primers specific to overlapping fragments of mtDNA. cfDNA samples were then sequenced using the Illumina MiSeq sequencing platform.

Results: We confirmed the presence of mtDNA, demonstrating that the full mitochondrial genome is in fact present within the cell-free plasma fraction of human blood. Sequencing identified the mitochondrial haplogroup matching with the tissue specimen for all patients.

Conclusion: We report the existence of full length mtDNA in cell-free human plasma that was successfully used to perform haplogroup matching. Clinical applications for this work include patient monitoring for heteroplasmy status after mitochondrially-targeted therapies or haplogroup monitoring as a measure of stem cell transplantation.

Keywords: Cell-free mitochondrial DNA; Mitochondrial disease; mtDNA Haplotype; mtDNA Mutations.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Aged
Aged, 80 and over
Case-Control Studies
Cell-Free Nucleic Acids / blood
Cell-Free Nucleic Acids / genetics*
DNA, Mitochondrial / blood
DNA, Mitochondrial / genetics*
Female
Haplotypes / genetics*
High-Throughput Nucleotide Sequencing / methods*
Humans
Male
Middle Aged
Mitochondrial Diseases / diagnosis*
Mitochondrial Diseases / genetics*

Substances

Cell-Free Nucleic Acids
DNA, Mitochondrial