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

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.