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Single Mitochondrion Sequencing Uncovers Widespread Heteroplasmy

NEW YORK (GenomeWeb) – Researchers have sequenced the genomes of single mitochondria to uncover widespread heteroplasmy.

A cell can contain hundreds or thousands of mitochondria, and each mitochondrion has its own genome, often in multiple copies. Researchers from the University of Pennsylvania plucked single mitochondria out of mouse and human neuronal cell lines for sequencing, as they reported yesterday in Cell Reports. They uncovered unexpectedly high levels of heteroplasmy, in which an organism has a mix of wild-type and mutated genomes.

Mutations in mitochondrial genomes contribute to a number of diseases, including myoclonic epilepsy, mitochondrial encephalomyopathy, and Leber's hereditary optic neuropathy. And as the point at which mitochondrial diseases emerge is linked in part to the frequency of those mutations, the Penn researchers noted that measuring and tracking heteroplasmy could help monitor disease progression.

"By being able to look at a single mitochondrion and compare mutational dynamics between mitochondria, we will be able to gauge the risk for reaching a threshold for diseases associated with increasing numbers of mitochondrial mutations," senior author and Penn researcher James Eberwine said in a statement.

Using MitoTracker Red dye, the researchers identified single mitochondria within primary mouse neuronal or astrocyte cultures and collected them via micropipette. Each mitochondrion was then lysed and its DNA PCR amplified twice before deep sequencing on the Illumina platform. In all, they sequenced 165 biologically unique single mitochondrion from mouse neuronal or astrocytes samples as well as 21 samples from populations of mitochondria isolated from tissues. After filtering, they were left with 118 samples from 103 from unique cells, and uncovered 3.9 SNV sites per mitochondrion, on average.

Even though the mice came from the same inbred line, the researchers noted a wide distribution of SNVs. Four samples in particular had higher-than-expected numbers of variants. Two of those mitochondrial samples were from the same cell and shared a unique variant.

Through an ANOVA analysis, the researchers found that while cell type and cell location had no effect on SNV numbers, but the mouse's mother did. This suggested to them that SNV positions arise randomly and then accumulate in different lineages.

The researchers further identified four sites within the mouse mitochondrial genome that harbored SNVs in four or more samples. Variants at two of these sites were predicted to be of moderate or high impact, they noted. One site fell within an alpha-helical-membrane spanning region of a subunit of cytcochrome oxidase and variants there could affect the protein's structure, while the other was within the gene encoding the mitochondrial tRNA for glutamine.

Eberwine and his colleagues similarly examined mitochondrion obtained from a human brain cell culture derived from the left frontal cortex of a glioblastoma patient. They extracted and analyzed mitochondria from the cells as they did for the mice, swapping in human primers.

In the human samples, they uncovered one significant SNV position that was present in a number of single mitochondria as well as the population samples they analyzed. When they compared the amount of variation in the human mitochondria to what they observed in the mice, the researchers noted that the mice had accumulated greater levels of variation, though they noted that the human sample size was small.

Still, this indicated to the researchers that different modes of vegetative segregation might be at work in mice and humans. Because of that, they further suggested that human-based model systems are needed to study mitochondrial genomics and diseases.

Eberwine added that what he and his colleagues found could eventually be helpful in slowing the rate that mtDNA mutations accumulate. "This roadmap of the location and number of mutations within the DNA of a mitochondrion and across all of a cell's mitochondria is where we need to start," he said.