Detection of Heteroplasmic Mitochondrial DNA in Single Mitochondria

Abstract

BackgroundMitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Leber's hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases.MethodologyThis work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrion's DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion.SignificanceMolecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria.

Sequence-Based Polymorphisms in the Mitochondrial D-Loop and Potential SNP Predictors for Chronic Dialysis

“…For example, elevated oxidative stress in chronic peritoneal dialysis patients may lead to alterations in the mtDNA copy number in peripheral leukocytes [33]. In our current study, the mtSNPs listed in Table 3 were homoplasmic, as revealed by sequencing chromatograms (data not shown) [58]–[60]. However, we cannot exclude the possibility that a minor fraction of heteroplasmic mutations, below the level of sensitivity of the sequencing method that we used, may be present.…”

Pervasive within-Mitochondrion Single-Nucleotide Variant Heteroplasmy as Revealed by Single-Mitochondrion Sequencing

“…While others have measured genetic variation of mtDNA isolated from single mitochondrion, these studies were limited to specific regions of the mtDNA, focused specifically on deletions, or measured a single mitochondrion isolated from a complex mixture of cell types (Cavelier et al, 2000; Reiner et al, 2010; Poe III et al, 2010)). This precludes assessment of total mutational burden across the entire genome for all genomes present.…”

Genetic Differentiation of the Mitochondrial Cytochrome Oxidase c Subunit I Gene in Genus Paramecium (Protista, Ciliophora)

“…Thus, there was little insight into the degree of divergence of genes at the level of the individual cell, a process termed as heteroplasmy. Heteroplasmy has been documented in various metazoan groups [18]–[25] and the kinetoplastid protist Trypanosoma cruzi [26] but not in ciliated protists. Therefore, it is not possible to conclusively determine whether the observed intra-specific divergence of mitochondrial genes in ciliates is genuine or the result of intra- individual genetic variation.…”