Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR

Abstract

Defects of mitochondrial DNA (mtDNA) are an important cause of disease and play a role in the ageing process. There are multiple copies of the mitochondrial genome in a single cell. In many patients with acquired or inherited mtDNA mutations, there exists a mixture of mutated and wild type genomes (termed heteroplasmy) within individual cells. As a biochemical and clinical defect is only observed when there are high levels of mutated mtDNA, a crucial investigation is to determine the level of heteroplasmic mutations within tissues and individual cells. We have developed an assay to determine the relative amount of deleted mtDNA using real-time fluorescence PCR. This assay detects the vast majority of deleted molecules, thus eliminating the need to develop specific probes. We have demonstrated an excellent correlation with other techniques (Southern blotting and three- primer competitive PCR), and have shown this technique to be sensitive to quantify the level of deleted mtDNA molecules in individual cells. Finally, we have used this assay to investigate patients with mitochondrial disease and shown in individual skeletal muscle fibres that there exist different patterns of abnormalities between patients with single or multiple mtDNA deletions. We believe that this technique has significant advantages over other methods to quantify deleted mtDNA and, employed alongside our method to sequence the mitochondrial genome from single cells, will further our understanding of the role of mtDNA mutations in human disease and ageing.

Pathological mechanisms underlying single large‐scale mitochondrial DNA deletions

“…Accordingly, we have shown that deletion level increases with higher degrees of OXPHOS deficiency across a total of 673 fibers from 6 patients. Whereas the deletion level recorded in deficient fibers was consistently high among all patients (>85%), the mean deletion level recorded in 236 respiratory‐normal fibers was 22.6% (SD = 23.4%), consistent with others studies recording an average deletion level of 21 to 31% 9, 12, 13. The variable deletion levels of the respiratory‐normal fiber groups recorded in the 6 patients reported here (and others in previous studies) reflect their different homogenate mtDNA deletion levels.…”

“…Fibers positive for both complex I and complex IV immunoreactivity (OXPHOS group 1), deemed respiratory‐”normal,” harbored variable deletion loads, as previously reported 9. In contrast, fibers classed as negative for both complex I and complex IV consistently showed the highest mtDNA deletion levels, with a notably narrow range of deletion level (see Table 2).…”

“…Although a specific deletion encompassing 4,977bp is commonly reported,6 single, large‐scale mtDNA deletions vary in size (between 1.3 and 10kb), removing a number of genes encoding both mt‐tRNAs and structural mRNAs 7, 8, 9, 10, 11. They are typically mutational events that occur during the replication/repair of the mitochondrial genome and are invariably heteroplasmic in human tissues ‐ a condition where both mutated and wild‐type mtDNA genomes coexist in the same cell.…”

“…They are typically mutational events that occur during the replication/repair of the mitochondrial genome and are invariably heteroplasmic in human tissues ‐ a condition where both mutated and wild‐type mtDNA genomes coexist in the same cell. In postmitotic tissues, such as skeletal muscle, the proportion of deleted to wild‐type mtDNA must exceed a reported threshold (50–90%)9, 10, 11, 12, 13 for the expression of a biochemical defect, leading to a marked mosaicism between different fibers and well‐described cytochrome c oxidase (COX)‐deficient, ragged‐red fibers 13…”

Quantitative analysis of mitochondrial DNA 4977-bp deletion in sporadic breast cancer and benign breast diseases

“…Several methods have been used previously to quantify the level of mtDNA 4977 mutation. Long range PCR, primer-shift PCR and semi-quantitative PCR are time-consuming and the results are sometimes not reproducible (18)(19)(20). Furthermore, these PCR methods use an endpoint determination that may not be truly quantitative because of plateau effects (21).…”