Our studies have shown that real-time PCR is a valuable technique in determining the level of deleted mtDNA molecules in single cells. In our hands the technique is reproducible even using very low quantities of mtDNA. The technique will be of value not only in the diagnosis of patients with suspected mtDNA deletions, but also investigating the role of clonal expansion of mtDNA deletions in ageing. These studies have been performed using a real-time PCR machine that is capable of detecting the amplification of a single target template in each tube. A further increase in sensitivity will be possible using a real-time PCR machine capable of detecting two such reactions in the same tube by the use of different fluorescent dyes.
Real-time PCR is only one of several methods that can be used to quantify the level of rearranged mtDNA molecules, but we believe it has significant advantages over other techniques. Southern blotting is the standard method to measure the level of the deletion in DNA extracted from tissues. The major disadvantage of Southern blotting is the large amount of DNA required for analysis, and it certainly is not a technique that can be used to investigate mtDNA deletions in single cells. In contrast, three-primer competitive PCR is a semi-quantitative method used to determine the level of a deletion in a single cell. The major disadvantage of this technique is that it is specific to an individual deletion and fresh primers and conditions have to be devised for each deletion to be detected. This may be possible when a patient has a single deletion but it is not possible to use this technique for the investigation of patients with multiple deletions or in the investigation of mtDNA deletions in ageing cells. For both Southern blotting and three-primer competitive PCR, we obtained a good correlation with the results using real-time PCR. The identification of deleted molecules in individual cells is also possible using techniques such as long-range PCR (30
) and primer-shift PCR (20
). These techniques are not quantitative and the value of simply detecting a deletion is limited. Whilst other workers have previously described a real-time PCR method to detect a common mtDNA deletion in blood DNA (33
), our specific aim was to apply this technique to the study of mtDNA rearrangements in individual cells, the level of the biochemical defect, and to confirm the validity of this approach using patient samples.
Although our original idea was merely to use the samples from patients to validate the technique of quantitative PCR, the technique has shown some very interesting differences between patients with single and multiple deletions. In all our patients with single deletions the COX-deficient cells had high levels of deletion removing ND4. In patients with multiple deletions this was not the case and all patients had COX-deficient cells that did not contain high levels of mitochondrial genome with a deletion removing ND4. Thus, this technique can rapidly differentiate between these two conditions. Whilst long-range PCR is also a very helpful technique for determining if there are single or multiple deletions, this technique can give misleading results when amplifying samples from older individuals due to the non-quantitative nature of the PCR.
Our findings in the patients with multiple mtDNA deletions were a little surprising. These are an interesting group of patients whose primary genetic defect is not in the mitochondrial genome. In these patients the defect is inherited in an autosomal manner and genetic defects have been identified in a number of genes involved in the maintenance of mtDNA, including ANT1 (34
), POLG (35
) and Twinkle (36
), a mitochondrial helicase. In these patients, different deletions clonally expand within individual cells to high levels, and thus result in COX deficiency. It has been stated that in patients with multiple mtDNA deletions ~96% of the deletions involve ND4; this was not found in our patients, which suggests that previous studies have underestimated the percentage of molecules in which the deletion involves other parts of the genome. Alternatively, as the primary defect often involves enzymes involved in mtDNA maintenance, it is possible that some of the other COX-deficient muscle fibres contain clonally expanded mtDNA point mutations. In none of the fibres without deleted mtDNA did we detect mtDNA depletion, as the mtDNA copy number was the same between fibres that contained deleted mtDNA species and those that did not.
There is a similarity between the situation in patients with multiple mtDNA deletions and the normal ageing process. In both groups there is the presence of COX-deficient cells. In ageing, somatic mutations occur, presumably due to a combination of DNA damage with inadequate repair. It is speculated that each COX-deficient cell should represent the clonal expansion of an individual mtDNA mutation (37
). Our development of single cell sequencing of the mitochondrial genome will allow us to detect clonal expansion of mtDNA point mutations. We believe real-time PCR will provide a technique to investigate for the possibility of clonal expansion of partially deleted mtDNA molecules.