Several conclusions can be drawn from this work. On the technical side, we have shown that the addition of a fluorescent‐labelled oligonucleotide primer in the last PCR cycle enables the accurate quantification of mtDNA heteroplasmy. Although the strongest correlation was between the undiluted and 1:2 diluted fluorescent products and the last cycle hot PCR, all the assays correlated well with an R2>0.95. We were surprised to find that the serial dilution of the fluorescent PCR products did not have a dramatic effect on the assay. This probably reflects the automated electrostatic injection methods used by most capillary‐based DNA analysers, which load a fixed amount of labelled DNA rather than a fixed volume of solution. It is possible that different results are obtained for manually loaded gel‐based systems. Arguably, the reproducibility of the fluorescent assay exceeds that of last cycle hot PCR (see the approximately 50% 3243A→G data in fig 1), and, given the inherent risks of using radiochemicals, last cycle fluorescent analysis should now be the benchmark for measuring mtDNA heteroplasmy.
Using the last cycle fluorescent PCR, we confirmed that the percentage of 3243A→G decreases in blood over time. The mean percentage decrease of 0.6% per year corresponds well to the largest previous study (0.69% per year; SD 0.61).6
In our study (fig 2), the greatest rate of decrease in the percentage of mutated mtDNA was seen in the younger subjects, consistent with an exponential loss of 3243A→G from the peripheral blood. With this in mind, reporting mean values for the percentage decrease could be misleading, and explains why the mean value in our adult study was significantly less than that found in one study of six subjects (1.12% per year; SD 0.65),7
which predominantly involved observations made over childhood (two‐sample t test comparing the data in table 1 with the data in Rahman et al7
The most striking novel finding is the profound depletion of mtDNA in blood leucocytes in patients harbouring this mutation. Our control data for the mean mtDNA copy number/leucocyte are remarkably similar to other published values based on real‐time PCR and other techniques,11,12
confirming that we were using a reliable assay. How can we therefore explain these observations? Given that the peripheral blood count was normal in each case, a change in the number of cell nuclei or cell types probably does not account for the profound depletion we have observed. From first principles, the depletion of mtDNA could be due to decreased rates of mtDNA synthesis relative to the intense cellular proliferation in leucocyte precursors. An alternative (and non‐exclusive) explanation is an increased loss of mtDNA from the precursors of the mature cells.
The peripheral leucocyte population is continuously recycled. Neutrophils are the largest single component (up to 80% of circulating leucocytes), maturing in the bone marrow for 11–12 days before being released into the circulation, where they remain for 6–8 h.13
The entire neutrophil population in the peripheral blood (approximately 5×1010
cells) is therefore replenished on a daily basis, requiring a massive amount of mtDNA replication to maintain normal mtDNA levels. This is sustained by a limited number of stem cells (1 in 104
to 1 in 105
marrow cells), which turn over at a low rate and repopulate the blood through a hierarchical cascade of cell divisions.14
It is therefore far more probable that any effect on mtDNA replication would manifest during the massive expansion of daughter cells leading to the formation of mature leucocytes, rather than in the stem‐cell population itself. Even a subtle decrease in the replication rate could lead to the dramatic depletion that we have observed in the peripheral blood. For example, if the mtDNA replication rate was a fraction f of the normal replication rate, then after a series of n cell divisions the leucocyte precursors would be depleted by a factor fn
. Twenty cell divisions (the number required for a million‐fold increase in cells) would give our observed depletion to 23% of the normal level with f
Could the bioenergetic defect be responsible for a relative decrease in rate of mtDNA synthesis in the leucocyte precursors? Although plausible, this explanation is difficult to reconcile with the increased amount of mtDNA seen in post‐mitotic tissues of subjects harbouring 3243A→G.15
In addition, if the loss of mtDNA were directly related to a defect of ATP synthesis, we would expect a closer relationship between the percentage level of mutated mtDNA and the degree of depletion. This was clearly not the case—for example, low amounts of mtDNA were found in a young subject with approximately 50% 3243A→G in blood and an older subject with approximately 5% 3243A→G in blood (figs 2 and 3). Recent in vitro studies on human NT2 teratocarcinoma cybrids harbouring 3243A→G (NT2.3243 cybrids) cast light on this issue.16
High percentages of 3243A→G were associated with a profound loss of mtDNA from the NT2.3243 cybrids. This seemed to be mutation specific, as the same depletion was not seen in NT2 cybrids homoplasmic for the 1555A→G mtDNA mutation. Moreover, the presence of a known suppressor mutation (12300G→A), which ameliorates the biochemical effects of 3243A→G at high levels, did not prevent the loss of mtDNA from the cybrid cell lines, indicating that the biochemical defect is not the principal factor behind the depletion. Remarkably, not all the cell lines with >99% 3243A→G lost their mtDNA, proving that the percentage of mutated mtDNA is not an absolute determinant of the depletion. Further work showed slow rates of segregation of the 3243A→G mutation in NT2.3243 cybrids, providing indirect evidence that functional or physical partitioning of mtDNA molecules contributes to the segregation process. These observations highlight the complex interplay between intercellular and intracellular signals that are involved in the mitotic segregation of the 3243A→G mutation.16
Similar mechanisms may be involved in rapidly dividing tissues in vivo, including the leucocyte population. It is intriguing that, as the percentage of mutated mtDNA in blood decreases in subjects harbouring the 3243A→G mutation (fig 2), there is an overall increase in the amount of mtDNA in blood, particularly wild‐type mtDNA (fig 4). This suggests a degree of recovery from the depletion as time passes, although the amount never reaches normal values.
An alternative explanation for the loss of one mtDNA genotype in blood is through immune surveillance mediated through an abnormal cell surface epitope. This hypothesis is supported by the description of a complex I (ND) gene variant in mice that forms a maternally transmitted major histocompatibility antigen.17
However, recent work on heteroplasmic mice has shown that immune‐mediated cell loss does not occur in heteroplasmic mice with the C57B/BALB genotype, and nuclear genes may be important.18
Finally, the depletion may be a secondary phenomenon, as in other mitochondrial disorders. Mutations in OPA1
cause the most common form of autosomal dominant optic atrophy. In a recent study, subjects with OPA1
mutations had markedly lower levels of mtDNA in leucocytes than controls.11
Given the central role of OPA1 in maintaining the structural integrity of mitochondria and mtDNA, it was suggested that the primary genetic defect led to the loss of both mitochondria and mitochondrial genomes. A similar structural disintegration of the mitochondrial network has been seen in subjects harbouring primary mtDNA defects, potentially explaining the observations we report here. By contrast, one study19
described a mild increase in the amount of mtDNA in leucocytes from subjects harbouring mtDNA mutations that cause Leber hereditary optic neuropathy. However, in contrast with the 3243A→G mutation, the biochemical defect in patients with Leber hereditary optic neuropathy is mild and sometimes undetectable.20,21
Further work will determine whether depletion of mtDNA is seen in other mtDNA disorders.
If the 3243A→G mutation is causing depletion through an effect on the structural integrity of mitochondria in leucocytes, why does this not occur in non‐dividing tissues such as skeletal muscle, where there tends to be a proliferation of mitochondria and mtDNA (supplementary table A online at www.jmg.bmjjournals.com/supplemental
)? At present, we can only speculate, but recent work in our laboratory has shown that, although initially the amount of skeletal muscle mtDNA is high in subjects with mtDNA mutations, there is a progressive loss of mtDNA over time.22
This could be partly due to muscle deconditioning, but it may be due to a more general process related to mitochondrial fragility. These findings have important implications for the molecular diagnosis of mitochondrial disorders—the presence of depletion may be a secondary phenomenon and the primary molecular defect may be a mtDNA mutation or a nuclear gene mutation not directly involved in the synthesis of mtDNA.
- The percentage of the 3243A→G MTTL1 mutation in blood decreases exponentially during life.
- Peripheral blood leucocytes in patients harbouring the 3243A→G mutation are profoundly depleted of mitochondrial DNA (mtDNA).
- Depletion of mtDNA is not always due to mutation of a nuclear gene involved in mtDNA maintenance.