Human Molecular Genetics, 2001, Vol. 10, No. 1 17-24
© 2001 Oxford University Press
Quantification and sequencing of somatic deleted mtDNA in single cells: evidence for partially duplicated mtDNA in aged human tissues
Beth Israel Deaconess Medical Center and Harvard Medical School, 77 Avenue Louis Pasteur, Room 921, Boston, MA 02115, USA
Received 23 June 2000; Revised and Accepted 8 November 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Single-cell PCR of the whole mitochondrial genome provides detailed information about intracellular clonal expansions of deleted mitochondrial DNA (
mtDNA), which contribute to aging of the muscle and possibly other tissues. Analysis of 
1400 cells from heart, diaphragm and skeletal muscle from 20 individuals without mitochondrial disease revealed that up to 25% of cells in a tissue sample may bear clonally expanded mtDNA. Sequence analysis of >50 clonal mtDNA reveals that about half of them lack the light strand origin of replication. This observation is puzzling since these molecules must have retained the ability to replicate in order to be able to undergo clonal expansion. We present evidence that such mtDNA molecules may in fact exist in the cell as partially duplicated mtDNA (pdmtDNA) previously described in certain mtDNA disorders. In contrast to the ‘originless’ mtDNA, the corresponding pdmtDNA do possess a light strand origin required for their propagation. Most pdmtDNA also possess an extra heavy strand origin, which may result in higher replication rate and thus provide a mechanism for expansion. Importantly, pdmtDNA are indistinguishable from mtDNA in PCR assays routinely used to detect somatic mtDNA deletions in tissues of normally aged individuals. These results indicate that a substantial proportion of age-related mtDNA deletions reported in the literature may exist as or be derived from pdmtDNA. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A well-documented age-dependent increase of the fraction of somatic deletions in mitochondrial DNA (mtDNA) has fueled a number of hypotheses postulating that these lesions might be causative in the aging of some tissues, especially of those with low proliferative potential and high energy demand, for example heart, skeletal muscle and brain (recently reviewed in ref. 1). Since each cell contains hundreds to hundreds of thousands of mtDNA copies, the physiological impact of deleted mtDNA molecules should critically depend on their distribution among cells. Indeed, if [at a given overall fraction of deleted mtDNA (
mtDNA) in a tissue] the deletions were uniformly distributed among cells, the damage would be minimal: the fraction of deletions per cell would not reach the physiological threshold value (2) and, even if it would, deletions with different portions of the genome removed would compensate for each other’s deficiencies. Conversely, if deletions of each particular kind accumulated to high proportions in certain cells, their deleterious effect would be maximized. Several independent approaches have convincingly demonstrated that, paradoxically, nature has realized this latter self-destructive scenario. Indeed, somatic mtDNA deletions appear to be clustered in single cells, apparently as clonal expansions of a single initial mutational event (3–6).
The finding that mtDNA in muscle tissues are represented by intracellular clones has substantially strengthened the mitochondrial theory of aging. It has been convincingly demonstrated that clonal expansions of mtDNA in skeletal muscle fibers contribute to sarcopenia, the general phenomenon of age-related loss of muscle fibers (7,8). The aging of certain areas of the brain, for example the substantia nigra, which in older persons present with a large proportion of dopaminergic neurons with focal mitochondrial defects (9) and high overall fractions of mtDNA (10), may also be caused by clonally expanded mtDNA. Moreover, it has been proposed that focal defects of mitochondrial metabolism caused by clonal distribution of mutant mtDNA may contribute to the age-related increase of systemic oxidative stress in the whole organism (11).
The importance of clonal mtDNA in the aging process calls for further studies of their abundance and mechanism of expansion. Single-cell PCR of the whole mitochondrial genome (6) is capable of measuring the whole spectrum of sizes of clonal expansions and accounts for almost all possible types of deletion. In the current communication, this approach is used to show that clonal deletions in individual human cardiomyocytes and muscle fiber segments are unexpectedly abundant. Furthermore, sequence analysis of clonal mtDNA appears to yield some clues regarding the mechanisms by which they may be generated and expanded. Specifically, partially duplicated mtDNA (pdmtDNA) may be responsible for clonal propagation of mtDNA deletions lacking the light strand origin of replication (OL). The pdmtDNAs were previously discovered (12) and studied in patients with various mitochondrial disorders (13–16) and in cell culture (17–19). As far as we know, this is the first report of a somatic partial duplication of mtDNA in normal aging.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Deleted mtDNA are distributed as intracellular clonal expansions
We studied cell-to-cell distribution of deletions in mtDNA by single-cell long-distance PCR of the whole mitochondrial genome. A typical single-cell PCR reaction yielded one band, corresponding to the full-length wild-type mtDNA, indicating that the cell was wild-type homoplasmic. In some cells, however, an additional band corresponding to a
mtDNA was observed, indicating that the cell contained heteroplasmic mtDNA. To eliminate the possibility of a PCR artifact or a random event, the PCR was repeated at least in triplicate from the same cell, and it was confirmed that the additional band was detected repeatedly. The length of the deletion varied from cell to cell, but generally only one deletion (or, rarely, a few) would be present. The only possible way for this to occur is through clonal expansion of a single initial mutational event. We therefore refer to these species as ‘clonally expanded mtDNA’.
Cells with clonal expansions of mtDNA are abundant
We have analyzed >1400 cells or fiber segments from 25 samples of skeletal muscle, heart and diaphragm procured from 20 individuals of various ages. Clonal mtDNA species were detected in 94 cells. A summary of the findings is presented in Figure 1. The heart data demonstrate a clear age-dependent increase in the fraction of cells with expansions. The diaphragm and skeletal muscle data are not conclusive with respect to age dependence, primarily due to the relatively narrow range of ages that were available for analysis. What is striking about the diaphragm and muscle data is that in some individuals the fraction of expansion-positive cells was as high as 25%.
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Primary structure of clonally expanded
mtDNA: a large proportion of mtDNAs lack OLOver 50 of the clonally expanded
mtDNAs were isolated and sequenced. The sequences of deletion junctions, ranged by the 5' breakpoint, are presented in Table 1 and Figure 2. The most notable observation regarding the primary structure of clonally expanded mtDNA is that almost half of the deletions (cells 32–58 in Table 1) are lacking OL, located at bp 5730–5760. This result seems to contradict our conclusion that the deletions are clonal. Indeed, clonality implies that the species are able to replicate, which most probably is not possible without OL (20; see, however, ref. 21 and Discussion). This apparent paradox prompted us to re-verify that the deletions were indeed clonal using independent approaches (see below). Another interesting observation is that a high proportion of breakpoints of deletions clustered around positions 3260 and 16070, which is consistent with earlier reports (22,23). It is intriguing that both positions are very close to the transcription termination sites for the two alternative heavy strand transcripts: the shorter ‘rRNA’ transcript (24) and the full-length transcript (25), respectively.
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Quantification of clonally expanded deletions by single-cell competitive PCR: deletions are indeed clonal
The crucial point in proving that a
mtDNA species has been clonally expanded would be to demonstrate, by accurate quantification, the presence of a significant number of mtDNA copies in a given cell. Long-distance PCR is not optimal for this because lower amplification efficiency and preferential damage of the longer wild-type DNA fragment results in a bias in favor of mtDNA. In competitive PCR, the use of short competitive templates, which differ from the target sequences by as little as a single base change, enables one to overcome both of these limitations. Two competitive PCR systems were used: one for the total number of mtDNAs per cell and the other specific for a particular mtDNA present in the cell under study, allowing one to calculate both the copy number and the fraction of mtDNA in a cell (see Materials and Methods).
Eight representative cells were chosen to confirm the presence of clonal expansions: four with and four without an OL. The primary long-distance PCR from these cells are shown in Figure 3, which demonstrates that each of the selected cells contained a clonal expansion of a single mtDNA. The results of competitive PCR in the same cells are shown in Table 2. As expected, comparison of Figure 3 and Table 2 shows that long-distance PCR overestimates the fraction of mtDNAs. For example, the wild-type band is almost absent in the long PCR of cell no. 9 (Fig. 3). In reality, the fraction of wild-type mtDNAs in this cell is 80% (Table 2). This bias is eliminated by the use of competitive PCR. As shown in Table 2, in most cases the cells contain very substantial numbers of mtDNAs, thus proving that the deletions are indeed clonally expanded. In conclusion, the absence of an OL does not prevent mtDNA from expanding.
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Single-cell limiting-dilution PCR: competitive PCR estimate confirmed
In limiting-dilution PCR, the diluted single-cell lysate was aliquoted at <1 copy/tube on average and subjected to multiple amplifications in parallel. A ‘positive’ PCR would indicate that at least one template copy was available for amplification in that particular PCR tube. The fraction of ‘positive’ reactions thus provides an estimate of the number of copies in the sample. Cell no. 9 (Table 2), i.e. the cell with the most prominent expansion of a
mtDNA lacking an OL (20 000 copies/cell) was chosen for the experiment. Similarly to the competitive PCR, limiting-dilution PCR gave an estimate of 20 000 copies of mtDNA per cell and thus confirmed that mtDNA in this cell clonally expanded despite the lack of an OL.
pdmtDNA is present in a cell with clonal expansion of mtDNA lacking an OL
The ability of mtDNA to replicate despite the lack of an OL prompted us to seek possible mechanisms. Notably, our PCR-based experimental procedure would be unable to distinguish between a mixture of mtDNA and full-length mtDNA on one side and a certain class of pdmtDNA (12) on the other. Indeed, a pdmtDNA would be resolved by PCR into a combination of a longer and a shorter PCR fragment, identical to those obtained in the case of a full-length–mtDNA mixture (Fig. 4, follow the arrowhead primers). We wondered, following Poulton et al. (13), whether at least some ‘mtDNAs’ actually existed in the cell in the form of a partial duplication (i.e. pdmtDNA) which does have an OL and thus should have no trouble replicating.
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Detection of pdmtDNA in single cells is technically challenging. Direct restriction mapping/hybridization used in other studies of pdmtDNA is not applicable in this case (there is too little DNA and initial PCR cannot be avoided). We designed a pair of primers located just outside the duplicated region of the suspected pdmtDNA from cell no. 9 (Fig. 4, arrows). If a pdmtDNA existed in the cell, it would yield, in addition to a normal-length fragment originating from the wild-type mtDNA, an
2-fold longer PCR fragment that includes the duplication. As shown in Figure 5A, the presence of a clear band of the expected size (5.4 kb) in PCR from cell no. 9 (lane 2), but not in a control PCR from a deletion-free cell (lane 1), is an indication that pdmtDNA is indeed present in the cell. The identity of the 5.4 kb band was confirmed by restriction analysis using KpnI and XbaI (Fig. 5B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Clonal
mtDNA are abundant in human muscle tissues: comparison with other approachesOur studies demonstrate that a very high percentage of cells in healthy aged human muscle tissues contains clonally expanded
mtDNA. Indeed, clonal expansions were detected in 10% of cells, and in a few cases in up to 25% of cells (Fig. 1). This is an underestimate, because some of cells with clonal expansions could have been missed due to lower sensitivity of our method for longer mtDNA. Other reports generally give much lower estimates of 1% and lower for the fraction of mtDNA in tissue homogenates (see for example refs 26,27). The two data sets cannot be compared directly for two reasons. First, in most reports, only a single type of deletion, usually the ‘common deletion’ (28), was quantified. In contrast, our data include almost all possible types of mtDNA bearing a heavy chain origin (OH). The ‘common deletion’ was a rare species, detected only once in >50 cells (Table 1, cell no. 46), which is consistent with other studies, in which a wide range of mtDNA was considered (29,30). Second, some of the expansions contained relatively low fractions of mtDNA (Table 2) so, although they were counted as expansions, their contribution to the overall mutant fraction in the tissue was small. On average, a clonal expansion represents 10% of the mtDNA content of the expansion-positive cell with the range from 100% to a few dozen molecules. The size distribution of clonal expansions in individual cells probably reflects the process of growth of these expansions from initial single mutational events to homoplasmy. Apparently, the process of expansion is driven by some favorable bias in turnover of mtDNA (31).
pdmtDNA as a possible means of clonal expansion
The finding that many of the clonally expanded mtDNAs lack an origin of replication is puzzling because mtDNA needs to replicate in order to expand clonally. Other researchers have also isolated mtDNA deletions lacking an OL (32,33), in some cases in multiple types and large amounts (30). To explain the existence of these species, some postulated originless replication (30,33). In contrast, others proposed the existence of a dimer in which two mtDNAs were fused in inverse orientation so that the second OH was functioning as the OL (34).
It has been proposed (13) that mtDNA lacking an OL found in certain myopathies may replicate as the corresponding partial duplications which do have an OL. We propose that the same mechanism could account for clonal expansion of mtDNA in individual cells during normal aging. Recent data suggest a possibility that the presence of more than one OH in a mtDNA molecule may confer replicative advantage (17,19,35). This phenomenon could account for the ability of pdmtDNA to clonally expand in the cell. This mechanism would predict a marked prevalence, among pdmtDNAs, of species with two or more OHs. It is reassuring that in search for mtDNAs lacking an OL, Melov et al. (33) were able to detect only those with spared OH. If those mtDNA in fact were pdmtDNA or their descendants, the pdmtDNA would have possessed two OHs, as predicted by the model. It should be noted, however, that a synchronous mtDNA replication mechanism has been recently postulated (21), which does not require an OL. This mechanism could have provided an alternative explanation for the ability of deletions lacking an OL to clonally expand in individual cells.
Detection of pdmtDNA in normal aging: technical difficulties solved by the single-cell approach
The assertion that pdmtDNA is common in aged human cells may seem to contradict the fact that, although reports of mtDNA are plentiful, no somatic partial duplications have been reported for normal aging, even though they are readily detected in certain myopathies. We believe that this is due to the inherent difficulty of detection of pdmtDNA in normal aging. There are two main obstacles. First, the fraction of a partial duplication of any particular type is very low. Note that myopathy samples used in previous studies of pdmtDNA contained anomalously high fractions of one particular rearrangement. It was possible therefore to detect duplication by direct restriction/Southern hybridization. In normal aging, however, the fraction of any given rearrangement in a tissue is much lower, and hybridization cannot be used as a detection method. In the case of deletions, this difficulty can be avoided by using PCR with deletion-specific primers, which enabled detection of age-associated deletions with very high sensitivity (28). Unfortunately (the second obstacle), it is inherently impossible to devise specific primers for a duplication. As illustrated in Figure 4, any pair of primers capable of amplifying a partial duplication (i.e. any pair of primers located outside the duplicated region) will also be able to amplify the wild-type mtDNA. Moreover, the product derived from the wild-type mtDNA will always be much shorter than that from the pdmtDNA and will out-perform the latter in PCR. Obviously, such a system cannot be used to detect pdmtDNA in the whole tissue, where wild-type mtDNA is present in huge excess.
Fortunately, the use of single cells permits one to overcome both problems. Indeed, in a particular cell, clonal expansion may render the fraction of a somatic partial duplication high enough to be detected, even though PCR with primers located outside the duplicated region is biased against duplication. This approach enabled us to detect pdmtDNA in a cell that was previously classified as containing a clonal mtDNA lacking an OL (Fig. 5), and to show that at least some cells presenting (in conventional assay) with clonally expanded mtDNA in fact contain pdmtDNA.
Biological consequences
There are a number of reports of pdmtDNA present in multiple tissues of individuals with mitochondrial diseases at relatively high fractions (12–14,16,32,36,37). In all cases, clinical phenotypes associated with the partial duplications were relatively mild, and notably included diabetes mellitus. There was a report of a man who died at age 57 and presented with anomalously high percentage (10–12%) of a duplication/triplication in muscle even though no neuromuscular disorder had been diagnosed (23). Interestingly, it has been reported that in some cases pdmtDNA induced catastrophic depletion of mtDNA in cultured cells (17). This mechanism could account for the depletion of mtDNA observed in individual cells in aging human tissues (3).
In conclusion, a cell-by-cell approach enabled us to show that clonal expansion of mtDNA is a frequent event in the cells of the aged human muscle tissues. A high proportion of mtDNAs lack an OL and their replication may be mediated by the pdmtDNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tissues
Human autopsy samples (collected within 24 h after death) and surgery discard tissue samples (collected within 4 h after surgery) were snap-frozen in liquid nitrogen and stored at –80°C. A total of 25 samples were procured from 20 individuals without any reported mitochondrial disorder. Thirteen heart samples (left ventricle, ages 31–109 years), seven diaphragm samples (ages 43–79 years) and five skeletal muscle samples (various locations, ages 16–89 years) were analyzed (
60 cells per tissue sample in most cases).
Long-distance PCR from individual cells
PCR from individual cells was performed as described elsewhere (6). Briefly, tissue was dissociated with collagenase, and single cardiomyocytes or muscle fiber segments 100 µm long (in the case of skeletal muscle and diaphragm) were collected into PCR tubes individually, using a glass capillary. The cells were lyzed with SDS–proteinase K, and 5–10% portions of the lysate were subjected to long-distance nested PCR.
Detection of mtDNA deletions and determination of the breakpoints
Single-cell PCR products were analyzed by gel electrophoresis in 0.7% agarose gel. mtDNAs were excised from the gel under the light of a 430 nm green laser (Casix) rather than UV light to preserve the integrity of long templates. The deletions were mapped by restriction analysis using digestion with a combination of AvaI, BclI and DraI and with BstNI alone. Breakpoint regions were amplified and sequenced.
Quantification of mtDNA in single cells by competitive PCR
Specific competitive templates for each deletion were created by introducing or destroying (via primer-directed single base change) the Tsp509I restriction site in deletion-specific PCR fragments 250–400 bp in length, including the breakpoint site of the corresponding deletion (see Table 2 for primer locations and nucleotide changes). In addition, a ‘universal’ competitive template that included a region present in all mtDNA and in the full-length mtDNA (bp 352–760 of mtDNA), was generated. To quantify a specific mtDNA in a given cell, one aliquot of a single-cell lysate (1/20 part) was mixed with a known number of copies of the specific competitive template and subjected to 60 cycles of PCR. The PCR products were digested with Tsp509I and gel separated to reveal the ratio of the competitive to the cellular template, which was used to calculate the copy number of the mtDNA. Similarly, another aliquot of the same lysate was mixed with the ‘universal’ competitive template, and the total copy number of mtDNA (deleted and wild-type) was determined and was further used to calculate mutant fraction of mtDNA.
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ACKNOWLEDGEMENTS |
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The authors thank Aubrey de Grey (University of Cambridge) for stimulating ideas and Peter Belenky (Brandeis University) for help in experiments. We are grateful to Dr Eric Schon for insightful and encouraging review of the manuscript. Some of the tissue samples used in this study were kindly provided by Drs Donald Johns, Tom Perls and Melissa Upton of Beth Israel Deaconess Medical Center, Boston, MA, or procured by the NCI Cooperative Human Tissue Network. This work was supported in part by grants from National Institutes of Health CA77044, AG08812 and AG13314.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 617 667 0973; Fax: +1 617 667 0980; Email: khrapko@hms.harvard.edu
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