Mutant mtDNA Molecules Research Articles

Study of the effect of the introduction of mitochondrial import determinants into the gRNA structure on the activity of the gRNA/SpCas9 complex in vitro.

It has long been known that defects in the structure of the mitochondrial genome can cause various neuromuscular and neurodegenerative diseases. Nevertheless, at present there is no effective method for treating mitochondrial diseases. The major problem with the treatment of such diseases is associated with mitochondrial DNA (mtDNA) heteroplasmy. It means that due to a high copy number of the mitochondrial genome, mutant copies of mtDNA coexist with wild-type molecules in the same organelle. The clinical symptoms of mitochondrial diseases and the degree of their manifestation directly depend on the number of mutant mtDNA molecules in the cell. The possible way to reduce adverse effects of the mutation is by shifting the level of heteroplasmy towards the wild-type mtDNA molecules. Using this idea, several gene therapeutic approaches based on TALE and ZF nucleases have been developed for this purpose. However, the construction of protein domains of such systems is rather long and laborious process. Meanwhile, the CRISPR/Cas9 system is fundamentally different from protein systems in that it is easy to use, highly efficiency and has a different mechanism of action. All the characteristics and capabilities of the CRISPR/Cas9 system make it a promising tool in mitochondrial genetic engineering. In this article, we demonstrate for the first time that the modification of gRNA by integration of specific mitochondrial import determinants in the gRNA scaffold does not affect the activity of the gRNA/Cas9 complex in vitro.

DNA-editing enzymes as potential treatments for heteroplasmic mtDNA diseases.

Mutations in the mitochondrial genome are the cause of many debilitating neuromuscular disorders. Currently, there is no cure or treatment for these diseases, and symptom management is the only relief doctors can provide. Although supplements and vitamins are commonly used in treatment, they provide little benefit to the patient and are only palliative. This is why gene therapy is a promising research topic to potentially treat and, in theory, even cure diseases caused by mutations in the mitochondrial DNA (mtDNA). Mammalian cells contain approximately a thousand copies of mtDNA, which can lead to a phenomenon called heteroplasmy, where both wild-type and mutant mtDNA molecules co-exist within the cell. Disease only manifests once the per cent of mutant mtDNA reaches a high threshold (usually >80%), which causes mitochondrial dysfunction and reduced ATP production. This is a useful feature to take advantage of for gene therapy applications, as not every mutant copy of mtDNA needs to be eliminated, but only enough to shift the heteroplasmic ratio below the disease threshold. Several DNA-editing enzymes have been used to shift heteroplasmy in cell culture and mice. This review provides an overview of these enzymes and discusses roadblocks of applying these to gene therapy in humans.

Mitochondrial Network State Scales mtDNA Genetic Dynamics

Mitochondrial DNA (mtDNA) mutations cause severe congenital diseases but may also be associated with healthy aging. mtDNA is stochastically replicated and degraded, and exists within organelles which undergo dynamic fusion and fission. The role of the resulting mitochondrial networks in the time evolution of the cellular proportion of mutated mtDNA molecules (heteroplasmy), and cell-to-cell variability in heteroplasmy (heteroplasmy variance), remains incompletely understood. Heteroplasmy variance is particularly important since it modulates the number of pathological cells in a tissue. Here, we provide the first wide-reaching theoretical framework which bridges mitochondrial network and genetic states. We show that, under a range of conditions, the (genetic) rate of increase in heteroplasmy variance and de novo mutation are proportionally modulated by the (physical) fraction of unfused mitochondria, independently of the absolute fission–fusion rate. In the context of selective fusion, we show that intermediate fusion:fission ratios are optimal for the clearance of mtDNA mutants. Our findings imply that modulating network state, mitophagy rate, and copy number to slow down heteroplasmy dynamics when mean heteroplasmy is low could have therapeutic advantages for mitochondrial disease and healthy aging.

Energetic costs of cellular and therapeutic control of stochastic mitochondrial DNA populations.

The dynamics of the cellular proportion of mutant mtDNA molecules is crucial for mitochondrial diseases. Cellular populations of mitochondria are under homeostatic control, but the details of the control mechanisms involved remain elusive. Here, we use stochastic modelling to derive general results for the impact of cellular control on mtDNA populations, the cost to the cell of different mtDNA states, and the optimisation of therapeutic control of mtDNA populations. This formalism yields a wealth of biological results, including that an increasing mtDNA variance can increase the energetic cost of maintaining a tissue, that intermediate levels of heteroplasmy can be more detrimental than homoplasmy even for a dysfunctional mutant, that heteroplasmy distribution (not mean alone) is crucial for the success of gene therapies, and that long-term rather than short intense gene therapies are more likely to beneficially impact mtDNA populations.

Mitochondrial DNA m.13514G>A heteroplasmy is associated with depressive symptoms in the elderly.

Mitochondrial DNA (mtDNA) heteroplasmy is a mixture of normal and mutated mtDNA molecules in a cell. High levels of heteroplasmy at several mtDNA sites in complex I lead to inherited neurological neurologic diseases and brain magnetic resonance imaging (MRI) abnormalities. Here, we test the hypothesis that mtDNA heteroplasmy at these complex I sites is associated with depressive symptoms in the elderly. We examined platelet mtDNA heteroplasmy for associations with depressive symptoms among 137 participants over age 70 from the community-based Health, Aging and Body Composition Study. Depressive symptoms were assessed using the 10-point version of the Center for Epidemiologic Studies Depression Scale (CES-D 10). Complete mtDNA sequencing was performed and heteroplasmy derived for 5 mtDNA sites associated with neurologic mitochondrial diseases and tested for associations with depressive symptoms. Of 5 candidate complex I mtDNA mutations examined for effects on depressive symptoms, increased heteroplasmy at m.13514A>G, ND5, was significantly associated with higher CES-D score (P=.01). A statistically significant interaction between m.13514A>G heteroplasmy and sex was detected (P=.04); in sex-stratified analyses, the impact of m.13514A>G heteroplasmy was stronger in male (P=.003) than in female (P=.98) participants. Men in highest tertile of mtDNA heteroplasmy exhibited significantly higher (P=.0001) mean±SE CES-D 10 scores, 5.37±0.58, when compared with those in the middle, 2.13±0.52, and lowest tertiles, 2.47±0.58. No associations between the 4 other candidate sites and depressive symptoms were observed. Increased mtDNA heteroplasmy at m.13514A>G is associated with depressive symptoms in older men. Heteroplasmy may represent a novel biological risk factor for depression.

Mitochondrial DNA Heteroplasmy Associations With Neurosensory and Mobility Function in Elderly Adults.

Mitochondrial DNA (mtDNA) heteroplasmy is a mixture of normal and mutated mtDNA molecules in a cell. High levels of heteroplasmy at specific mtDNA sites lead to inherited mitochondrial diseases with neurological, sensory, and movement impairments. Here we test the hypothesis that heteroplasmy levels in elderly adults are associated with impaired function resembling mild forms of mitochondrial disease. We examined platelet mtDNA heteroplasmy at 20 disease-causing sites for associations with neurosensory and mobility function among 137 participants from the community-based Health, Aging, and Body Composition Study. Elevated mtDNA heteroplasmy at four mtDNA sites in complex I and tRNA genes was nominally associated with reduced cognition, vision, hearing, and mobility: m.10158T>C with Modified Mini-Mental State Examination score (p = .009); m.11778G>A with contrast sensitivity (p = .02); m.7445A>G with high-frequency hearing (p = .047); and m.5703G>A with 400 m walking speed (p = .007). These results indicate that increased mtDNA heteroplasmy at disease-causing sites is associated with neurosensory and mobility function in older persons. We propose the novel use of mtDNA heteroplasmy as a simple, noninvasive predictor of age-related neurologic, sensory, and movement impairments.

Context-Dependent Role of Mitochondrial Fusion-Fission in Clonal Expansion of mtDNA Mutations

The accumulation of mutant mitochondrial DNA (mtDNA) molecules in aged cells has been associated with mitochondrial dysfunction, age-related diseases and the ageing process itself. This accumulation has been shown to often occur clonally, where mutant mtDNA grow in number and overpopulate the wild-type mtDNA. However, the cell possesses quality control (QC) mechanisms that maintain mitochondrial function, in which dysfunctional mitochondria are isolated and removed by selective fusion and mitochondrial autophagy (mitophagy), respectively. The aim of this study is to elucidate the circumstances related to mitochondrial QC that allow the expansion of mutant mtDNA molecules. For the purpose of the study, we have developed a mathematical model of mitochondrial QC process by extending our previous validated model of mitochondrial turnover and fusion-fission. A global sensitivity analysis of the model suggested that the selectivity of mitophagy and fusion is the most critical QC parameter for clearing de novo mutant mtDNA molecules. We further simulated several scenarios involving perturbations of key QC parameters to gain a better understanding of their dynamic and synergistic interactions. Our model simulations showed that a higher frequency of mitochondrial fusion-fission can provide a faster clearance of mutant mtDNA, but only when mutant–rich mitochondria that are transiently created are efficiently prevented from re-fusing with other mitochondria and selectively removed. Otherwise, faster fusion-fission quickens the accumulation of mutant mtDNA. Finally, we used the insights gained from model simulations and analysis to propose a possible circumstance involving deterioration of mitochondrial QC that permits mutant mtDNA to expand with age.

A magic bullet to specifically eliminate mutated mitochondrial genomes from patients' cells

When mitochondrial diseases result from mutations found in the mitochondrial DNA, engineered mitochondrial-targeted nucleases such as mitochondrial-targeted zinc finger nucleases are shown to specifically eliminate the mutated molecules, leaving the wild-type mitochondrial DNA intact to replicate and restore normal copy number. In this issue, Gammage and colleagues successfully apply this improved technology on patients' cells with two types of genetic alterations responsible for neuropathy ataxia and retinitis pigmentosa (NARP) syndrome and Kearns Sayre syndrome and progressive external ophthalmoplegia (PEO).

Whole blood genome-wide expression profiling and network analysis suggest MELAS master regulators

Background: The heteroplasmic mitochondrial DNA (mtDNA) mutation A3243G causes the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome as one of the most frequent mitochondrial diseases. The process of reconfiguration of nuclear gene expression profile to accommodate cellular processes to the functional status of mitochondria might be a key to MELAS disease manifestation and could contribute to its diverse phenotypic presentation.Objective: To determine master regulatory protein networks and disease-modifying genes in MELAS syndrome.Methods: Analyses of whole blood transcriptomes from 10 MELAS patients using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses.Results: Hierarchical cluster analysis elucidated that the relative abundance of mutant mtDNA molecules is decisive for the nuclear gene expression response. Further analyses confirmed not only transcription factors already known to be involved in mitochondrial diseases (such as TFAM), but also detected the hypoxia-inducible factor 1 complex, nuclear factor Y and cAMP responsive element-binding protein-related transcription factors as novel master regulators for reconfiguration of nuclear gene expression in response to the MELAS mutation. Correlation analyses of gene alterations and clinico-genetic data detected significant correlations between A3243G-induced nuclear gene expression changes and mutant mtDNA load as well as disease characteristics. These potential disease-modifying genes influencing the expression of the MELAS phenotype are mainly related to clusters primarily unrelated to cellular energy metabolism, but important for nucleic acid and protein metabolism, and signal transduction.Discussion: Our data thus provide a framework to search for new pathogenetic concepts and potential therapeutic approaches to treat the MELAS syndrome.

Evaluating Mutant mtDNA Tissue Distribution in Early Fetal Development

Pathogenic mutations in mitochondrial DNA (mtDNA) result in a wide spectrum of disorders with a prevalence of over 1 in 5,000. The thousands of mtDNA copies that are present in every cell are virtually identical in unaffected individuals, but in individuals with mtDNA-related disease there may be a mixture of normal and mutated mtDNA molecules (heteroplasmy) which can vary among tissues. Prenatal (PND) and preimplantation (PGD) diagnosis of many types of mtDNA diseases are unreliable, since it is hard to predict with certainty the way in which heteroplasmic mtDNA mutations segregate within tissues. This makes the prediction of recurrence risk immensely difficult. Monnot et al. (Hum Mutat 32:116–125, 2011) perform an elegant study examining the segregation of one of the most common mtDNA mutations (m.3243A>G, causing MELAS) throughout the entire embryofetal development period. To determine whether m.3243A>G segregation is governed by random drift in somatic tissues during early development, the authors quantified the levels of mutant to wild-type mtDNA. Their results demonstrated a uniform tissue distribution of mtDNA mutant load in somatic tissues from the developing fetus sampled between 12–25 weeks, indicating a lack of selection. The authors also measured mtDNA mutant load in individual blastomeres in a large number of samples from embryos of a single patient and showed that there is very little inter-blastomere variability. Understanding this segregation pattern is of critical importance to the success of PGD, whereby the biopsied blastomere has to be representative of the genetic composition of the embryo as a whole. Should this distribution not be uniform, then there is the possibility of misdiagnosis, resulting in either eligible embryos being unnecessarily discarded or the birth of a severely affected individual. Given the uncertainties in reproductive counselling for mitochondrial disorders, such studies as this are vital for improving the reliability of prenatal and preimplantation diagnosis.

Genome-wide expression profiling of A3243G MELAS patients in quest for potential disease-modifying target genes

Mitochondrial encephalomyopathies represent an expanding group of clinical as well as genetical heterogeneous disorders caused by mitochondrial DNA (mtDNA) mutations or nuclear gene defects. The heteroplasmic mtDNA mutation at np 3243 (3243 A>G) in the tRNALeu(UUR) gene gives rise to one of the most frequent mitochondrial diseases, the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes). Affected patients present with a broad spectrum of clinical phenotypes, including features that comprise the name of the disorder such as mitochondrial encephalomyopathy, lactic acidosis and strokes, but diabetes mellitus and hearing loss can also be part of the phenotype. As the percentage of mutant genomes did not prove to be directly correlated to the course and phenotype of disease, other yet unknown factors must be involved. Recent studies have shown that eukaryotic cells are able to reconfigure their nuclear gene expression profile to accommodate various cellular processes to the functional status of mitochondria. This process might be a key to MELAS disease manifestation and could, moreover, contribute to the phenotypic variability. We used Affymetrix oligonucleotide microarrays to analyze global gene expression changes in blood samples of ten MELAS patients compared to matched controls. Acquired data were then subjected to hierarchical clustering in order to match clinical and genetical data with expression patterns. We thereby elucidated that the relative abundance of mutant mtDNA molecules is relevant for the nuclear gene expression profile. Within this context, we identified potential disease-modifying nuclear target genes and clarified how their expression correlates with the patient's profile and the clinical outcome of MELAS syndrome. In conclusion, we elucidated A3243G-related changes in blood mRNAs that distinguish MELAS patients from controls. Moreover, we demonstrated how these modifications in mRNA expression correlate with the patient's load of mutant mtDNA, clinical outcome and disease duration. Altogether, the herein presented results may provide first biomarkers for MELAS syndrome, thereby pioneering towards potential therapeutic targets.

Human Mitochondrial DNA Nucleoids Are Linked to Protein Folding Machinery and Metabolic Enzymes at the Mitochondrial Inner Membrane

Mitochondrial DNA (mtDNA) is packaged into bacterial nucleoid-like structures, each containing several mtDNA molecules. The distribution of nucleoids during mitochondrial fission and fusion events and during cytokinesis is important to the segregation of mitochondrial genomes in heteroplasmic cells bearing a mixture of wild-type and mutant mtDNA molecules. We report fractionation of HeLa cell mtDNA nucleoids into two subsets of complexes that differ in their sedimentation velocity and their association with cytoskeletal proteins. Pulse labeling studies indicated that newly replicated mtDNA molecules are evenly represented in the rapidly and slowly sedimenting fractions. Slowly sedimenting nucleoids were immunoaffinity purified using antibodies to either of two abundant mtDNA-binding proteins, TFAM or mtSSB. These two different immunoaffinity procedures yielded very similar sets of proteins, with 21 proteins in common, including most of the proteins previously shown to play roles in mtDNA replication and transcription. In addition to previously identified mitochondrial proteins, multiple peptides were observed for one novel DNA metabolic protein, the DEAH-box helicase DHX30. Antibodies raised against a recombinant fragment of this protein confirmed the mitochondrial localization of a specific isoform of DHX30.

Apoptosis in mitochondrial myopathies is linked to mitochondrial proliferation

Increased susceptibility to apoptosis has been shown in many models of mitochondrial defects but its relevance to human diseases is still discussed. We addressed the presence of apoptosis in muscle from patients with mitochondrial DNA (mtDNA) disorders. Taking advantage of the mosaic pattern of muscle morphological anomalies associated with heteroplasmic mtDNA alterations, we have used an in situ approach to address the relationship between apoptosis and respiratory defect, mitochondrial proliferation and mutation load. Different patterns of mitochondrial morphological alterations were provided by the analysis of muscles with large mtDNA deletion (16 cases) or with the MELAS mutation (4 cases). The patient's age at biopsy ranged from 0.4 to 66 years and the muscle mutant mtDNA proportion from 32 to 82%. Apoptotic muscle fibres were observed in a small proportion of muscle fibres of 16 out of the 20 biopsies by three different detection methods for different steps of apoptosis: caspase 3 activation, fragmentation of nuclear DNA [terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay] or overexpression of the pro-apoptotic factor Bax. Analysis of apoptotic features in parallel to cytochrome c oxidase (COX) and succinate dehydrogenase activity of more than 34,000 individual muscle fibres showed that apoptosis occurred only in muscle fibres with mitochondrial proliferation (ragged red fibres, RRF) irrespective of their COX activity. Molecular analyses of single muscle fibres evidenced that, as expected, the presence of COX defect was associated with higher proportion of mutant mtDNA and lower amount of normal mtDNA. Within COX-defective fibres, the presence of mitochondrial proliferation was associated with increase of the mtDNA content but without change in the ratio between normal and mutant mtDNA molecules, thus showing that mitochondrial proliferation was accompanied by similar amplification of normal and mutant mtDNA molecules. Within RRF, apoptosis was associated with higher mutation proportion, suggesting that it was provoked by severe respiratory defect in the same time as increased mitochondrial mass. In conclusion, apoptosis most probably contributes to mitochondrial pathology. It is tightly linked to mitochondrial proliferation and high mutation load. When considering training therapeutics, one will have to take into account the possibility to induce apoptosis in parallel to mitochondrial proliferation.

Disruption of Fusion Results in Mitochondrial Heterogeneity and Dysfunction

Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Paradoxically, cells invest many resources to maintain tubular mitochondrial morphology, when reducing both fusion and fission simultaneously achieves the same end. This observation suggests a requirement for mitochondrial fusion, beyond maintenance of organelle morphology. Here, we show that cells with targeted null mutations in Mfn1 or Mfn2 retained low levels of mitochondrial fusion and escaped major cellular dysfunction. Analysis of these mutant cells showed that both homotypic and heterotypic interactions of Mfns are capable of fusion. In contrast, cells lacking both Mfn1 and Mfn2 completely lacked mitochondrial fusion and showed severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Disruption of OPA1 by RNAi also blocked all mitochondrial fusion and resulted in similar cellular defects. These defects in Mfn-null or OPA1-RNAi mammalian cells were corrected upon restoration of mitochondrial fusion, unlike the irreversible defects found in fzodelta yeast. In contrast, fragmentation of mitochondria, without severe loss of fusion, did not result in such cellular defects. Our results showed that key cellular functions decline as mitochondrial fusion is progressively abrogated.

Mitochondrial DNA as a Cancer Biomarker

As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases.

Single cell quantification of the 8993T > G NARP mitochondrial DNA mutation by fluorescent PCR

When a mitochondrial DNA (mtDNA) mutation is identified, the reliable and sensitive quantification of the mutation load is a prerequisite for evaluating the feasibility of prenatal/pregestational diagnosis of the disease. We have developed a quantification assay of the 8993T > G NARP mutation using semi-quantitative fluorescent PCR. The test was reproducible and the experimental values were linear even at extremely low concentrations of mutant mtDNA molecules, making quantification of the mutant load in individual cells feasible (including blastomeres). Studying single circulating lymphocytes from a single NARP 8993T > G patient, we found a broad distribution of the disease causing mutation (0–44%) supporting the remarkable variability of heteroplasmy at the cellular level. This observation and the experimental approach reported here should be relevant to either prenatal or preimplantation diagnosis.

Live now--pay by ageing: high performance mitochondrial activity in youth and its age-related side effects.

Radical oxygen species are a byproduct of normal energy metabolism in mitochondria. The short-lived radicals cause damage to their immediate surrounding, i.e. the mitochondria. While most of this damage will be removed by normal mitochondrial turnover, damage to mitochondrial DNA (mtDNA) can persist and may accumulate with age. Recent evidence indicates that mutant mtDNA molecules can accumulate within individual cells, potentially hampering mitochondrial function.

Clonally expanded mtDNA point mutations are abundant in individual cells of human tissues.

Using single-cell sequence analysis, we discovered that a high proportion of cells in tissues as diverse as buccal epithelium and heart muscle contain high proportions of clonal mutant mtDNA expanded from single initial mutant mtDNA molecules. We demonstrate that intracellular clonal expansion of somatic point mutations is a common event in normal human tissues. This finding implies efficient homogenization of mitochondrial genomes within individual cells. Significant qualitative differences observed between the spectra of clonally expanded mutations in proliferating epithelial cells and postmitotic cardiomyocytes suggest, however, that either the processes generating these mutations or mechanisms driving them to homoplasmy are likely to be fundamentally different between the two tissues. Furthermore, the ability of somatic mtDNA mutations to expand (required for their phenotypic expression), as well as their apparently high incidence, reinforces the possibility that these mutations may be involved actively in various physiological processes such as aging and degenerative disease. The abundance of clonally expanded point mutations in individual cells of normal tissues also suggests that the recently discovered accumulation of mtDNA mutations in tumors may be explained by processes that are similar or identical to those operating in the normal tissue.

Mitochondrial genome instability in human cancers.

Malfunction of mismatch repair (MMR) genes produces nuclear genome instability (NGI) and plays an important role in the origin of some hereditary and sporadic human cancers. The appearance of non-inherited microsatellite alleles in tumor cells (microsatellite instability, MSI) is one of the expressions of NGI. We present here data showing mitochondrial genome instability (mtGI) in most of the human cancers analyzed so far. The mtDNA markers used were point mutations, length-tract instability of mono- or dinucleotide repeats, mono- or dinucleotide insertions or deletions, and long deletions. Comparison of normal and tumoral tissues from the same individual reveals that mt-mutations may show as homoplasmic (all tumor cells have the same variant haplotype) or as heteroplasmic (tumor cells are a mosaic of inherited and acquired variant haplotypes). Breast, colorectal, gastric and kidney cancers exhibit mtGI with a pattern of mt-mutations specific for each tumor. No correlation between NGI and mtGI was found in breast, colorectal or kidney cancers, while a positive correlation was found in gastric cancer. Conversely, germ cell testicular cancers lack mtGI. Damage by reactive oxygen species (ROS), slipped-strand mispairing (SSM) and deficient repair are the causes explaining the appearance of mtGI. The replication and repair of mtDNA are controlled by nuclear genes. So far, there is no clear evidence linking MMR gene malfunction with mtGI. Polymerase gamma (POLgamma) carries out the mtDNA synthesis. Since this process is error-prone due to a deficiency in the proofreading activity of POLgamma, this enzyme has been assumed to be involved in the origin of mt-mutations. Somatic cells have hundreds to thousands of mtDNA molecules with a very high rate of spontaneous mutations. Accordingly, most somatic cells probably have a low frequency of randomly mutated mtDNA molecules. Most cancers are of monoclonal origin. Hence, to explain the appearance of mtGI in tumors we have to explain why a given variant mt-haplotype expands and replaces part of (heteroplasmy) or all (homoplasmy) wild mt-haplotypes in cancer cells. Selective and/or replicative advantage of some mutations combined with a severe bottleneck during the mitochondrial segregation accompanying mitosis are the mechanisms probably involved in the origin of mtGI.

Functional mitochondrial heterogeneity in heteroplasmic cells carrying the mitochondrial DNA mutation associated with the MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes).

Most mitochondrial DNA (mtDNA) alterations associated with human disorders are heteroplasmic, i.e. mutant mtDNA molecules coexist with normal ones within the cell. We addressed the possibility of intermitochondrial exchanges through histologic analyses of cybrid clones with increasing proportion of the MELAS (A3243G) mtDNA transfer RNA point mutation. MtDNA-dependent cytochrome c oxidase activity and protein composition as well as mitochondrial membrane potential appeared heterogeneous in individual cells from clonal heteroplasmic cell populations on the basis of confocal and electron microscopy. The number of defective cells increased with increasing mutation load. We conclude that in the presence of a heteroplasmic mtDNA mutation in the cell type that we studied, intermitochondrial molecular exchanges cannot provide an efficient even distribution of the complementing molecules such as wild-type mtDNA, transfer RNA, or protein. Mitochondria in these heteroplasmic cells cannot, therefore, be considered a single functional unit.