Gimap3 regulates tissue-specific mitochondrial DNA segregation

Mitochondrial DNA (mtDNA) sequence variants segregate in mutation and tissue-specific manners, but the mechanisms remain unknown. The segregation pattern of pathogenic mtDNA mutations is a major determinant of the onset and severity of disease. Using a heteroplasmic mouse model, we demonstrate that Gimap3, an outer mitochondrial membrane GTPase, is a critical regulator of this process in leukocytes. Gimap3 is important for T cell development and survival, suggesting that leukocyte survival may be a key factor in the genetic regulation of mtDNA sequence variants and in modulating human mitochondrial diseases.     

Rapid mitochondrial DNA segregation in primate preimplantation embryos precedes somatic and germline bottleneck

The timing and mechanisms of mitochondrial DNA (mtDNA) segregation and transmission in mammals are poorly understood. Genetic bottleneck in female germ cells has been proposed as the main phenomenon responsible for rapid intergenerational segregation of heteroplasmic mtDNA. We demonstrate here that mtDNA segregation occurs during primate preimplantation embryogenesis resulting in partitioning of mtDNA variants between daughter blastomeres. A substantial shift toward homoplasmy occurred in fetuses and embryonic stem cells (ESCs) derived from these heteroplasmic embryos. We also observed a wide range of heteroplasmic mtDNA variants distributed in individual oocytes recovered from these fetuses. Thus, we present here evidence for a previously unknown mtDNA segregation and bottleneck during preimplantation embryo development, suggesting that return to the homoplasmic condition can occur during development of an individual organism from the zygote to birth, without a passage through the germline.     

Mitochondrial gene segregation in mammals: is the bottleneck always narrow?

Summary The segregation of a heteroplasmic silent polymorphism in the mitochondrial ND6 gene has been followed in a human maternal lineage comprising eight individuals and spanning three generations. Heteroplasmy persisted in all eight maternally related family members. More importantly, the frequencies of the two alleles showed relatively little variation among individuals or between generations. In contrast to the findings in other mammalian lineages, the present results indicate relatively slow mitochondrial gene segregation. A narrow bottleneck in the number of mitochondrial DNA (mtDNA) molecules, which occurs at some stage of oogenesis, has been advanced to explain rapid mammalian mitochondrial gene segregation. It is suggested here that the segregation of mitochondrial genes may be more complex than initially envisaged, and that models need to be developed that account for both rapid and slow segregation. One possibility, which reconciles both physical and genetic studies of mammalian mtDNA, is that the unit of mitochondrial segregation is the organelle itself, each containing multiple mtDNA molecules.     

Heteroplasmic mitochondrial DNA variants in cardiovascular diseases

Mitochondria are implicated in the pathogenesis of cardiovascular diseases (CVDs) but the reasons for this are not well understood. Maternally-inherited population variants of mitochondrial DNA (mtDNA) which affect all mtDNA molecules (homoplasmic) are associated with cardiometabolic traits and the risk of developing cardiovascular disease. However, it is not known whether mtDNA mutations only affecting a proportion of mtDNA molecules (heteroplasmic) also play a role. To address this question, we performed a high-depth (~1000-fold) mtDNA sequencing of blood DNA in 1,399 individuals with hypertension (HTN), 1,946 with ischemic heart disease (IHD), 2,146 with ischemic stroke (IS), and 723 healthy controls. We show that the per individual burden of heteroplasmic single nucleotide variants (mtSNVs) increases with age. The age-effect was stronger for low-level heteroplasmies (heteroplasmic fraction, HF, 5–10%), likely reflecting acquired somatic events based on trinucleotide mutational signatures. After correcting for age and other confounders, intermediate heteroplasmies (HF 10–95%) were more common in hypertension, particularly involving non-synonymous variants altering the amino acid sequence of essential respiratory chain proteins. These findings raise the possibility that heteroplasmic mtSNVs play a role in the pathophysiology of hypertension.     

Mitochondrial Genotype Segregation in a Mouse Heteroplasmic Lineage Produced by Embryonic Karyoplast Transplantation

Mitochondrial genotypes have been shown to segregate both rapidly and slowly when transmitted to consecutive generations in mammals. Our objective was to develop an animal model to analyze the patterns of mammalian mitochondrial DNA (mtDNA) segregation and transmission in an intraspecific heteroplasmic maternal lineage to investigate the mechanisms controlling these phenomena. Heteroplasmic progeny were obtained from reconstructed blastocysts derived by transplantation of pronuclear-stage karyoplasts to enucleated zygotes with different mtDNA. Although the reconstructed zygotes contained on average 19% mtDNA of karyoplast origin, most progeny contained fewer mtDNA of karyoplast origin and produced exclusively homoplasmic first generation progeny. However, one founder heteroplasmic adult female had elevated tissue heteroplasmy levels, varying from 6% (lung) to 69% (heart), indicating that stringent replicative segregation had occurred during mitotic divisions. First generation progeny from the above female were all heteroplasmic, indicating that, despite a meiotic segregation, they were derived from heteroplasmic founder oocytes. Some second and third generation progeny contained exclusively New Zealand Black/BINJ mtDNA, suggesting, but not confirming, an origin from an homoplasmic oocyte. Moreover, several third to fifth generation individuals maintained mtDNA from both mouse strains, indicating a slow or persistent segregation pattern characterized by diminished tissue and litter variability beyond second generation progeny. Therefore, although some initial lineages appear to segregate rapidly to homoplasmy, within two generations other lineages transmit stable amounts of both mtDNA molecules, supporting a mechanism where mitochondria of different origin may fuse, leading to persistent intraorganellar heteroplasmy.     

Analysis of heteroplasmy in the major noncoding region of mitochondrial DNA in the blood and atherosclerotic plaques of carotid arteries

For identification of somatic mitochondrial DNA (mtDNA) mutations, the mtDNA major noncoding region (D-loop) sequence in blood samples and carotid atherosclerosis plaques from patients with atherosclerosis was analyzed. Five point heteroplasmic positions were observed in 4 of 23 individuals (17%). Only in two cases could heteroplasmy have resulted from somatic mutation, whereas three heteroplasmic positions were found in both vascular tissue and blood. In addition, length heteroplasmy in a polycytosine stretches was registered at nucleotide positions 303–315 in 16 individuals, and also in the 16184–16193 region in four patients. The results suggest that somatic mtDNA mutations can occur during atherosclerosis, but some heteroplasmic mutations may appear in all tissues, possibly being inherited.     

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.     

Selection against pathogenic mtDNA mutations in a stem cell population leads to the loss of the 3243A-->G mutation in blood

The mutation 3243A-->G is the most common heteroplasmic pathogenic mitochondrial DNA (mtDNA) mutation in humans, but it is not understood why the proportion of this mutation decreases in blood during life. Changing levels of mtDNA heteroplasmy are fundamentally related to the pathophysiology of the mitochondrial disease and correlate with clinical progression. To understand this process, we simulated the segregation of mtDNA in hematopoietic stem cells and leukocyte precursors. Our observations show that the percentage of mutant mtDNA in blood decreases exponentially over time. This is consistent with the existence of a selective process acting at the stem cell level and explains why the level of mutant mtDNA in blood is almost invariably lower than in nondividing (postmitotic) tissues such as skeletal muscle. By using this approach, we derived a formula from human data to correct for the change in heteroplasmy over time. A comparison of age-corrected blood heteroplasmy levels with skeletal muscle, an embryologically distinct postmitotic tissue, provides independent confirmation of the model. These findings indicate that selection against pathogenic mtDNA mutations occurs in a stem cell population.     

Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases.     

Inheritance of mitochondrial DNA recombinants in double-heteroplasmic families: potential implications for phylogenetic analysis

Recently, somatic recombination of human mitochondrial DNA (mtDNA) was discovered in skeletal muscle. To determine whether recombinant mtDNA molecules can be transmitted through the germ line, we investigated two families, each harboring two inherited heteroplasmic mtDNA mutations. Using allele-specific polymerase chain reaction and single-cell and single-molecule mutational analyses, we discovered, in both families, all four possible allelic combinations of the two heteroplasmic mutations (tetraplasmy), the hallmark of mtDNA recombination. We strongly suggest that these recombinant mtDNA molecules were inherited rather than de novo generated somatically, because they (1) are highly abundant and (2) are present in different tissues of maternally related family members, including young individuals. Moreover, the comparison of the complete mtDNA sequence of one of the families with database sequences revealed an irregular, nontreelike pattern of mutations, reminiscent of a reticulation. We therefore propose that certain reticulations of the human mtDNA phylogenetic tree might be explained by recombination of coexisting mtDNA molecules harboring multiple mutations.     

Quantitative Changes in Gimap3 and Gimap5 Expression Modify Mitochondrial DNA Segregation in Mice

Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment.     

Mitochondrial DNA Variant Discovery and Evaluation in Human Cardiomyopathies through Next-Generation Sequencing

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We “shotgun” sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche''s 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300× average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20× while heteroplasmic variants required >200× coverage. Several Sanger “misses” were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.     

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA

Studies in vitro have shown that a respiratorydeficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (GA) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (⁰) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.     

Sequence analysis of the complete mitochondrial genome in patients with mitochondrial encephaloneuromyopathies lacking the common pathogenic DNA mutations

The purpose of this study was to identify novel mitochondrial deoxyribonucleic acid (mtDNA) mutations in a series of patients with clinical and/or morphological features of mitochondrial dysfunction, but still no genetic diagnosis. A heterogeneous group of clinical disorders is caused by mutations in mtDNA that damage respiratory chain function of cell energy production. We developed a method to systematically screen the entire mitochondrial genome. The sequence-data were obtained with a rapid automated system. In the six mitochondrial genomes analysed we found 20 variants of the revised Cambridge reference sequence [Nat. Genet. 23 (1999) 147]. In skeletal muscle nineteen novel mtDNA variants were homoplasmic, suggesting secondary pathogenicity or co-responsibility in determination of the disease. In one patient we identified a novel heteroplasmic mtDNA mutation which presumably has a pathogenic role. This screening is therefore useful to extend the mtDNA polymorphism database and should facilitate definition of disease-related mutations in human mtDNA.     

Genotypic stability, segregation and selection in heteroplasmic human cell lines containing np 3243 mutant mtDNA

The mitochondrial genotype of heteroplasmic human cell lines containing the pathological np 3243 mtDNA mutation, plus or minus its suppressor at np 12300, has been followed over long periods in culture. Cell lines containing various different proportions of mutant mtDNA remained generally at a consistent, average heteroplasmy value over at least 30 wk of culture in nonselective media and exhibited minimal mitotic segregation, with a segregation number comparable with mtDNA copy number (>/=1000). Growth in selective medium of cells at 99% np 3243 mutant mtDNA did, however, allow the isolation of clones with lower levels of the mutation, against a background of massive cell death. As a rare event, cell lines exhibited a sudden and dramatic diversification of heteroplasmy levels, accompanied by a shift in the average heteroplasmy level over a short period (<8 wk), indicating selection. One such episode was associated with a gain of chromosome 9. Analysis of respiratory phenotype and mitochondrial genotype of cell clones from such cultures revealed that stable heteroplasmy values were generally reestablished within a few weeks, in a reproducible but clone-specific fashion. This occurred independently of any straightforward phenotypic selection at the individual cell-clone level. Our findings are consistent with several alternate views of mtDNA organization in mammalian cells. One model that is supported by our data is that mtDNA is found in nucleoids containing many copies of the genome, which can themselves be heteroplasmic, and which are faithfully replicated. We interpret diversification and shifts of heteroplasmy level as resulting from a reorganization of such nucleoids, under nuclear genetic control. Abrupt remodeling of nucleoids in vivo would have major implications for understanding the developmental consequences of heteroplasmy, including mitochondrial disease phenotype and progression.     

Reactive oxygen species stimulate mitochondrial allele segregation toward homoplasmy in human cells

Mitochondria that contain a mixture of mutant and wild-type mitochondrial (mt) DNA copies are heteroplasmic. In humans, homoplasmy is restored during early oogenesis and reprogramming of somatic cells, but the mechanism of mt-allele segregation remains unknown. In budding yeast, homoplasmy is restored by head-to-tail concatemer formation in mother cells by reactive oxygen species (ROS)–induced rolling-circle replication and selective transmission of concatemers to daughter cells, but this mechanism is not obvious in higher eukaryotes. Here, using heteroplasmic m.3243A > G primary fibroblast cells derived from MELAS patients treated with hydrogen peroxide (H₂O₂), we show that an optimal ROS level promotes mt-allele segregation toward wild-type and mutant mtDNA homoplasmy. Enhanced ROS level reduced the amount of intact mtDNA replication templates but increased linear tandem multimers linked by head-to-tail unit-sized mtDNA (mtDNA concatemers). ROS-triggered mt-allele segregation correlated with mtDNA-concatemer production and enabled transmission of multiple identical mt-genome copies as a single unit. Our results support a mechanism by which mt-allele segregation toward mt-homoplasmy is mediated by concatemers.     

Homopolymeric tract heteroplasmy in mtDNA from tissues and single oocytes: support for a genetic bottleneck.

While mtDNA polymorphisms at single base positions are common, the overwhelming majority of the mitochondrial genomes within a single individual are usually identical. When there is a point-mutation difference between a mother and her offspring, there may be a complete switching of mtDNA type within a single generation. It is generally assumed that there is a genetic bottleneck whereby a single or small number of founder mtDNA(s) populate the organism, but it is not known at which stages the restriction/amplification of mtDNA subtype(s) occur, and this uncertainty impedes antenatal diagnosis for mtDNA disorders. Length polymorphisms in homopolymeric tracts have been demonstrated in the large noncoding region of mtDNA. We have developed a new method, T-PCR (trimmed PCR), to quantitate heteroplasmy for two of these tracts (D310 and D16189). D310 variation is sufficient to indicate clonal origins of tissues and single oocytes. Tissues from normal individuals often possessed more than one length variant (heteroplasmy). However, there was no difference in the pattern of the length variants between somatic tissues in any control individual when bulk samples were taken. Oocytes from normal women undergoing in vitro fertilization were frequently heteroplasmic for length variants, and in two cases the modal length of the D310 tract differed in individual oocytes from the same woman. These data suggest that a restriction/amplification event, which we attribute to clonal expansion of founder mtDNA(s), has occurred by the time oocytes are mature, although further segregation may occur at a later stage. In contrast to controls, the length distribution of the D310 tract varied between tissues in a patient with heteroplasmic mtDNA rearrangements, suggesting that these mutants influence segregation. These findings have important implications for the genetic counselling of patients with pathogenic mtDNA mutations.     

Inheritance of mitochondrial disorders

Over the last decade there have been major advances in our understanding of the genetic basis of mitochondrial disease, enabling genetic counseling for patients with autosomal dominant and autosomal recessive disorders. Genetic counseling for patients with mitochondrial DNA (mtDNA) mutations is less well established. Approximately one-third of adults with a mtDNA disorder are sporadic cases, usually due to a single deletion of mtDNA. About two-thirds of adults with mtDNA disease harbor a maternally transmitted point mutation. The recurrence risks are well documented for homoplasmic mtDNA mutations causing Leber hereditary optic neuropathy, but the situation is less clear for families with heteroplasmic mtDNA disorders. Two large studies have shown that for some heteroplasmic point mutations there appears to be a relationship between the percentage level of mutant mtDNA in a mother's blood and her risk of having clinically affected offspring. The situation is less clear for other point mutations, some of which may cause sporadic disease. Recent evidence has cast light on the general principles behind the transmission of heteroplasmic mtDNA point mutations, which may be important for genetic counseling in the future.