Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome

Mitochondrial mutations, an important cause of incurable human neuromuscular diseases, are mostly heteroplasmic: mutated mitochondrial DNA is present in cells simultaneously with wild-type genomes, the pathogenic threshold being generally >70% of mutant mtDNA. We studied whether heteroplasmy level could be decreased by specifically designed oligoribonucleotides, targeted into mitochondria by the pathway delivering RNA molecules in vivo. Using mitochondrially imported RNAs as vectors, we demonstrated that oligoribonucleotides complementary to mutant mtDNA region can specifically reduce the proportion of mtDNA bearing a large deletion associated with the Kearns Sayre Syndrome in cultured transmitochondrial cybrid cells. These findings may be relevant to developing of a new tool for therapy of mtDNA associated diseases.     

Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes.

Many human mitochondrial diseases are associated with defects in the mitochondrial DNA (mtDNA). Mutated and wild-type forms of mtDNA often coexist in the same cell in a state called heteroplasmy. Here, we report the isolation of a Caenorhabditis elegans strain bearing the 3.1-kb uaDf5 deletion that removes 11 genes from the mtDNA. The uaDf5 deletion is maternally transmitted and has been maintained for at least 100 generations in a stable heteroplasmic state in which it accounts for approximately 60% of the mtDNA content of each developmental stage. Heteroplasmy levels vary between individual animals (from approximately 20 to 80%), but no observable phenotype is detected. The total mtDNA copy number in the uaDf5 mutant is approximately twice that of the wild type. The maternal transmission of the uaDf5 mtDNA is controlled by at least two competing processes: one process promotes the increase in the average proportion of uaDf5 mtDNA in the offspring, while the second promotes a decrease. These two forces prevent the segregation of the mtDNAs to homoplasmy.     

Absolute quantitation of a heteroplasmic mitochondrial DNA deletion using a multiplex three-primer real-time PCR assay

Quantitation of wild-type and deleted mitochondrial DNA (mtDNA) coexisting within the same cell (a.k.a., heteroplasmy) is important in mitochondrial disease and aging. We report the development of a multiplex three-primer PCR assay that is capable of absolute quantitation of wild-type and deleted mtDNA simultaneously. Molecular beacons were designed to hybridize with either type of mtDNA molecule, allowing real-time detection during PCR amplification. The assay is specific and can detect down to six copies of mtDNA, making it suitable for single-cell analyses. The relative standard deviation in the threshold cycle number is approximately 0.6%. Heteroplasmy was quantitated in individual cytoplasmic hybrid cells (cybrids), containing a large mtDNA deletion, and bulk cell samples. Individual cybrid cells contained 100-2600 copies of wild-type mtDNA and 950-4700 copies of deleted mtDNA, and the percentage of heteroplasmy ranged from 43+/-16 to 95+/-16%. The average amount of total mtDNA was 3800+/-1600 copies/cybrid cell, and the average percentage of heteroplasmy correlated well with the bulk cell sample. The single-cell analysis also revealed that heteroplasmy in individual cells is highly heterogeneous. This assay will be useful for monitoring clonal expansions of mtDNA deletions and investigating the role of heteroplasmy in cell-to-cell heterogeneity in cellular models of mitochondrial disease and aging.     

Tissular distribution of heteroplasmy and ultrastructural studies of mitochondria from a Drosophila subobscura mitochondrial deletion mutant

A mutant strain of Drosophila subobscura possesses two mitochondrial genome types: a minority population (20%) identical to the wild strain mtDNA (15.9 kb), and a largely predominant population (80%) of shorter genomes (10.9 kb), presenting a deletion of more than 30% of its coding region. Study of tissular distribution of heteroplasmy shows it to be identical — about 80% — in the head (nervous tissue) and thorax (muscles). On the other hand, a lower percentage (64%) is observed in the ovaries. The strain is apparently unaffected despite this massive loss of genes, coding for four tRNA and for complex I and III subunits. Contrary to observations of similar situations in man, the mutant strain shows no accumulation or structurally abnormal mitochondria. Furthermore, cytochemical studies fail to detect mitochondria devoid of cytochrome oxidase activity (COX^?). Finally, mitoribosome populations are identical in mitochondria from both strains. These results suggest that, in the mutant strain, there are no mitochondria containing deleted genomes only: heteroplasmy would thus be intramitochondrial.     

Heteroplasmy in bovine fetuses produced by intra- and inter-subspecific somatic cell nuclear transfer: neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood

Varying degrees of mitochondrial DNA (mtDNA) heteroplasmy have been observed in nuclear transfer embryos, fetuses, and offspring, but the mechanisms leading to this condition are unknown. We have generated a clone of 12 bovine somatic cell nuclear transfer fetuses, using nuclear donor cells, recipient oocytes, and recipient heifers with defined mtDNA genotypes, to study nuclear-mitochondrial interactions and the origins of mtDNA heteroplasmy. Embryos were reconstructed from granulosa cells with Bos taurus mtDNA type A and recipient oocytes collected from three different maternal lineages with B. taurus mtDNA type B, B. taurus mtDNA type C, or B. indicus mtDNA. Sequence differences in the control region (CR) of B. taurus mtDNAs ranged from 6 to 11 nucleotides and differences between B. taurus and B. indicus CRs from 45 to 50 nucleotides. Fetuses were recovered from recipient heifers with B. taurus mtDNA type B on Day 80 after nuclear transfer (eight B. taurus A/B, two B. taurus A/C, and two B. taurus A/B. indicus). Agarose gel analysis of the CR by polymerase chain reaction-based restriction fragment length polymorphism failed to detect nuclear donor mtDNA in 11 investigated tissues of 10 viable fetuses and in DNA samples of two fetuses in resorption (one B. taurus A/B and one B. taurus A/C). A more sensitive analysis of 1801 plasmid clones with CR inserts derived from tissues of a B. taurus A/B. indicus fetus detected no or very low levels of heteroplasmy (0.5-0.7%). However, the analyses detected considerable amounts ( approximately 2.5% and 5%) of recipient heifer mtDNA in blood samples from two fetuses. Our data do not suggest a replicative advantage of somatic nuclear donor cell mtDNA in bovine transmitochondrial clones produced with oocytes from domestic forms of the same or a different aurochs (B. primigenius) subspecies. Detection of mtDNA from the recipient animal in the circulation of two fetuses points to leakage of the placental barrier, mimicking heteroplasmy.     

Sequence homology at the breakpoint and clinical phenotype of mitochondrial DNA deletion syndromes

Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders. Large mtDNA deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the deletion compared to mtDNA background. The youngest patient group (<6 years old) showed a diffused pattern of deletion distribution in size and locations, with a significantly lower sequence homology flanking the deletion, and the highest percentage of deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (<6 years old) carry the 5 kb common deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the deletion flanking regions is a consistent feature of mtDNA deletions. Decreased levels of sequence homology and increased levels of deletion mutant heteroplasmy appear to correlate with earlier onset and more severe disease with multisystem involvement.     

Modulating heteroplasmy

Patients with mitochondrial DNA (mtDNA) disease usually harbor a mixture of mutant and wild-type mtDNA (a state termed heteroplasmy), and the clinical features of the disease depend on the percentage of mutant mtDNA (the "mutation load") in vulnerable tissues. Factors that modulate the mutation load are poorly understood, but recent work has started to unravel the mechanisms. In certain circumstances heteroplasmy might be regulated at the level of the individual mitochondrial genome.     

The nuclear genome is involved in heteroplasmy control in a mitochondrial mutant strain of Drosophila subobscura.

Most (78%) mitochondrial genomes in the studied mutant strain of Drosophila subobscura have undergone a large-scale deletion (5 kb) in the coding region. This mutation is stable, and is transmitted intact to the offspring. This animal model of major rearrangements of mitochondrial genomes can be used to analyse the involvement of the nuclear genome in the production and maintenance of these rearrangements. Successive backcrosses between mutant strain females and wild-type males yield a biphasic change in heteroplasmy level: (a) a 5% decrease in mutated genomes per generation (from 78 to 55%), until the nuclear genome is virtually replaced by the wild-type genome (seven to eight crosses); and (b) a continuous decrease of 0.5% per generation when the nuclear context is completely wild-type. In parallel with these changes, NADH dehydrogenase activity, which is halved in the mutant strain (five subunits of this complex are affected by the mutation), gradually increases and stabilizes near the wild-type activity. A return to a nuclear context is accompanied by the opposite phenomena: progressive increase in heteroplasmy level and stabilization at the value seen in the wild-type strain and a decrease in the activity of complex I. These results indicate that the nuclear genome plays an important role in the control of heteroplasmy level and probably in the production of rearranged genomes.     

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere(NT-B) and fibroblast(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p<0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p<0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.     

Metabolically induced heteroplasmy shifting and l-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS

The m.3243A>G variant in the mitochondrial tRNA(Leu(UUR)) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood. We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A>G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition of l-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition of l-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting and l-arginine therapy may constitute promising therapeutic strategies against MELAS.     

Transmission of mitochondrial DNA in pigs and progeny derived from nuclear transfer of Meishan pig fibroblast cells

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Here, we examined the transmission of mtDNA from NT pigs to their progeny. NT pigs were created by microinjection of Meishan pig fetal fibroblast nuclei into enucleated oocytes (maternal Landrace background). Transmission of donor cell (Meishan) mtDNA was analyzed using 4 NT pigs and 25 of their progeny by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, PCR-RFLP, and a specific PCR to detect Meishan mtDNA single nucleotide polymorphisms (SNP-PCR). In the blood and hair root of NT pigs, donor mtDNAs were not detected by PCR-SSCP and PCR-RFLP, but detected by SNP-PCR. These results indicated that donor mtDNAs comprised between 0.1% and 1% of total mtDNA. Only one of the progeny exhibited heteroplasmy with donor cell mtDNA populations, ranging from 0% to 44% in selected tissues. Additionally, other progeny of the same heteroplasmic founder pig were analyzed, and 89% (16/18) harbored donor cell mtDNA populations. The proportion of donor mtDNA was significantly higher in liver (12.9 +/- 8.3%) than in spleen (5.0 +/- 3.9%), ear (6.7 +/- 5.3%), and blood (5.8 +/- 3.7%) (P < 0.01). These results demonstrated that donor mtDNAs in NT pigs could be transmitted to progeny. Moreover, once heteroplasmy was transmitted to progeny of NT-derived pigs, it appears that the introduced mitochondrial populations become fixed and maternally-derived heteroplasmy was more readily maintained in subsequent generations.     

The distribution of mitochondrial DNA heteroplasmy due to random genetic drift

Cells containing pathogenic mutations in mitochondrial DNA (mtDNA) generally also contain the wild-type mtDNA, a condition called heteroplasmy. The amount of mutant mtDNA in a cell, called the heteroplasmy level, is an important factor in determining the amount of mitochondrial dysfunction and therefore the disease severity. mtDNA is inherited maternally, and there are large random shifts in heteroplasmy level between mother and offspring. Understanding the distribution in heteroplasmy levels across a group of offspring is an important step in understanding the inheritance of diseases caused by mtDNA mutations. Previously, our understanding of the heteroplasmy distribution has been limited to just the mean and variance of the distribution. Here we give equations, adapted from the work of Kimura on random genetic drift, for the full mtDNA heteroplasmy distribution. We describe how to use the Kimura distribution in mitochondrial genetics, and we test the Kimura distribution against human, mouse, and Drosophila data sets.     

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.     

Mitochondrial biochemical activities and heteroplasmy evolution in established D. subobscura cell line

Summary A mutant strain of drosophila (D. subobscura) has two types of mitochondrial genomes: a small population (20%) identical to that of the wild strain (15.9 kb) and a predominant population (80%) which has undergone a 5-kb deletion affecting more than 30% of the coding zone. Two cell lines were established from homogenates of embryos from mutant and wild strains. The activities of the respiratory complexes measured in the different cell lines are much lower than in the flies, indicating a glycolytic metabolism. Various modifications of the medium composition did not change this metabolic pathway. The mutant cell line has two types of populations of mitochondrial genomes and the heteroplasmy is equivalent to that measured in the mutant strain. However, the biochemical characteristics differ from those observed in the flies (i.e., the decrease of complex I and III activities), and the various systems of compensation for the consequences of the deletion that are showed in the mutant strain are no longer observed. Furthermore, in contrast with observations made on mutant flies, the heteroplasmy appears unstable in the mutant cell lines: after 60 or so generations, it progressively decreases until it disappears completely. The limited importance of mitochondrial energy metabolism in cells may explain the low impact of the mutation on the established cell line, in contrast to what is seen in the mutant strain.     

Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection

Laser-capture microdissection was coupled with PCR to define the mitochondrial genotype of aged muscle fibers exhibiting mitochondrial enzymatic abnormalities. These electron transport system (ETS) abnormalities accumulate with age, are localized segmentally along muscle fibers, are associated with fiber atrophy and may contribute to age-related fiber loss. DNA extracted from single, 10 µm thick, ETS abnormal muscle fibers, as well as sections from normal fibers, served as templates for PCR-based deletion analysis. Large mitochondrial (mt) DNA deletion mutations (4.4–9.7 kb) were detected in all 29 ETS abnormal fibers analyzed. Deleted mtDNA genomes were detected only in the regions of the fibers with ETS abnormalities; adjacent phenotypically normal portions of the same fiber contained wild-type mtDNA. In addition, identical mtDNA deletion mutations were found within different sections of the same abnormal region. These findings demonstrate that large deletion mutations are associated with ETS abnormalities in aged rat muscle and that, within a fiber, deletion mutations are clonal. The displacement of wild-type mtDNAs with mutant mtDNAs results in concomitant mitochondrial enzymatic abnormalities, fiber atrophy and fiber breakage.     

Familial mitochondrial encephalomyopathy (MERRF): genetic, pathophysiological, and biochemical characterization of a mitochondrial DNA disease

A large MERRF pedigree permitted the direct testing of the predictions for a mitochondrial DNA (mtDNA) mutation. A mtDNA mutation was demonstrated by proving maternal inheritance and by identifying specific deficiencies in muscle energetics and mitochondrial respiratory complexes I and IV. mtDNA heteroplasmy (a mixture of mutant and wild-type mtDNAs) was demonstrated by showing variation in the mitochondrial energetic capacity between family members. The phenotypic consequences of differential tissue-specific reliance on mitochondrial ATP was shown by correlating individual respiratory deficiency with the nature and severity of patients' clinical manifestations. The observed spectrum of clinical manifestations resulting from this heteroplasmic mtDNA mutation implies that mtDNA disease may be much more prevalent than previously anticipated.     

Deletion-mutant mtDNA increases in somatic tissues but decreases in female germ cells with age

The proportions of mutant and wild-type mtDNA are crucial in determining the severity of mitochondrial diseases. It has been generally considered that deletion-mutant mtDNA has replication advantages and accumulates with time. Here, we examine the tissue-by-tissue proportions of mutant mtDNA with a 4696-bp deletion (DeltamtDNA) and wild-type mtDNA in mitochondrial disease model mice (mito-mice). Comparison of the proportions of DeltamtDNA in each tissue at various ages showed that the rate of accumulation of DeltamtDNA differed among tissues. The heart, skeletal muscles, kidney, liver, testis, and ovary showed increases in the proportion of DeltamtDNA with age, but the pancreas, spleen, brain, and blood showed only a slight or no increase in proportion. In contrast to the somatic tissues, however, the germ cells of female mito-mice and resultant offspring showed a strong decrease in DeltamtDNA with maternal age. The decrease was so acute that some offspring showed complete disappearance of DeltamtDNA, even though their elder brothers and sisters had high proportions of DeltamtDNA. Female germ cells have a machinery that prevents the inheritance of defective mtDNA to the following generation since germ cells are kept for a long time until they are ovulated.     

线粒体DNA的异质性

哺乳动物线粒体DNA（mitochondrial DNA, mtDNA）位于线粒体.当细胞中mtDNA发生突变时,就会出现野生型与突变型mtDNA的共存.这种情况被称为mtDNA异质性.从mtDNA异质性的形成到在表型上引起相应的病变是一个复杂的过程.mtDNA异质性是如何形成和其在特异组织的增殖复制,mtDNA异质性的变化对个体的影响,如何提高mtDNA突变负荷检测的精度和灵敏度都是近些年的研究热点.本文对mtDNA异质性的检测、遗传、组织特异性以及其相关的疾病等方面进行了阐述.     

Genetic Analysis of Systematic Mitochondrial Heteroplasmy in Rabbits

One unusual property of rabbit mitochondrial DNA (mtDNA) is the existence of repeated 153-bp motifs in the vicinity of the replication origin of its H strand. Furthermore, every individual is heteroplasmic: it carries mtDNA molecules with a variable number of repeats. A systematic study of 8 females and their progeny has been devised to analyze mtDNA transmission through generations. The results suggest that three mechanisms are acting simultaneously. (1) Genetic drift in the germ line is revealed by the evolution of heteroplasmy when two major molecular forms are present in a female. (2) A high mutation rate (around 10(-2) per animal generation) generating molecular diversity, by deletion and addition of repeated units, is required to explain the observation of heteroplasmy in every individual. Moreover, the rates of mutation from the most frequent type to the other types are unequal. The deletion of one unit is more frequent than a deletion of two units, which is in turn more frequent than a deletion of three. (3) Selection for shorter molecules in somatic cells is probable. The frequency distribution of mtDNA types depends on the organ analyzed (kidney-spleen and liver vs. gonads).