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.     

线粒体DNA异质性

线粒体 DNA（mitochondrial DNA,mtDNA）是线粒体内最重要的遗传物质。mtDNA 突变普 遍存在,突变型 mtDNA 与野生型 mtDNA 共存的现象被称为 mtDNA 异质性。mtDNA 异质性与衰老和多种疾病密切相关。mtDNA异质性特性、mtDNA 异质性与衰老和疾病相关性以及线粒体疾病的治疗等都是近年来遗传学研究的热点。本文从 mtDNA 异质性的动态变化、组织特异性、mtDNA 异质性与疾病以及线粒体疾病的治疗等方面对 mtDNA 异质性进行综述。     

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype–phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNA^Lys mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.     

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.     

Detection of mutations in mitochondrial DNA by droplet digital PCR

Mutations in mitochondrial DNA (mtDNA) may result in various pathological processes. Detection of mutant mtDNAs is a problem for diagnostic practice that is complicated by heteroplasmy – a phenomenon of the inferring presence of at least two allelic variants of the mitochondrial genome. Also, the level of heteroplasmy largely determines the profile and severity of clinical manifestations. Here we discuss detection of mutations in heteroplasmic mtDNA using up-todate methods that have not yet been introduced as routine clinical assays. These methods can be used for detecting mutations in mtDNA to verify diagnosis of “mitochondrial disease”, studying dynamics of mutant mtDNA in body tissues of patients, as well as investigating structural features of mtDNAs. Original data on allele-specific discrimination of m.11778G>A mutation by droplet digital PCR are presented, which demonstrate an opportunity for simultaneous detection and quantitative assessment of mutations in mtDNAs.     

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.     

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.     

Mitochondrial genome and human mitochondrial diseases

Today there are described more than 400 point mutations and more than hundred of structural rearrangements of mitochondrial DNA associated with characteristic neuromuscular and other mitochondrial syndromes, from lethal in the neonatal period of life to the disease with late onset. The defects of oxidative phosphorylation are the main reasons of mitochondrial disease development. Phenotypic diversity and phenomenon of heteroplasmy are the hallmark of mitochondrial human diseases. It is necessary to assess the amount of mutant mtDNA accurately, since the level of heteroplasmy largely determines the phenotypic manifestation. In spite of tremendous progress in mitochondrial biology since the cause-and-effect relations between mtDNA mutation and the human diseases was established over 20 years ago, there is still no cure for mitochondrial diseases.     

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.     

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.     

Searching for a needle in the haystack: comparing six methods to evaluate heteroplasmy in difficult sequence context

Mitochondrial DNA (mtDNA) mutations have been involved in disease, aging and cancer and furthermore exploited for evolutionary and forensic investigation. When investigating mtDNA mutations the peculiar aspects of mitochondrial genetics, such as heteroplasmy and threshold effect, require suitable approaches which must be sensitive enough to detect low-level heteroplasmy and, precise enough to quantify the exact mutational load. In order to establish the optimal approach for the evaluation of heteroplasmy, six methods were experimentally compared for their capacity to reveal and quantify mtDNA variants. Drawbacks and advantages of cloning, Fluorescent PCR (F-PCR), denaturing High Performance Liquid Chromatography (dHPLC), quantitative Real-Time PCR (qRTPCR), High Resolution Melting (HRM) and 454 pyrosequencing were determined. In particular, detection and quantification of a mutation in a difficult sequence context were investigated, through analysis of an insertion in a homopolymeric stretch (m.3571insC).     

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.     

线粒体DNA的异质性与检测方法

线粒体DNA的异质性在生物学、医学、法医、生物技术等领域有重要的应用。本文从自然发生(包括体细胞突变、父系渗入和杂交)和人工产生(包括转基因、细胞融合、核移植和转线粒体)两大方面系统地介绍了线粒体DNA异质性的产生机制及其遗传。并介绍了线粒体DNA异质性的检测方法,已知突变位点mtDNA异质性的检测方法有原位PCR技术、PCR-RFLP和实时荧光定量PCR技术,未知突变位点mtDNA异质性的检测方法有长PCR技术、时相温度梯度凝胶电泳(TTGE)和变性高效液相色谱(DHPLC)等。最后对克隆生物中线粒体异质性检测的应用实例作了介绍。     

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.     

Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography

In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1–2 kb, digested in fragments of 90–600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA^Leu(UUR) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA^Leu(UUR) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.     

Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing

Disease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations.     

Assaying the probabilities of obtaining maternally inherited heteroplasmy as the basis for modeling OXPHOS diseases in animals

Gross alterations in cell energy metabolism underlie manifestations of hereditary OXPHOS (oxidative phosphorylation) diseases, many of which depend on proportion of mutant mitochondrial DNA (mtDNA) in tissues. An animal model of OXPHOS disease with maternal inheritance of mitochondrial heteroplasmy might help understanding the peculiarities of abnormal mtDNA distribution and its effect on pre- and postnatal development. Previously we obtained mice that carry human mtDNA in some tissues. It co-existed with murine mtDNA (heteroplasmy) and was transmitted maternally to the progeny of animals developed from zygotes injected with human mitochondria. To analyze the probability of obtaining heteroplasmic mice we increased the number of experiments with early embryos and obtained more specimens from F1. About 33% of zygotes injected with human mtDNA developed into post-implantation embryos (7th-13th days). Lower amount of such developed into neonate mice (ca. 21%). Among post-implantation embryos and in generations F0 and F1 percentages of human mtDNA-carriers were ca. 14-16%. Such percentages are sufficient for modeling maternally inherited heteroplasmy in small animal groups. More data are needed to understand the regularities of anomalous mtDNA distribution among cells and tissues and whether heart and muscles frequently carrying human mtDNA in our experiments are particularly susceptible to heteroplasmy.     

The implications of mitochondrial DNA copy number regulation during embryogenesis

Mutations of mitochondrial DNA (mtDNA) cause a wide array of multisystem disorders, particularly affecting organs with high energy demands. Typically only a proportion of the total mtDNA content is mutated (heteroplasmy), and high percentage levels of mutant mtDNA are associated with a more severe clinical phenotype. MtDNA is inherited maternally and the heteroplasmy level in each one of the offspring is often very different to that found in the mother. The mitochondrial genetic bottleneck hypothesis was first proposed as the explanation for these observations over 20 years ago. Although the precise bottleneck mechanism is still hotly debated, the regulation of cellular mtDNA content is a key issue. Here we review current understanding of the factors regulating the amount of mtDNA within cells and discuss the relevance of these findings to our understanding of the inheritance of mtDNA heteroplasmy.     

Gene therapy for mitochondrial disease by delivering restriction endonucleaseSmaI into mitochondria

The restriction endonucleaseSmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993TG mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F₀F₁-ATPase. Our ultimate goal is to applySmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed theSmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by theSmaI enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targetedEcoRI induced a decrease in cytochromec oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases.