Mutations in mtDNA: Are We Scraping the Bottom of the Barrel?

The small, maternally inherited mtDNA has turned out to be a Pandora's box of pathogenic mutations: 12 years into the era of "mitochondrial medicine," about 100 pathogenic point mutations and innumerable rearrangements have been associated with a bewildering variety of multisystemic as well as tissue-specific human diseases. After reviewing the principles of mitochondrial genetics, we compare and contrast the clinical and pathological features of disorders due to mutations in genes affecting mitochondrial protein synthesis with those of mutations in protein-coding genes. In contrast to the striking progress in our understanding of etiology, pathogenesis is only partially explained by the rules of mitochondrial genetics and remains largely terra incognita. We review recent progress in prenatal diagnosis and epidemiology. Therapy is still woefully inadequate, but a number of promising approaches are being developed.     

Mitochondrial Encephalomyopathies: Defects of Nuclear DNA

The term "mitochondrial diseases" encompasses a heterogeneous group of disorders in which a primary mitochondrial dysfunction is suspected or proven by morphologic, genetic, or biochemical criteria. Clinically, these progressive disorders usually affect muscle, either alone (mitochondrial myopathies) or in combination with other systems, most often brain (encephalomyopathies). Mitochondria are unique among intracellular organelles in that mitochondrial proteins are encoded by two genomes, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). The vast majority of mitochondrial proteins are encoded by the nuclear genome, whereas mtDNA (a circular, double stranded 16.5 kb molecule) encodes only 13 polypeptides, all of them subunits of respiratory chain complexes. In addition to structural genes, mtDNA also codes for 22 transfer RNAs and two ribosomal RNAs. Our understanding of mitochondrial diseases has grown at an impressive rate in the past few years, and most of the progress has been in the area of mtDNA genetics, where several mtDNA mutations have been associated with specific diseases (reviewed in this issue by Zeviani et al.). In comparison, our understanding of mitochondrial disorders due to nDNA lesions has lagged behind and, to date, molecular defects of nuclear genes have been documented in only a few patients. We will review which alterations in the nuclear genome can cause mitochondrial disorders and which criteria are useful in identifying such mutations. While several examples will be provided, this is not intended as a complete review of the subject.     

Transmission of the human mitochondrial genome

The segregation and transmission of mitochondrial genomes in humans are complicated processes, but are particularly important for understanding the inheritance and clinical abnormalities of mitochondrial disorders. This review describes three aspects of mitochondrial genetics. First, that the segregation and transmission of mitochondrial (mt)DNA molecules are likely to be determined by their physical association within the organelles and by the dynamics of mitochondrial structure and subcellular organization. Second, that the transmission of heteroplasmic mtDNA sequence changes from one generation to the next often involves rapid shifts in allele frequency. For >20 years, the standard explanation has been that there is a developmental bottleneck in which, at some stage of oogenesis, there is a reduction in the effective number of mitochondrial units of inheritance. The third aspect is that ongoing analyses of the segregation and transmission of pathogenic mtDNA mutations indicate the operation of multiple genetic processes. Thus, the segregation and transmission of mtDNA mutations occurs predominantly, but not exclusively, under conditions of random genetic drift. However, there is also evidence for bias due to incomplete ascertainment of pedigrees and for negative selection of pathogenic mutations in rapidly dividing somatic tissues such as the white blood cell population.     

Mutation screening of the mitochondrial genome using denaturing high-performance liquid chromatography

Over 170 known mutations of the mitochondrial genome are responsible for disease. Due to the unique features of mitochondrial genetics, such patients are clinically diverse and difficult to diagnose. As pathogenic mitochondrial DNA (mtDNA) mutations are mostly heteroplasmic, denaturing high-performance liquid chromatography (DHPLC) could be used to detect these heteroplasmic species and therefore act as a rapid screening test for mtDNA mutations. The entire mitochondrial genome was amplified by PCR in 40 overlapping regions. In addition, known mtDNA mutants were constructed for each of these regions using a PCR-based site-directed mutagenesis approach. These mutants were used as positive controls and showed a detection limit of 3-10% heteroplasmy by DHPLC (depending on the specific mutation) compared to 40% for conventional sequencing. To further validate the screening test, mtDNA from 17 patients with seven different pathogenic mutations was used to compare mutation detection by DHPLC and conventional sequencing. DHPLC had a sensitivity of 88% compared to 82% for sequencing. This increased to 100% sensitivity for DHPLC when excluding the m.8993T>G mutation. DHPLC analysis is therefore a sensitive, rapid and cost-effective method to screen for mutations in the mitochondrial genome. The role of pyrosequencing in the quantitation of mutant load for known mtDNA mutations was highlighted using the m.3243A>G mutation as an illustrative example. Pyrosequencing analysis was able to discriminate samples containing as little as 5% heteroplasmy and proved to be an accurate and reproducible method for estimation of mutant load.     

Mitochondrial DNA deletion mutations in patients with neuropsychiatric symptoms

It has been suggested that mitochondrial dysfunction is important in the pathogenesis of psychiatric disorders such as depression, schizophrenia and dementia. We report herein three adult patients exhibiting such psychiatric symptoms as the core manifestation, accompanied by various degrees of myopathic symptoms. Pathological findings in biopsied skeletal muscle were compatible with mitochondrial myopathy in all cases. Maternal inheritance was not apparent in all three cases; however, two patients were born to consanguineous parents. Mutation analysis on the mitochondrial DNA (mtDNA) and seven nuclear genes, in which pathogenic mutations are known to cause mtDNA deletions, was performed. MtDNA deletion mutations were identified in skeletal muscles of all patients. Neither pathogenic mutations nor copy number variation was identified among the nuclear genes. Although further studies are needed, the molecular pathways inducing mitochondrial abnormalities may be implicated in a variety of psychiatric conditions.     

The pathophysiology of mitochondrial disease as modeled in the mouse

It is now clear that mitochondrial defects are associated with a plethora of clinical phenotypes in man and mouse. This is the result of the mitochondria''s central role in energy production, reactive oxygen species (ROS) biology, and apoptosis, and because the mitochondrial genome consists of roughly 1500 genes distributed across the maternal mitochondrial DNA (mtDNA) and the Mendelian nuclear DNA (nDNA). While numerous pathogenic mutations in both mtDNA and nDNA mitochondrial genes have been identified in the past 21 years, the causal role of mitochondrial dysfunction in the common metabolic and degenerative diseases, cancer, and aging is still debated. However, the development of mice harboring mitochondrial gene mutations is permitting demonstration of the direct cause-and-effect relationship between mitochondrial dysfunction and disease. Mutations in nDNA-encoded mitochondrial genes involved in energy metabolism, antioxidant defenses, apoptosis via the mitochondrial permeability transition pore (mtPTP), mitochondrial fusion, and mtDNA biogenesis have already demonstrated the phenotypic importance of mitochondrial defects. These studies are being expanded by the recent development of procedures for introducing mtDNA mutations into the mouse. These studies are providing direct proof that mtDNA mutations are sufficient by themselves to generate major clinical phenotypes. As more different mtDNA types and mtDNA gene mutations are introduced into various mouse nDNA backgrounds, the potential functional role of mtDNA variation in permitting humans and mammals to adapt to different environments and in determining their predisposition to a wide array of diseases should be definitively demonstrated.     

A functionally dominant mitochondrial DNA mutation

Mutations in mitochondrial DNA (mtDNA) tRNA genes can be considered functionally recessive because they result in a clinical or biochemical phenotype only when the percentage of mutant molecules exceeds a critical threshold value, in the range of 70-90%. We report a novel mtDNA mutation that contradicts this rule, since it caused a severe multisystem disorder and respiratory chain (RC) deficiency even at low levels of heteroplasmy. We studied a 13-year-old boy with clinical, radiological and biochemical evidence of a mitochondrial disorder. We detected a novel heteroplasmic C>T mutation at nucleotide 5545 of mtDNA, which was present at unusually low levels (<25%) in affected tissues. The pathogenic threshold for the mutation in cybrids was between 4 and 8%, implying a dominant mechanism of action. The mutation affects the central base of the anticodon triplet of tRNA(Trp) and it may alter the codon specificity of the affected tRNA. These findings introduce the concept of dominance in mitochondrial genetics and pose new diagnostic challenges, because such mutations may easily escape detection. Moreover, similar mutations arising stochastically and accumulating in a minority of mtDNA molecules during the aging process may severely impair RC function in cells.     

Two pathogenic point mutations exist in the authentic mitochondrial genome, not in the nuclear pseudogene

Technical advancements in molecular genetics have shown various mitochondrial DNA (mtDNA) abnormalities in patients with mitochondrial myopathies. Recently, it has been revealed that, in these patients, the nuclear DNA carries sequences similar to those of the mtDNA (nuclear pseudogene) and it has several point mutations previously reported to be pathogenic. We verified the existence of the T3250C and T3291C mutations, which we have found in patients with mitochondrial myopathy, in the authentic mitochondrial genome. A long polymerase chain reaction provides a powerful tool for avoiding nuclear pseudogene amplification and for ruling out ambiguity in the detection of the mutation for diagnosis. Received: August 2, 2000 / Accepted: August 30, 2000     

Mitochondrial DNA background modulates the assembly kinetics of OXPHOS complexes in a cellular model of mitochondrial disease

Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disorder, is mostly due to three mitochondrial DNA (mtDNA) mutations in respiratory chain complex I subunit genes: 3460/ND1, 11778/ND4 and 14484/ND6. Despite considerable clinical evidences, a genetic modifying role of the mtDNA haplogroup background in the clinical expression of LHON remains experimentally unproven. We investigated the effect of mtDNA haplogroups on the assembly of oxidative phosphorylation (OXPHOS) complexes in transmitochondrial hybrids (cybrids) harboring the three common LHON mutations. The steady-state levels of respiratory chain complexes appeared normal in mutant cybrids. However, an accumulation of low molecular weight subcomplexes suggested a complex I assembly/stability defect, which was further demonstrated by reversibly inhibiting mitochondrial protein translation with doxycycline. Our results showed differentially delayed assembly rates of respiratory chain complexes I, III and IV amongst mutants belonging to different mtDNA haplogroups, revealing that specific mtDNA polymorphisms may modify the pathogenic potential of LHON mutations by affecting the overall assembly kinetics of OXPHOS complexes.     

Respiratory chain complex I deficiency caused by mitochondrial DNA mutations

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.     

Placing mitochondrial DNA mutations within the progression model of type I endometrial carcinoma

Mitochondrial DNA (mtDNA) mutations have been described in almost all types of cancer. However, their exact role and timing of occurrence during tumor development and progression are still a matter of debate. A Vogelstein-like model of progression is well established for endometrial carcinoma (EC), however, mtDNA has been scarcely investigated in these tumors despite the fact that mitochondrial biogenesis increase has been shown to be a hallmark of type I EC. Here, we screened a panel of 23 type I EC tissues and matched typical hyperplasia for mutations in mtDNA and in four oncosupressors/oncogenes, namely PTEN, KRAS, CTNNB1 and TP53. Overall, mtDNA mutations were identified in 69% of cases, while mutational events in nuclear genes occurred in 56% of the cases, indicating that mtDNA mutations may precede the genetic instability of these genes canonically involved in progression from hyperplasia to tumor. Protein expression analysis revealed an increase in mitochondrial biogenesis and activation of oxidative stress response mechanisms in tumor tissues, but not in hyperplasia, in correlation with the occurrence of pathogenic mtDNA mutations. Our results point out an involvement of mtDNA mutations in EC progression and explain the increase in mitochondrial biogenesis of type I EC. Last, since mtDNA mutations occur after hyperplasia, their potential role in contributing to genetic instability may be envisioned.     

线粒体遗传与人类系统性高血压

线粒体DNA(mitochondrial DNA,mtDNA)具有母系遗传的特点,由mtDNA突变引起的家族性线粒体疾病常累及心、脑、骨骼肌等高能耗器官.近年来,研究者发现部分原发性高血压患者具有典型的母系遗传特点,从而证实并丰富了mtDNA突变在母系遗传性高血压中的作用.然而,一些根本的共性问题仍然有待于进一步的研究与探讨.本文就线粒体基因组的进化、mtDNA的遗传方式、mtDNA突变在母系遗传性高血压中的分子机理进行综述,并对今后的研究方向提出设想.     

Generation of trans-mitochondrial mice carrying homoplasmic mtDNAs with a missense mutation in a structural gene using ES cells

Generation of various kinds of trans-mitochondrial mice, mito-mice, each carrying mtDNAs with a different pathogenic mutation, is required for precise investigation of the pathogenesis of mitochondrial diseases. This study used two respiration-deficient mouse cell lines as donors of mtDNAs with possible pathogenic mutations. One cell line expressed 45-50% respiratory activity due to mouse mtDNAs with a T6589C missense mutation in the COI gene (T6589C mtDNA) and the other expressed 40% respiratory activity due to rat (Rattus norvegicus) mtDNAs in mouse cells. By cytoplasmic transfer of these mtDNAs to mouse ES cells, we isolated respiration-deficient ES cells. We obtained chimeric mice and generated their F(6) progeny carrying mouse T6589C mtDNAs by its female germ line transmission. They were respiration-deficient and thus could be used as models of mitochondrial diseases caused by point mutations in mtDNA structural genes. However, chimeric mice and mito-mice carrying rat mtDNAs were not obtained, suggesting that significant respiration defects or some deficits induced by rat mtDNAs in mouse ES cells prevented their differentiation to generate mice carrying rat mtDNAs.     

Whole mitochondrial genome screening in maternally inherited non-syndromic hearing impairment using a microarray resequencing mitochondrial DNA chip

Mitochondrial DNA (mtDNA) mutations have been implicated in non-syndromic hearing loss either as primary or as predisposing factors. As only a part of the mitochondrial genome is usually explored in deafness, its prevalence is probably under-estimated. Among 1350 families with non-syndromic sensorineural hearing loss collected through a French collaborative network, we selected 29 large families with a clear maternal lineage and screened them for known mtDNA mutations in 12S rRNA, tRNASer(UCN) and tRNALeu(UUR) genes. When no mutation could be identified, a whole mitochondrial genome screening was performed, using a microarray resequencing chip: the MitoChip version 2.0 developed by Affymetrix Inc. Known mtDNA mutations was found in nine of the 29 families, which are described in the article: five with A1555G, two with the T7511C, one with 7472insC and one with A3243G mutation. In the remaining 20 families, the resequencing Mitochip detected 258 mitochondrial homoplasmic variants and 107 potentially heteroplasmic variants. Controls were made by direct sequencing on selected fragments and showed a high sensibility of the MitoChip but a low specificity, especially for heteroplasmic variations. An original analysis on the basis of species conservation, frequency and phylogenetic investigation was performed to select the more probably pathogenic variants. The entire genome analysis allowed us to identify five additional families with a putatively pathogenic mitochondrial variant: T669C, C1537T, G8078A, G12236A and G15077A. These results indicate that the new MitoChip platform is a rapid and valuable tool for identification of new mtDNA mutations in deafness.     

Mitochondrial deafness mutations reviewed

The first molecular defect for nonsyndromic hearing loss was identified in 1993, and was a mitochondrial mutation. Since then a number of inherited mitochondrial DNA (mtDNA) mutations have been implicated in hearing loss, and acquired mtDNA mutations have been proposed as one of the causes of the hearing loss associated with aging, presbyacusis. These molecular findings have raised as many questions as they have answered, however, since the pathophysiology between the mutations and the clinical phenotype remains poorly understood. This mini-review will, after a short background review of mitochondrial genetics, (1) outline the different mtDNA mutations associated with inherited syndromic, nonsyndromic, and ototoxic hearing loss, (2) summarize the data on acquired mtDNA mutations and their possible association with presbyacusis, (3) describe the biochemical consequences of the inherited mtDNA mutations, (4) suggest the clinical implications of the identification of these mutations, and (5) discuss the penetrance and tissue specificity of the hearing associated mtDNA mutations.     

Novel non‐neutral mitochondrial DNA mutations found in childhood acute lymphoblastic leukemia

Mitochondria produce adenosine triphosphate (ATP) for energy requirements via the mitochondrial oxidative phosphorylation (OXPHOS) system. One of the hallmarks of cancer is the energy shift toward glycolysis. Low OXPHOS activity and increased glycolysis are associated with aggressive types of cancer. Mitochondria have their own genome (mitochondrial DNA [mtDNA]) encoding for 13 essential subunits of the OXPHOS enzyme complexes. We studied mtDNA in childhood acute lymphoblastic leukemia (ALL) to detect potential pathogenic mutations in OXPHOS complexes. The whole mtDNA from blood and bone marrow samples at diagnosis and follow‐up from 36 ALL patients were analyzed. Novel or previously described pathogenic mtDNA mutations were identified in 8 out of 36 patients. Six out of these 8 patients had died from ALL. Five out of 36 patients had an identified poor prognosis genetic marker, and 4 of these patients had mtDNA mutations. Missense or nonsense mtDNA mutations were detected in the genes encoding subunits of OXPHOS complexes, as follows: MT‐ND1, MT‐ND2, MT‐ND4L and MT‐ND6 of complex I; MT‐CO3 of complex IV; and MT‐ATP6 and MT‐ATP8 of complex V. We discovered mtDNA mutations in childhood ALL supporting the hypothesis that non‐neutral variants in mtDNA affecting the OXPHOS function may be related to leukemic clones.     

Biochemical analysis of human POLG2 variants associated with mitochondrial disease

Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic.     

Gene expression profile in dilated cardiomyopathy caused by elevated frequencies of mitochondrial DNA mutations in the mouse heart.

BACKGROUND: Elevated mitochondrial DNA (mtDNA) mutations are associated with aging and age-related diseases, but their pathogenic potential is unclear. METHODS: We performed expression profiling using an Incyte cDNA array of a mouse model of elevated mtDNA mutations wherein random mutations accumulate specifically in the heart. At frequencies of about 1 mutation/10,000 base pairs, these mice show apoptosis of cardiomyocytes and development of four-chamber dilated cardiomyopathy. RESULTS: Significant Analysis of Microarrays (SAM) revealed that 117 genes were altered in their expression in the transgenic (Tg) heart at a threshold of less than one false positive, of which 34 were up-regulated and 83 were down-regulated. Some of the changes were confirmed by Northern and Western blots. By classification of these genes into functional categories, we identified changes that reflected cardiac pathology. The results indicated that cardiomyopathy caused by mtDNA mutations was largely characterized by gene expression changes indicative of increased fibrosis and cardiac remodeling of the extracellular matrix. Few changes were observed, suggesting an alteration in either mitochondrial energy production or generation of increased oxidative stress. CONCLUSIONS: Elevated frequencies of mtDNA mutations in the mouse heart lead to gene expression changes that are associated with remodeling of the extracellular matrix. Because cardiomyocytic death by apoptosis is also a feature of the dilated cardiomyopathy evident in these mice, extracellular remodeling may be a response to apoptotic signaling originating from the mitochondria with mtDNA mutations.     

Role of mitochondrial mutations in cancer

A role for mitochondria in cancer causation has been implicated through identification of mutations in the mitochondrial DNA (mtDNA) and in nuclear-encoded mitochondrial genes. Although many mtDNA mutations were detected in common tumors, an unequivocal causal link between heritable mitochondrial abnormalities and cancer is provided only by the germ line mutations in the nuclear-encoded genes for succinate dehydrogenase (mitochondrial complex II) and fumarate hydratase (fumarase). The absence of evidence for highly penetrant tumors caused by inherited mtDNA mutations contrasts with the frequent occurrence of mtDNA mutations in many different tumor types. Thus, either the majority of diverse mtDNA mutations observed in tumors are not important for the process of carcinogenesis or that they play a common oncogenic role.     

Mitochondrial DNA somatic mutations (point mutations and large deletions) and mitochondrial DNA variants in human thyroid pathology: a study with emphasis on Hürthle cell tumors

In an attempt to progress in the understanding of the relationship of mitochondrial DNA (mtDNA) alterations and thyroid tumorigenesis, we studied the mtDNA in 79 benign and malignant tumors (43 Hürthle and 36 non-Hürthle cell neoplasms) and respective normal parenchyma. The mtDNA common deletion (CD) was evaluated by semiquantitative polymerase chain reaction. Somatic point mutations and sequence variants of mtDNA were searched for in 66 tumors (59 patients) and adjacent parenchyma by direct sequencing of 70% of the mitochondrial genome (including all of the 13 OXPHOS system genes). We detected 57 somatic mutations, mostly transitions, in 34 tumors and 253 sequence variants in 59 patients. Follicular and papillary carcinomas carried a significantly higher prevalence of non-silent point mutations of complex I genes than adenomas. We also detected a significantly higher prevalence of complex I and complex IV sequence variants in the normal parenchyma adjacent to the malignant tumors. Every Hürthle cell tumor displayed a relatively high percentage (up to 16%) of mtDNA CD independently of the lesion's histotype. The percentage of deleted mtDNA molecules was significantly higher in tumors with D-loop mutations than in mtDNA stable tumors. Sequence variants of the ATPase 6 gene, one of the complex V genes thought to play a role in mtDNA maintenance and integrity in yeast, were significantly more prevalent in patients with Hürthle cell tumors than in patients with non-Hürthle cell neoplasms. We conclude that mtDNA variants and mtDNA somatic mutations of complex I and complex IV genes seem to be involved in thyroid tumorigenesis. Germline polymorphisms of the ATPase 6 gene are associated with the occurrence of mtDNA CD, the hallmark of Hürthle cell tumors.