Mitochondrial DNA and disease

Mitochondrial DNA (mtDNA) defects are a relatively common cause of inherited disease and have been implicated in both ageing and cancer. MtDNA encodes essential subunits of the mitochondrial respiratory chain and defects result in impaired oxidative phosphorylation (OXPHOS). Similar OXPHOS defects have been shown to be present in a number of neurodegenerative conditions, including Parkinson's disease, as well as in normal ageing human tissues. Additionally, a number of tumours have been shown to contain mtDNA mutations and an altered metabolic phenotype. In this review we outline the unique characteristics of mitochondrial genetics before detailing important pathological features of mtDNA diseases, focusing on adult neurological disease as well as the role of mtDNA mutations in neurodegenerative diseases, ageing and cancer.     

The pathophysiology of mitochondrial biogenesis: towards four decades of mitochondrial DNA research

Mitochondria are with very few exceptions ubiquitous organelles in eukaryotic cells where they are essential for cell life and death. Mitochondria play a central role not only in a variety of metabolic pathways including the supply of the bulk of cellular ATP through oxidative phosphorylation (OXPHOS), but also in complex processes such as development, apoptosis, and aging. Mitochondria contain their own genome that is replicated and expressed within the organelle. It encodes 13 polypeptides all of them components of the OXPHOS system, and thus, the integrity of the mitochondrial DNA (mtDNA) is critical for cellular energy supply. In the past 12 years more than 50 point mutations and around 100 rearrangements in the mtDNA have been associated with human diseases. Also in recent years, several mutations in nuclear genes that encode structural or regulatory factors of the OXPHOS system or the mtDNA metabolism have been described. The development of increasingly powerful techniques and the use of cellular and animal models are opening new avenues in the study of mitochondrial medicine. The detailed molecular characterization of the effects produced by different mutations that cause mitochondrial cytopathies will be critical for designing rational therapeutic strategies for this group of devastating diseases.     

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.     

Nuclear DNA origin of cytochrome c oxidase deficiency in Leigh's syndrome: genetic evidence based on patient's-derived rho{degrees} transformants

Defects of the respiratory chain carrying out oxidative phosphorylation(OXPHOS) are the biochemical hallmark of human mitochondrialdisorders. Faulty OXPHOS can be due to mutations in either nuclearor mitochondrial genes, that are involved in the synthesis ofindividual respiratory subunits or in their post-translationalcontrol. The most common mitochondrial disorder of infancy andchildhood is Leigh's syndrome, a severe encephalopathy, oftenassociated with a defect of cytochrome c oxidase (COX). In orderto demonstrate which genome is primarily involved in COX-deficient(COX(^–))-Leigh's syndrome, we generated two lines of transmitochondrialcybrids. The first was obtained by fusing nuclear DNA-less cytoplastsderived from normal fibroblasts, with mitochondrial DNA-less(rho°) transformant fibroblasts derived from a patient withCOX(^–)-Leigh's syndrome. The second cybrid line was obtainedby fusing rho° cells derived from 143B.TK^– human osteosarcomacells, with cytoplasts derived from the same patient. The firstcybrid line showed a specific and severe COX(-) phenotype, whilein the second all the respiratory chain complexes, includingCOX, were normal. These results indicate that the COX defectin our patient is due to a mutation of a nuclear gene. The useof cybrids obtained from ‘customized’, patient-derivedrho° cells can have wide applications in the identificationof respiratory chain defects originated by nuclear DNA—encodedmutations, and in the study of nuclear DNA-mitochondrial DNAinteractions.     

Clinical spectrum and diagnosis of mitochondrial disorders

Respiratory chain deficiencies have long been regarded as neuromuscular diseases mostly originating from mutations in the mitochondrial DNA. Actually, oxidative phosphorylation, i.e., adenosine triphosphate (ATP) synthesis-coupled electron transfer from substrate to oxygen through the respiratory chain, does not only occur in the neuromuscular system. For this reason, a respiratory chain deficiency can theoretically give rise to any symptom, in any organ or tissue, at any age and with any mode of inheritance, owing to the dual genetic origin of respiratory chain enzymes (nuclear DNA and mitochondrial DNA). In recent years, it has become increasingly clear that genetic defects of oxidative phosphorylation account for a large variety of clinical symptoms in both childhood and adulthood. Diagnosis of a respiratory chain deficiency is difficult initially when only one symptom is present, and easier when additional, seemingly unrelated, symptoms are observed. The clinical heterogeneity is echoed by the genetic heterogeneity illustrated by the increasing number of nuclear genes that have been shown to be involved in these diseases. In the absence of clear-cut genotype-phenotype correlations and in front of the large number of possibly involved genes, biochemical analyses are still the cornerstone of the diagnosis of this condition.     

Mitochondria and degenerative disorders

In mammalian cells, mitochondria provide energy from aerobic metabolism. They play an important regulatory role in apoptosis, produce and detoxify free radicals, and serve as a cellular calcium buffer. Neurodegenerative disorders involving mitochondria can be divided into those caused by oxidative phosphorylation (OXPHOS) abnormalities either due to mitochondrial DNA (mtDNA) abnormalities, e.g., chronic external ophthalmoplegia, or due to nuclear mutations of OXPHOS proteins, e.g., complex I and II associated with Leigh syndrome. There are diseases caused by nuclear genes encoding non-OXPHOS mitochondrial proteins, such as frataxin in Friedreich ataxia (which is likely to play an important role in mitochondrial-cytosolic iron cycling), paraplegin (possibly a mitochondrial ATP-dependent zinc metalloprotease of the AAA-ATPases in hereditary spastic paraparesis), and possibly Wilson disease protein (an abnormal copper transporting ATP-dependent P-type ATPase associated with Wilson disease). Huntingon disease is an example of diseases with OXPHOS defects associated with mutations of nuclear genes encoding non-mitochondrial proteins such as huntingtin. There are also disorders with evidence of mitochondrial involvement that cannot as yet be assigned. These include Parkinson disease (where a complex I defect is described and free radicals are generated from dopamine metabolism), amyotrophic lateral sclerosis, and Alzheimer disease, where there is evidence to suggest mitochondrial involvement perhaps secondary to other abnormalities.     

Mitochondrial and nuclear DNA defects in Saccharomyces cerevisiae with mutations in DNA polymerase gamma associated with progressive external ophthalmoplegia

A number of nuclear mutations have been identified in a variety of mitochondrial diseases including progressive external ophthalmoplegia (PEO), Alpers syndrome and other neuromuscular and oxidative phosphorylation defects. More than 50 mutations have been identified in POLG, which encodes the human mitochondrial DNA (mtDNA) polymerase gamma, PEO and Alpers patients. To rapidly characterize the effects of these mutations, we have developed a versatile system that enables the consequences of homologous mutations, introduced in situ into the yeast mtDNA polymerase gene MIP1, to be evaluated in vivo in haploid and diploid cells. Overall, distinct phenotypes for expression of each of the mip1-PEO mutations were observed, including respiration-defective cells with decreased viability, dominant-negative mutant polymerases, elevated levels of mitochondrial and nuclear DNA damage and chromosomal mutations. Mutations in the polymerase domain caused the most severe phenotype accompanied by loss of mtDNA and cell viability, whereas the mutation in the exonuclease domain showed mild dominance with loss of mtDNA. Interestingly, the linker region mutation caused elevated mitochondrial and nuclear DNA damage. The cellular processes contributing to these observations in the mutant yeast cells are potentially relevant to understanding the pathologies observed in human mitochondrial disease patients.     

The transmission of OXPHOS disease and methods to prevent this

Diseases owing to defects of oxidative phosphorylation (OXPHOS) affect approximately 1 in 8,000 individuals. Clinical manifestations can be extremely variable and range from single-affected tissues to multisystemic syndromes. In general, tissues with a high energy demand, like brain, heart and muscle, are affected. The OXPHOS system is under dual genetic control, and mutations in both nuclear and mitochondrial genes can cause OXPHOS diseases. The expression and segregation of mitochondrial DNA (mtDNA) mutations is different from nuclear gene defects. The mtDNA mutations can be either homoplasmic or heteroplasmic and in the latter case disease becomes manifest when the mutation exceeds a tissue-specific threshold. This mutation load can vary between tissues and often an exact correlation between mutation load and phenotypic expression is lacking. The transmission of mtDNA mutations is exclusively maternal, but the mutation load between embryos can vary tremendously because of a segregational bottleneck. Diseases by nuclear gene mutations show a normal Mendelian inheritance pattern and often have a more constant clinical manifestation. Given the prevalence and severity of OXPHOS disorders and the lack of adequate therapy, existing and new methods for the prevention of transmission of OXPHOS disorders, like prenatal diagnosis (PND), preimplantation genetic diagnosis (PGD), cytoplasmic transfer (CT) and nuclear transfer (NT), are technically and ethically evaluated.     

New insights into structure and function of mitochondria and their role in aging and disease

This review covers some novel findings on mitochondrial biochemistry and discusses diseases due to mitochondrial DNA mutations as a model of the changes occurring during physiological aging. The random collision model of organization of the mitochondrial respiratory chain has been recently challenged on the basis of findings of supramolecular organization of respiratory chain complexes. The source of superoxide in Complex I is discussed on the basis of laboratory experiments using a series of specific inhibitors and is presumably iron sulfur center N2. Maternally inherited diseases due to mutations of structural genes in mitochondrial DNA are surveyed as a model of alterations mimicking those occurring during normal aging. The molecular defects in senescence are surveyed on the basis of the "Mitochondrial Theory of Aging", establishing mitochondrial DNA somatic mutations, caused by accumulation of oxygen radical damage, to be at the basis of cellular senescence. Mitochondrial production of reactive oxygen species increases with aging and mitochondrial DNA mutations and deletions accumulate and may be responsible for oxidative phosphorylation defects. Evidence is presented favoring the mitochondrial theory, with primary mitochondrial alterations, although the problem is made more complex by changes in the cross-talk between nuclear and mitochondrial DNA.     

The role of mitochondria in the pathogenesis of neurodegenerative diseases

A growing body of evidence indicates that mitochondrial dysfunction may play an important role in the pathogenesis of many neurodegenerative disorders. Because mitochondrial metabolism is not only the principal source of high energy intermediates, but also of free radicals, it has been suggested that inherited or acquired mitochondrial defects could be the cause of neuronal degeneration as a consequence of energy defects and oxidative damage. Mitochondrial respiratory chain dysfunction has been reported in association with primary mitochondrial DNA abnormalities, and also as a consequence of mutations in nuclear genes directly involved in mitochondrial functions, such as SURF1, frataxin, and paraplegin. Defects of oxidative phosphorylation and increased free radical production have also been observed in diseases that are not due to primary mitochondrial abnormalities. In these cases, the mitochondrial dysfunction is likely to be an epiphenomenon, which, nevertheless, could be of importance in precipitating a cascade of events leading to cell death. In either case, understanding the role of mitochondria in the pathogenesis of neurodegenerative diseases could be important for the development of therapeutic strategies in these disorders.     

Clinical differences in patients with mitochondriocytopathies due to nuclear versus mitochondrial DNA mutations

Defects in oxidative phosphorylation (OXPHOS) are genetically unique because the different components involved in this process, respiratory chain enzyme complexes (I, III, and IV) and complex V, are encoded by nuclear and mitochondrial genome. The objective of the study was to assess whether there are clinical differences in patients suffering from OXPHOS defects caused by nuclear or mitochondrial DNA (mtDNA) mutations. We studied 16 families with > or = two siblings with a genetically established OXPHOS deficiency, four due to a nuclear gene mutation and 12 due to a mtDNA mutation. Siblings with a nuclear gene mutation showed very similar clinical pictures that became manifest in the first years (ranging from first months to early childhood). There was a severe progressive course. Seven of the eight children died in their first decade. Conversely, siblings with a mtDNA mutation had clinical pictures that varied from almost alike to very distinct. They became symptomatic at an older age (ranging from childhood to adulthood), with the exception of defects associated with Leigh or Leigh-like phenotype. The clinical course was more gradual and relatively less severe; four of the 26 patients died, one in his second year, another in her second decade and two in their sixth decade. There are differences in age at onset, severity of clinical course, outcome, and intrafamilial variability in patients affected of an OXPHOS defect due to nuclear or mtDNA mutations. Patients with nuclear mutations become symptomatic at a young age, and have a severe clinical course. Patients with mtDNA mutations show a wider clinical spectrum of age at onset and severity. These differences may be of importance regarding the choice of which genome to study in affected patients as well as with respect to genetic counseling.     

Identification of the gene encoding the human mitochondrial RNA polymerase (h-mtRPOL) by cyberscreening of the Expressed Sequence Tags database

A gene cloning strategy based on the screening of the Expressed Sequence Tags database (dbEST) using sequences of mitochondrial housekeeping proteins of yeast was employed to identify the cDNA encoding the precursor of the human mitochondrial RNA polymerase (h- mtRPOL). The 3831 bp h-mtRPOL cDNA is located on chromosome 19p13.3 and encodes a protein of 1230 amino acid residues. The protein sequence shows significant homologies with sequences corresponding to mitochondrial RNA polymerases from lower eukaryotes, and to RNA polymerases from several bacteriophages. The mitochondrial RNA polymerase carries out the central activity of mitochondrial gene expression and, by providing the RNA primers for replication- initiation, is also implicated in the maintenance and propagation of the mitochondrial genome. Genes involved in the control of mtDNA replication and gene expression are attractive candidates for human disorders due to abnormalities of nucleo-mitochondrial intergenomic signalling. The availability of the h-mtRPOL cDNA will allow us to test its role in mitochondrial pathology. In addition, we propose the 'cyberscreening' of dbEST, based on yeast/human cross-species comparison, as a powerful, simple, rapid and inexpensive method, that may accelerate several-fold the molecular dissection of the human mitochondrial proteome.      

Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency

NADH-ubiquinone oxidoreductase (complex I) deficiency is amongst the most encountered defects of the mitochondrial oxidative phosphorylation (OXPHOS) system and is associated with a wide variety of clinical signs and symptoms. Mutations in complex I nuclear structural genes are the most common cause of isolated complex I enzyme deficiencies. The cell biological consequences of such mutations are poorly understood. In this paper we have used blue native electrophoresis in order to study how different nuclear mutations affect the integrity of mitochondrial OXPHOS complexes in fibroblasts from 15 complex I-deficient patients. Our results show an important decrease in the levels of intact complex I in patients harboring mutations in nuclear-encoded complex I subunits, indicating that complex I assembly and/or stability is compromised. Different patterns of low molecular weight subcomplexes are present in these patients, suggesting that the formation of the peripheral arm is affected at an early assembly stage. Mutations in complex I genes can also affect the stability of other mitochondrial complexes, with a specific decrease of fully-assembled complex III in patients with mutations in NDUFS2 and NDUFS4. We have extended this analysis to patients with an isolated complex I deficiency in which no mutations in structural subunits have been found. In this group, we can discriminate between complex I assembly and catalytic defects attending to the fact whether there is a correlation between assembly/activity levels or not. This will help us to point more selectively to candidate genes for pathogenic mutations that could lead to an isolated complex I defect.     

Analysis of mitochondrial DNA sequences in patients with isolated or combined oxidative phosphorylation system deficiency

Background

Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA.

Objective

To analyse the sequences of the mtDNA coding region in 25 patients with OXPHOS system deficiency to identify the underlying genetic defect.

Results

Three novel non‐synonymous substitutions in protein‐coding genes, 4681T→C in MT‐ND2, 9891T→C in MT‐CO3 and 14122A→G in MT‐ND5, and one novel substitution in the 12S rRNA gene, 686A→G, were found. The definitely pathogenic mutation 3460G→A was identified in an 18‐year‐old woman who had severe isolated complex I deficiency and progressive myopathy.

Conclusions

Bioinformatic analyses suggest a pathogenic role for the novel 4681T→C substitution found in a boy with Leigh''s disease. These results show that the clinical phenotype caused by the primary Leber''s hereditary optic neuropathy mutation 3460G→A is more variable than has been thought. In the remaining 23 patients, the role of mtDNA mutations as a cause of the OXPHOS system deficiency could be excluded. The deficiency in these children probably originates from mutations in the nuclear genes coding for respiratory enzyme subunits or assembly factors.The oxidative phosphorylation (OXPHOS) system consists of five enzyme complexes composed of >70 subunits encoded by the nuclear genome and 13 subunits encoded by mitochondrial DNA (mtDNA). Both isolated and combined enzyme complex deficiencies have been reported in children with various clinical phenotypes. Defects in the OXPHOS system are common causes of inborn errors in energy metabolism, with an estimated incidence of 1 per 10 000 live births.¹ The inheritance pattern is autosomal recessive in most cases, but autosomal dominant and X‐chromosomal inheritance has also been described. Maternal inheritance points to a mutation in mtDNA as the cause of the disease.²More than 2000 human mtDNA‐coding region sequences have been reported since 2000, and about half of these sequences are from Europeans.^3,4,5,6,7,8 The total number of non‐synonymous mutations leading to an amino acid replacement in mtDNA of European origin has been estimated to be 1081, but as many as 18 100 sequences should be analysed to identify 95% of these substitutions.⁹ Sequencing of the complete mtDNA from patients with an OXPHOS system deficiency will evidently lead to the identification of novel pathogenic mutations. This approach has already yielded several novel mutations in MT‐ND genes so far, and some of them—for example, 10191T→C and 14487T→C—may not be uncommon causes of disease.^10,11

Key points

• Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA. The sequence of mtDNA‐coding region was analysed in 25 patients with OXPHOS system deficiency to identify the underlying genetic defect.
• 4681T→C, a novel substitution in MT‐ND2, was found in a patient with Leigh''s disease. Further analyses suggested a pathogenic role for this substitution.
• 3460G→A, one of the mutations causing Leber''s hereditary optic neuropathy, was identified in a patient with progressive myopathy. The finding suggests that the clinical phenotype caused by this mutation is more variable than what has been known.

There is a growing need to analyse complete mtDNA sequences with a high throughput and in a cost‐efficient manner. We analysed the entire coding region of mtDNA in 28 patients (consisting of children and young adults) with OXPHOS system deficiency using a protocol consisting of conformation‐sensitive gel electrophoresis (CSGE) of amplified mtDNA fragments and subsequent sequencing of those fragments that differed in mobility in CSGE. Obtained sequences were compared with previously reported mtDNA sequences to identify haplotype‐specific or novel variants, and to detect possible sequencing errors.¹² The quality of the sequences was confirmed by comparison of the sequences obtained using the CSGE protocol with those obtained using direct mtDNA sequencing, and by correct identification of three samples with a known pathogenic mutation. Three novel non‐synonymous substitutions and one novel rRNA substitution were detected, and their pathogenic potential was estimated on several criteria.