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Mitochondrial DNA mutations in human disease
Author: Robert W. Taylor
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"� 2005 Nature Publishing Group Mitochondrial Research Group, School of Neurology, Neurobiology and Psychiatry, The Medical School, University of Newcastle upon Tyne, NE2 4HH, United Kingdom. Correspondence to D.M.T. e-mail: d.m.turnbull@ncl. ac.uk doi:10.1038/nrg1606 MITOCHONDRIAL DNA MUTATIONS IN HUMAN DISEASE Robert W. Taylor and Doug M. Turnbull Abstract | The human mitochondrial genome is extremely small compared with the nuclear genome, and mitochondrial genetics presents unique clinical and experimental challenges. Despite the diminutive size of the mitochondrial genome, mitochondrial DNA (mtDNA) mutations are an important cause of inherited disease. Recent years have witnessed considerable progress in understanding basic mitochondrial genetics and the relationship between inherited mutations and disease phenotypes, and in identifying acquired mtDNA mutations in both ageing and cancer. However, many challenges remain, including the prevention and treatment of these diseases. This review explores the advances that have been made and the areas in which future progress is likely. Until recently, mitochondrial genetics and diseases that are due to defects in mitochondrial DNA (mtDNA) were often considered to be curiosities, outside main- stream genetics. These views were not helped by the different genetic rules by which mtDNA is inherited compared with nuclear DNA, the presence of sev- eral mtDNA copies in individual cells and a belief that mtDNA disease is rare. In addition, the clinical syndromes caused by mtDNA mutations have vari- able phenotypes and are often described by instantly forgettable eponyms or acronyms 1 . These views per- haps ignore the fact that the mitochondrial genome is central to the study of evolutionary genetics, has an important role in forensic medicine 2 and that the human, bovine and mouse mitochondrial genomes were the first mammalian genomes to be completely sequenced 3?5 . The increasing ease with which the mitochondrial genome can be analysed, and the availability of a con- sensus human sequence 6 , have both helped to recog- nize mtDNA disorders as a frequent cause of genetic disease. It is difficult to estimate the true prevalence of mtDNA disease owing to its many clinical guises, presentations and the involvement of numerous causative mutations. Data on a single-point mutation (3243A>G) in Finland indicate that approximately 1 in 6,000 individuals are affected, whereas estimates from the British population intimate that about 1 in 3,500 people either have mtDNA disease or are at risk of developing it 7,8 . These estimates do not include the recent apparent association of mtDNA mutations and common clinical features (for example, hypertension) 9 , which indicate that the incidence could be higher still. In addition, there is increasing evidence from animal models 10 and human studies 11 that acquired mtDNA mutations and mitochondrial dysfunction are involved in ageing and age-related diseases such as diabetes 12,13 . Although our present knowledge already indicates that mtDNA mutations are an important cause of disease, the true impact of these mutations on human health remains to be determined. This review focuses on abnormalities of the mito- chondrial genome in relation to human disease. Mitochondrial disease can also arise from nuclear gene disorders because most proteins involved in mitochondrial metabolism and all those involved in mtDNA maintenance are nuclear-encoded. Mutations in these nuclear genes can mimic the features seen in patients with mtDNA defects, and indeed some nuclear genetic disorders result in secondary abnormalities of the mitochondrial genome. Excellent reviews describe the defects that are due to mutations in nuclear genes that are involved in mitochondrial oxidative metabo- lism 1,14,15 . Here we review our current understanding of the role of mtDNA in human disease and highlight areas that are either controversial or merit further stud- ies. We first review the basic features of mitochondrial genetics. Much progress has been made in this area NATURE REVIEWS | GENETICS VOLUME 6 | MAY 2005 | 389 REVIEWS � 2005 Nature Publishing Group CO 2 OXPHOS complexes Acetyl-CoA Carbohydrate Fatty acids ATP 16S rRNA 12S rRNA D-loop Cyt b ND1 ND2 COI COII ATPase COIII ND3 ND4L ND4 ND5 ND6 V F T L I M W D K 8 6 Q A N C Y S TCA cycle ?-oxidation Light strand Heavy strand Mitochondrial DNA Mitochondrial DNA P O H O L E OXPHOS complexes Mitochondrion H S L G R ab ATP and a clear understanding is important to further our knowledge of the diagnosis, pathogenesis and treat- ment of mtDNA disease. We then turn to clinical syn- dromes that are due to mtDNA mutations, recognizing that mtDNA mutations might cause various clinical features, which often makes diagnosis a considerable challenge. Even when diagnosed, there is no cura- tive treatment for patients who have mtDNA disease. Nonetheless, there are several experimental possibili- ties to either prevent transmission or to correct the genetic defect and these are discussed here. Although models of mtDNA disease exist, these are predomi- nantly cell-based rather than transgenic mouse mod- els. Some valuable models are available, but few mimic the characteristics of pathogenic mtDNA mutations. We conclude by considering the possible role of the mitochondrial genome in ageing, which, despite much speculation, has only recently been supported by firm evidence from an animal model. Mitochondrial genetics: the basics Mitochondria are found in all nucleated cells and are the principal generators of cellular ATP by oxidative phosphorylation (OXPHOS), incorporating the elec- tron-transferring respiratory chain (complexes I?IV) and the ATP synthase (complex V). Mitochondria are the only location of extra-chromosomal DNA within the cell (except in plant chloroplasts), and they are under the dual genetic control of both nuclear DNA and the mitochondrial genome. The mitochondrial genome consists of a multicopy, circular dsDNA molecule (16.6 kb in humans), which encodes 13 essential polypep- tides of the OXPHOS system and the necessary RNA machinery (2 rRNAs and 22 tRNAs) for their transla- tion within the organelle (FIG. 1). The remaining protein subunits that make up the respiratory-chain complexes, together with those required for mtDNA maintenance, are nuclear-encoded, synthesized on cytoplasmic ribos- omes, and are specifically targeted and sorted to their correct location within the organelle. Mitochondrial genetics is different from Mendelian genetics in almost every aspect, from the uniparental inheritance of disease mutations, to the presence of many copies of the genome within a single cell and the basic mechanisms that underlie replication and control of transcription uniF6AETABLE 1uniF6AF. The differences between the two genetic systems that are in human cells are prob- ably a relic of evolution 16 , but lead to some fascinating biology that dictates the functional consequences of mtDNA mutations. Replication, transcription and translation of mito- chondrial DNA. Both replication and transcription are important in terms of understanding the development of mtDNA mutations and their biochemical consequences. There is now good evidence to indicate that the minimal mitochondrial replisome 17 consists of several proteins. These include: the heterodimeric mtDNA polymerase ?, which consists of a catalytic subunit with proof-read- ing ability (PolgA) and a processivity subunit (PolgB) 18 , Twinkle, which has 5??3? DNA helicase activity 19 , and a mitochondrial single-stranded binding protein. Together with mitochondrial transcription factor A (TFAM), these proteins associate with mtDNA to form structures Figure 1 | The role of the mitochondrial genome in energy generation. a | This highlights the importance of the mitochondrial genome in contributing polypeptide subunits to the five enzyme complexes that comprise the oxidative phosphorylation (OXPHOS) system within the inner mitochondrial membrane ? the site of ATP synthesis. The reoxidation of reducing equivalents (NADH (reduced nicotinamide adenine dinucleotide) and FADH 2 (reduced flavin adenine dinucleotide)) that are produced by the oxidation of carbohydrates (the tricarboxylic acid (TCA) cycle) and fatty acids (?-oxidation) is coupled to the generation of an electrochemical gradient across the inner mitochondrial membrane, which is harnessed by the ATP synthase to drive the formation of ATP. b | A map of the human mitochondrial genome. The genes that encode the subunits of complex I (ND1?ND6 and ND4L) are shown in blue; cytochrome c oxidase (COI?COIII) is shown in red; cytochrome b of complex III is shown in green; and the subunits of the ATP synthase (ATPase 6 and 8) are shown in yellow. The two ribosomal RNAs (rRNAs; 12S and 16S, shown in purple) and 22 tRNAs, indicated by black lines and denoted by their single letter code, which are required for mitochondrial protein synthesis are also shown. The displacement loop (D-loop), or non-coding control region, contains sequences that are vital for the initiation of both mtDNA replication and transcription, including the proposed origin of heavy-strand replication (shown as O H ). The origin of light-strand replication is shown as O L . 390 | MAY 2005 | VOLUME 6 www.nature.com/reviews/genetics REVIEWS � 2005 Nature Publishing Group NUCLEOID A dynamic complex that consists of several copies of mitochondrial DNA and key maintenance proteins within the organelle. POLYCISTRONIC A form of gene organization that results in transcription of an mRNA that codes for multiple gene products, each of which is independently translated from the mRNA. that are termed NUCLEOIDS, which are believed to be the units of mtDNA transmission and inheritance. The mode of mtDNA replication has recently become the subject of much debate. On the basis of ultrastructural and biochemical analyses, mtDNA replication had been considered to occur by a strand- displacement model 20 , in which replication of the leading (heavy (H)) strand occurs first, initiating at a specific site (called O H ) in the non-coding control region (FIG. 1). When leading-strand replication has reached two-thirds around the genome, the origin of replication (O L ) on the lagging (light (L)) strand is exposed, allowing L-strand synthesis to occur in the opposite direction. Recent studies using two-dimen- sional gel electrophoresis challenge this model by providing evidence, in human, mouse and avian tis- sues, of a more conventional replication mechanism in which leading-lagging strand DNA replication is coordinated 21?23 . The two modes of replication have been hotly debated 24?26 and the recent description of another D-loop origin of replication adds further interest to this debate 27 . Nascent chains that start at this origin do not terminate prematurely and seem to be mainly responsible for mtDNA maintenance under steady-state conditions. Our understanding of the transcriptional machin- ery in mammalian mitochondria has improved, pre- dominantly owing to the identification of specific proteins and the development of an in vitro system to dissect out the regulatory features 28 . In human mtDNA, transcriptional initiation can occur on both strands. Transcription from the mitochondrial promoters produces a POLYCISTRONIC precursor RNA that is then processed to produce individual tRNA and mRNA molecules 29,30 . To initiate transcription, the dedicated mitochondrial RNA polymerase (POLRMT) requires TFAM, and either mitochondrial transcription fac- tor B1 (TFB1M) or B2 (TFB2M) 31,32 . Recent evidence shows that TFAM induces a structural change of the light-strand promoter that is required for POLRMT- dependent promoter recognition 33 . The importance of mitochondrial transcription to cellular dysfunc- tion as a result of pathogenic mtDNA mutations is a neglected area of research that might give important insights into some of the tissue-specific or mutation- specific effects. The mRNAs for the 13 mtDNA-encoded OXPHOS proteins are translated on mitochondrial ribosomes. Mutations in mitochondrial tRNA or rRNA genes affect pathology by disrupting mitochondrial transla- tion 34 and recent research has concentrated on identify- ing nuclear factors, including mitochondrial ribosomal proteins (MRPs), that are essential for this process. The first mutations in these nuclear genes, including MRPS16 (mitochondrial ribosomal protein S16) 35 and EFG1 (officially known as GFM1; G elongation fac- tor, mitochondrial 1) 36 have recently been described in consanguineous families that have generalized mito- chondrial translation defects. Homoplasmy and heteroplasmy. The polyploid nature of the mitochondrial genome ? up to several thou- sand copies per cell ? gives rise to an important feature of mitochondrial genetics, homoplasmy and heteroplasmy. In simple terms, homoplasmy is when all copies of the mitochondrial genome are identical; Table 1 | Comparison between the human nuclear and mitochondrial genomes* Characteristic Nuclear genome Mitochondrial genome Size ~3.3 x 10 9 bp 16,569 bp Number of DNA molecules per cell 23 in haploid cells; 46 in diploid cells Several thousand copies per cell (polyploidy) Number of genes encoded ~20,000?30,000 37 (13 polypeptides, 22 tRNAs and 2 rRNAs) Gene density ~1 per 40,000 bp 1 per 450 bp Introns Frequently found in most genes Absent Percentage of coding DNA ~3% ~93% Codon usage The universal genetic code AUA codes for methionine; TGA codes for tryptophan; AGA and AGG specify stop codons Associated proteins Nucleosome-associated histone proteins and non-histone proteins No histones; but associated with several proteins (for example, TFAM) that form nucleoids Mode of inheritance Mendelian inheritance for autosomes and the X chromosome; paternal inheritance for the Y chromosome Exclusively maternal Replication Strand-coupled mechanism that uses DNA polymerases ? and ? Strand-coupled and strand-displacement models; only uses DNA polymerase ? Transcription Most genes are transcribed individually All genes on both strands are transcribed as large polycistrons Recombination Each pair of homologues recombines during the prophase of meiosis There is evidence that recombination occurs at a cellular level but little evidence that it occurs at a population level *Table modified from REF. 171 ? (1999) John Wiley and Sons. TFAM, mitochondrial transcription factor A; rRNA, ribosomal RNA. NATURE REVIEWS | GENETICS VOLUME 6 | MAY 2005 | 391 REVIEWS � 2005 Nature Publishing Group abe cd TRANSMITOCHONDRIAL CYBRID Derived from the term ?cytoplasmic hybrid?. A cell line that is used to study the pathophysiological consequences of any given mitochondrial DNA mutation in a control nuclear background. Cybrids are generated by fusing patient cytoplasts (enucleated cells that contain mitochondria) with a cell line that lacks mitochondrial DNA. heteroplasmy is when there is a mixture of two or more mitochondrial genotypes. The value of these terms is apparent when we consider mtDNA mutations that lead to disease. Some mutations affect all copies of the mitochondrial genome (homoplasmic mutation), whereas others are only present in some copies of the mitochondrial genome (heteroplasmic mutation). In the presence of heteroplasmy, there is a threshold level of mutation that is important for both the clinical expression of the disease and for biochemical defects, as routinely demonstrated by the cytochemical assess- ment of cytochrome c oxidase (COX; complex IV) activity in an individual cell (FIG. 2). Numerous single- cell and TRANSMITOCHONDRIAL CYBRID CELL studies have shown that the mutated form is functionally recessive and that a biochemical phenotype is associated with high levels of mutation above a crucial threshold 37 . The concept of homoplasmy is more apparent than real. In most individuals there is no evidence of hetero- plasmy, but all available evidence indicates that mtDNA is constantly undergoing mutation, with clonal expan- sion or loss of either point mutations or deletions 38,39 . Because these acquired mutations occur at random, all acquired mutations will be present at a low level and therefore might not be detected in a tissue homogenate or blood sample. Perhaps we should only use ?homo- plasmy? to describe a state in which we cannot detect these acquired mutations, although it would be naive to think that all mitochondrial genomes within an indi- vidual, tissue or even a cell are identical. Inheritance of mitochondrial DNA. The standard model of mtDNA inheritance is that it is transmitted strictly through the maternal line 40 and that mtDNA lineages are therefore clonal. However, this model has recently been challenged. Low levels of paternal transmission of mtDNA have been observed in crosses between mouse species, but not within species 41 , although further stud- ies showed that this paternal mtDNA was not transmit- ted to the subsequent generation 42 . In addition, there is evidence that recombination has contributed to the dis- tribution of mtDNA polymorphisms within the human population 43 . Although recombination might occur at the cellular level 44,45 , the occurrence of recombination within the population is contentious 46 . Studies of a patient with mitochondrial myopathy ? who carried a novel, 2-bp pathogenic deletion in the NADH (reduced nicotinamide adenine dinucleotide) dehydrogenase subunit 2 (ND2) gene in his muscle mtDNA ? have also challenged the maternal inheritance of mtDNA 47 . Using haplotype analysis, this sporadic mutation was present in paternally derived mtDNA, although the deletion itself was not evident in the father?s mtDNA. With the exception of skeletal muscle, all other tis- sues contained a single, maternally derived mtDNA haplotype. Subsequent studies of other patients Figure 2 | Cytochrome c oxidase deficiency in mitochondrial DNA-associated disease and ageing. Transverse tissue sections that are reacted for both cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) activities sequentially, with COX-positive cells shown in brown and COX-deficient cells shown in blue. a | Skeletal muscle from a patient with a heteroplasmic mitochondrial tRNA point mutation. The section shows a typical ?mosaic? pattern of COX activity, with many muscle fibres harbouring levels of mutated mtDNA that are above the crucial threshold to produce a functional enzyme complex. b | Cardiac tissue (left ventricle) from a patient with a homoplasmic tRNA mutation that causes hypertrophic cardiomyopathy, which demonstrates an absence of COX in most cells. c | A section of cerebellum from a patient with an mtDNA rearrangement that highlights the presence of COX-deficient neurons. d,e | Tissues that show COX deficiency that is due to clonal expansion of somatic mtDNA mutations within single cells ? a phenomenon that is seen in both post-mitotic cells (d; extraocular muscles) and rapidly dividing cells (e; colonic crypt) in ageing humans. 392 | MAY 2005 | VOLUME 6 www.nature.com/reviews/genetics REVIEWS � 2005 Nature Publishing Group Fertilization Primordial germ cell containing mutant mtDNA Primary oocytes Oocyte maturation and mtDNA amplification Mature oocytes Low level of mutation (unaffected offspring) Intermediate level of mutation (mildly affected offspring) High level of mutation (affected offspring) Mutant mitochondrion Normal mitochondrion Nucleus LEBER HEREDITARY OPTIC NEUROPATHY A mitochondrial disease that is characterized by optic nerve dysfunction, which leads to bilateral visual failure in young adults. GENETIC BOTTLENECK A temporary reduction in population size that causes the loss of genetic variation. with mitochondrial myopathies have not shown any evidence of paternal transmission 48?50 , even when assisted reproduction techniques were applied 51,52 . Mitochondrial DNA transmission. The current genetic advice is that fathers with mtDNA mutations are at no risk of transmitting the defect to their offspring. Maternal transmission of mutated mtDNA occurs, but the risk depends on the type of mutation and possibly the segregation of the mutation within maternal tis- sues. Identifying specific mtDNA mutations and inves- tigating family members for evidence of transmission will give guidance to the likelihood of transmission through the germline. Detailed studies of large patient cohorts provide invaluable information on the risk of transmission. A recent analysis of a single, large-scale mtDNA deletion in 226 families, showed that the risk of recurrence in the offspring of an affected mother was 4.11% uniF6AEREF. 53uniF6AF. If the mother was unaffected, then there was no record of affected siblings, which indi- cates that the risk of recurrence is negligible. Homoplasmic mtDNA mutations are transmitted to all maternal offspring. However, the genetic advice given to these families is not straightforward. Most patients with LEBER HEREDITARY OPTIC NEUROPATHY (LHON; see also Online links box) have homoplasmic mtDNA mutations. Although all offspring inherit the mutation, only some will develop the disease. Approximately 50% of males, but only 10% of females, develop impaired vision 54 , which certainly indicates that nuclear genetic factors are important in the expression of the dis- ease. Another homoplasmic mtDNA mutation, the 1555A>G 12S ribosomal RNA (RNR1) mutation 55 , is an important cause of post-lingual deafness. Its clinical expression depends on the administration of amino- glycoside antibiotics, which highlights the importance of environmental factors in the expression of homo- plasmic mtDNA disease. Although it is obvious that genetic counselling has to be tailored to specific muta- tions, further information is required to determine the risk for individual defects. The transmission of heteroplasmic mtDNA point mutations is even more complex. Nuclear genetic 56 and environmental factors almost certainly affect expression of the disease phenotype. In addition, there is a GENETIC BOTTLENECK during development, and the amount of mutated mtDNA that is transmitted to the offspring is variable 57 (FIG. 3). Because many of the clinical features depend on the relative proportions of mutated versus wild-type mtDNA, the outcome for each pregnancy remains difficult to predict 58 . Clinical features of human mtDNA disease Mitochondria are vital components of all nucleated cells. Therefore, it is not surprising that mtDNA dis- eases affect many tissues and that the clinical features Figure 3 | The mitochondrial genetic bottleneck. During the production of primary oocytes, a selected number of mitochondrial DNA (mtDNA) molecules are transferred into each oocyte. Oocyte maturation is associated with the rapid replication of this mtDNA population. This restriction-amplification event can lead to a random shift of mtDNA mutational load between generations and is responsible for the variable levels of mutated mtDNA observed in affected offspring from mothers with pathogenic mtDNA mutations. Mitochondria that contain mutated mtDNA are shown in red, those with normal mtDNA are shown in green. NATURE REVIEWS | GENETICS VOLUME 6 | MAY 2005 | 393 REVIEWS � 2005 Nature Publishing Group DIPLOPIA Double vision; derived from the Greek diplous, meaning double, and ops, meaning eye. ATAXIA The loss of the ability to coordinate muscular movement. DYSPHAGIA A difficulty in swallowing. PTOSIS The abnormal lowering or drooping of the upper eyelid that is caused by muscle weakness. DYSTONIA A neurological movement disorder that is characterized by involuntary muscle contractions that might cause twisting or jerking movements of the body. PANCYTOPAENIA A deficiency of all blood cells including red cells, white cells and platelets. OPHTHALMOPLEGIA Weakness of one or more of the muscles that control eye movement. PEARSON SYNDROME A severe disease during infancy that affects bone marrow and pancreas function owing to large-scale rearrangements of the mitochondrial genome. are so variable uniF6AEBOX 1uniF6AF. Such diversity makes it difficult to define the impact of mtDNA mutations on human health, however, stratifying the clinical features into the following groups defines the extent of the problem: classic mtDNA syndromes, clinical syndromes with a high risk of mtDNA mutations, involvement in com- mon disease phenotypes and mtDNA as a predisposi- tion for common disease and ageing. Clinical features are specific and clinical diagnosis is straightforward for patients with the classic syndromes. However, in many patients, the clinical symptoms might be non-specific and mtDNA disease is only one of several possible diagnoses. Important clues to a pos- sible mitochondrial origin for the symptoms include a maternal family history and the involvement of several tissues uniF6AEBOX 2uniF6AF. For a large number of patients, diagnosis depends on the results of several lines of investigation. This includes histochemical and biochemical stud- ies to determine the precise nature of the respiratory chain defect, analysis of the mitochondrial genome for common mutations and, finally, complete genome sequencing to look for rare or novel mutations 59 . The mitochondrial genome is highly variable and the ease of whole-genome sequencing does not resolve some diag- nostic dilemmas because the interpretation of a novel sequence change can be difficult in relation to poten- tial pathogenicity 60 . Accurate diagnosis of an mtDNA defect is essential because of the consequences for both the patient and the family members. Classic syndromes. Genetic defects of the human mito- chondrial genome were first described in 1988 uniF6AEREFS 61,62uniF6AF and arose from the investigation of two syndromes ? Kearns?Sayre syndrome (KSS) and LHON. In KSS, single, large-scale mtDNA deletions (usually sporadic) were detected in muscle biopsies, and mitochondrial ultrastructural and cytochemical abnormalities in the muscle were apparent. For LHON, a strict maternal pattern of inheritance was evident and point mutations involving the ND family of genes that encode subunits of complex I were identified. Since 1988, several muta- tions 63 of the mitochondrial genome have been identi- fied and associated with disease uniF6AETABLE 2uniF6AF. Clinical syndromes that have a high probability of mitochondrial DNA involvement. Over the past decade, we have become aware of several clinical syndromes that might be associated with mtDNA mutations. The increasing recognition of mtDNA involvement in dis- ease is partially due to the relative ease of sequencing the mitochondrial genome, although defining patho- genicity of specific base substitutions can be difficult 60 . Examples of such diseases include progressive external OPHTHALMOPLEGIA 64 , PEARSON SYNDROME 65 , LEIGH SYNDROME 66,67 (see also Online links box), exercise-induced muscle pain, fatigue and RHABDOMYOLYSIS 68 (see also Online links box), and amino-glycoside-induced hearing loss 55 . For some of these conditions, such as progressive external ophthalmoplegia, mtDNA disease is the most likely cause, whereas in others, such as Leigh syndrome, there is a long list of potential genetic causes. Involvement in common disease phenotypes. The principal difficulty for clinicians is that patients with mtDNA disease rarely have a classic phenotype and mtDNA enters into the differential diagnosis of many Box 1 | Common features of mitochondrial DNA-associated diseases Adult ? Neurological: migraine | strokes | epilepsy | dementia | myopathy | peripheral neuropathy | DIPLOPIA | ATAXIA | speech disturbances | sensorineural deafness ? Gastrointestinal: constipation | irritable bowel | DYSPHAGIA ? Cardiac: heart failure | heart block | cardiomyopathy ? Respiratory: respiratory failure | nocturnal hypoventilation | recurrent aspiration | pneumonia ? Endocrinal: diabetes | thyroid disease | parathyroid disease | ovarian failure ? Ophthalmological: optic atrophy | cataract | ophthalmoplegia | PTOSIS Paediatric ? Neurological: epilepsy | myopathy | psychomotor retardation | ataxia | spasticity | DYSTONIA | sensorineural deafness ? Gastrointestinal: vomiting | failure to thrive | dysphagia ? Cardiac: biventricular hypertrophic cardiomyopathy | rhythm abnormalities ? Respiratory: central hypoventilation | apnoea ? Haematological: anaemia | PANCYTOPAENIA ? Renal: renal tubular defects ? Liver: hepatic failure ? Endocrinal: diabetes | adrenal failure ? Ophthalmological: optic atrophy Box 2 | Guidelines for the recognition of patients with mitochondrial DNA disease A list of the clinical guidelines that have been generated from our experience of investigating and managing patients with mtDNA mutations. ? Maternal inheritance ? Recognition of classic syndromes; for example, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke- like episodes (MELAS) or chronic progressive external ophthalmoplegia (CPEO) ? Recognition of characteristic clinical features; for example, biventricular cardiac hypertrophy ? Involvement of many organ systems; for example, diabetes and deafness ? Specific combinations of symptoms; for example, strokes and migraine and seizures and ataxia ? Abnormal clinical or laboratory investigations; for example, lactic acidaemia in children, characteristic magnetic- resonance-imaging findings, abnormal muscle biopsy that is associated with RAGGEDuniF6BARED FIBRES or cytochrome c oxidase-deficient fibres 394 | MAY 2005 | VOLUME 6 www.nature.com/reviews/genetics REVIEWS � 2005 Nature Publishing Group LEIGH SYNDROME A disease that affects the brainstem and basal ganglia and is characterised by defects in mitochondrial oxidative phosphorylation. RHABDOMYOLYSIS The breakdown of muscle fibres owing to injury, toxins or metabolic disease, which leads to high concentrations of myoglobin in both plasma and urine RAGGEDuniF6BARED FIBRES The pathological hallmark of mtDNA disease that is characterized by the subsarcolemmal accumulation of abnormal mitochondria in the muscle fibre, which stains red with a Gomori trichrome stain. common clinical syndromes. A good example of a common condition in which mtDNA is a potential cause is diabetes, the most common metabolic dis- ease to affect humans 69 . mtDNA disease certainly is associated with diabetes, but these patients represent a tiny proportion of those affected with diabetes 70?72 . Because several mtDNA mutations cause diabetes 63 it is clearly impractical and uneconomical to screen all patients with diabetes for causative mutations. From the list of common clinical features seen in patients with mtDNA disease uniF6AEBOX 1uniF6AF, it is obvious that it is difficult to decide which patients to investigate for possible mtDNA disease. Further difficulties in this area have been high- lighted by a recent report that describes a clear associa- tion between members of a large family that harbour a homoplasmic mitochondrial tRNA mutation and a metabolic syndrome that is characterized by hyperten- sion, hypomagnesaemia and hypercholesterolaemia 9 . Because conditions such as hypertension and hyperc- holesterolaemia affect many individuals 73 , the number of patients with mtDNA disease is potentially very high. However, it must be kept in the context that most reports describe isolated families. In epidemiological surveys of populations 74 , the incidence of mtDNA mutations within a specific disease phenotype 75 is small. Other fac- tors that make the diagnosis of mtDNA difficult include the specific combination of clinical symptoms uniF6AEBOX 2uniF6AF; for example, mitochondrial diabetes is often accompa- nied by deafness 70 , and stroke-like episodes are usually associated with migraine-like symptoms. There is no doubt that many patients with mtDNA mutations go undiagnosed for years and many are probably never recognized. Increased awareness among physicians and paediatricians is important, as is the investigation of patients in specialist centres that are able to provide a complete range of diagnostic tests. Mitochondrial DNA variants as a predisposition for common disease. There has been considerable interest in the possibility that mtDNA variants might predispose Table 2 | Clinical disorders that are caused by mutations in mitochondrial DNA Mitochondrial DNA disorder Clinical phenotype mtDNA genotype Gene Status Inheritance Reference Kearns?Sayre syndrome Progressive myopathy, ophthalmoplegia, cardiomyopathy A single, large-scale deletion Several deleted genes Heteroplasmic Usually sporadic 61,158 CPEO Ophthalmoplegia A single, large-scale deletion Several deleted genes Heteroplasmic Usually sporadic 61, 64 Pearson syndrome Pancytopoenia, lactic acidosis A single, large-scale deletion Several deleted genes Heteroplasmic Usually sporadic 65 MELAS Myopathy, encephalopathy lactic acidosis, stroke-like episodes 3243A>G; 3271T>C Individual mutations TRNL1 ND1 and ND5 Heteroplasmic Heteroplasmic Maternal Maternal 159 160, 161 MERRF Myoclonic epilepsy, myopathy 8344A>G; 8356T>C TRNK Heteroplasmic Maternal 162 NARP Neuropathy, ataxia, retinitis pigmentosa 8993T>G ATP6 Heteroplasmic Maternal 163 MILS Progressive brain-stem disorder 8993T>C ATP6 Heteroplasmic Maternal 67 MIDD Diabetes, deafness 3243A>G TRNL1 Heteroplasmic Maternal 164 LHON Optic neuropathy 3460G>A 11778G>A 14484T>C ND1 ND4 ND6 Hetero- or homoplasmic Hetero- or homoplasmic Hetero- or homoplasmic Maternal Maternal Maternal 165 62 166 Myopathy and diabetes Myopathy, weakness, diabetes 14709T>C TRNE Hetero- or homoplasmic Maternal 167,168 Sensorineural hearing loss Deafness 1555A>G Individual mutations RNR1 TRNS1 Homoplasmic Hetero- or homoplasmic Maternal Maternal 55 169,170 Exercise intolerance Fatigue, muscle weakness Individual mutations CYB Heteroplasmic Sporadic 68 Fatal, infantile encephalopathy; Leigh/Leigh-like syndrome Encephalopathy, lactic acidosis 10158T>C; 10191T>C ND3 Heteroplasmic Sporadic 66 ATP6, ATPase 6; CPEO, chronic progressive external ophthalmoplegia; CYB, cytochrome b; LHON, Leber hereditary optic neuropathy; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes; MERRF, myoclonic epilepsy and ragged-red fibres; MIDD, maternally-inherited diabetes and deafness; MILS, maternally-inherited Leigh syndrome; ND1,3?6, NADH dehydrogenase subunits 1,3?6; NARP, neurogenic weakness, ataxia and retinitis pigmentosa; RNR1, 12S ribosomal RNA; TRNE,TRNK,TRNL1,TRNS1, mitochondrial tRNAs. NATURE REVIEWS | GENETICS VOLUME 6 | MAY 2005 | 395 REVIEWS � 2005 Nature Publishing Group CYTOPLAST An enucleated donor cell that contains patient mitochondria and that is used to generate cybrid fusions. to common diseases; for example, diabetes, Alzheimer disease (AD) and Parkinson disease (PD) 76 . The known association of diabetes with mtDNA mutations and the involvement of reactive oxygen species in the aetiology of AD and PD has encouraged a large number of stud- ies. The first approach involves identifying candidate pathogenic mutations by comparing mtDNA sequences between patients and control groups. Unfortunately, interpreting the results is limited by the relatively small number of individual mtDNA sequences in each group and by the high mutation rate of mtDNA, which results in a high frequency of rare variants or private polymor- phisms 77 . Some variants are more common than oth- ers. For example, the 16189 variant, a variable length polycytosine tract in the non-coding control region, has been associated with several late-onset, multifactorial disorders, including susceptibility to cardiomyopathy 78 and type 2 diabetes 79 , and has been proposed to affect mtDNA replication 79 . However, because this variant occurs frequently within the population, concerns arise (that are similar to the ?haplogroup? studies) over the control cohorts that are chosen for such population- based surveys. Human populations can be divided into several mtDNA haplogroups that are based on specific SNPs, reflecting mutations accumulated by a discrete maternal lineage 80 . Therefore, haplogroup association studies have been used to define the role of mtDNA mutations in complex diseases. In this approach, disease and control mtDNA sequence sets are assigned to haplo- groups, and the distribution of haplogroups is compared. A significant difference is interpreted to indicate that the mtDNA ?background? has an effect on expression or penetrance of the clinical abnormalities. Various results have been reported for both AD and PD 81?84 . Given the inconsistency among different studies, it is difficult to conclude unequivocally that the risk of either PD or AD is influenced by mitochondrial haplogroups. Some of the inconsistency could be due to population-substructure differences between disease and control groups. It is dif- ficult to ?match? groups, even when one tries to control nationality and ethnicity 85 . Models of mitochondrial DNA disease The inability to genetically engineer or transform the mitochondrial genome in vitro or in vivo has severely hampered efforts to study the relationship between mtDNA genotype and clinical phenotype in animal models that bear heteroplasmic, pathogenic mtDNA mutations. Despite this, much has been learned of the biochemical and cellular consequences of spe- cific mtDNA mutations through the generation of transmitochondrial cytoplasmic cybrid cells. In this system, immortalized human cell lines that have been completely depleted of their endogenous mtDNA (?� cells) are repopulated with exogenous, patient mitochondria. As ?� cells have no functional respiratory chain and are dependent on pyruvate and uridine for growth, the loss of either of these two metabolic requirements can be used to select for transformants that harbour complementing (exogenous) mtDNA 86 . This elegant system allows the functional and physiological consequences of different levels of heteroplasmy of certain mtDNA mutations to be tested 87,88 . In addition, it has also proved useful in determining the genetic origin of certain mitochondrial disorders 89,90 , the effect of nuclear background on the segregation of pathogenic mtDNA mutations 91 and in identifying the first tRNA suppressor mutation in human mitochondria 92 . It has also been used to test rational, genetic therapies and more recently, to demonstrate the presence of heterolo- gous mtDNA recombination in human cells 44 . The generation of heteroplasmic mice that harbour pathogenic mtDNA mutations was frustratingly diffi- cult. Heteroplasmic mice were first generated by fusing zygotes that carried one mtDNA haplotype with enu- cleated embryos that carried a different haplotype 93,94 , although these mice (BALB and NZB) harboured only neutral mtDNA variants. Further advances were made with the generation of chimeric, heteroplasmic chloramphenicol resistant (CAP R ) mice, which could transmit mtDNA mutations to subsequent generations and showed signs of mitochondrial dysfunction 95,96 . By fusing CYTOPLASTS that harbour high levels of a somatic mtDNA rearrangement to one-cell embryos, Hayashi and colleagues generated a model that has a pathogenic deletion or duplication 97 of the mitochondrial genome, which was transmitted through the germ line. These mice showed muscle and cardiac defects, but also, more prominently, renal failure together with lactic acidosis, anaemia and a mosaic distribution of COX deficiency. Human mtDNA-rearrangement mutations rarely result in renal disease, so this experiment highlighted the potential for species-specific symptoms and signs. Although subsequent studies that use this mouse model have improved our understanding of mitochondrial seg- regation and disease pathogenesis 98 , no reported stud- ies specifically address the development of potential therapies. The generation of mouse models that target nuclear genes involved in mtDNA maintenance or replication, such as the Tfam knockout mouse, has proved to be a more successful approach 99?103 . Using the Cre-loxP recombination system, Larsson and colleagues dis- rupted the Tfam gene in selected mouse tissues, yield- ing models that provide clues to potentially important factors in the development of specific clinical features associated with mtDNA disease. For example, the mice generated by the postnatal disruption of Tfam in neurons of the hippocampus and neocortex develop a late-onset neurodegeneration, but there is minimal cell loss until the mice developed seizures 101 . This finding indicates that stress or neuronal discharge resulted in an acute energy crisis, which highlights the importance of management issues such as the effective control of seizures in patients with mtDNA disease. Management of mtDNA diseases Although defining the clinical features and establish- ing the diagnosis of mtDNA disease remain consider- able challenges, the development of effective curative treatment is also a challenging hurdle for experimental 396 | MAY 2005 | VOLUME 6 www.nature.com/reviews/genetics REVIEWS � 2005 Nature Publishing Group SATELLITE CELLS Quiescent cells that are located between the basal lamina and the plasmalemma of the muscle fibre, and are a main contributor to postnatal muscle growth. ALLOTOPIC EXPRESSION The expression of a gene in a different cellular compartment to its target location. In the context described here, it is the recoding of a mitochondrial gene to allow it to be expressed in the nucleus. The subsequent conjugation of a mitochondrial- targeting sequence promotes import and localization of the gene product to the organelle. AMNIOCENTESIS An aspiration of cells from the amniotic sac, using a needle, for biochemical and genetic analysis. CHORIONIC VILLUS BIOPSY A placental biopsy that is carried out in early pregnancy to collect fetal tissue for genetic and biochemical analysis. POLAR BODY A small haploid cell that is produced during oogenesis and that does not develop into a functional ovum. scientists. However, there are important issues asso- ciated with the clinical management of patients and many of these issues have recently been discussed 104 . Early detection of treatable symptoms is crucial. Mitochondrial DNA mutation levels can be manipu- lated through exercise. In healthy individuals, lack of exercise (deconditioning) leads to an overall reduction in mitochondrial enzyme activity, whereas endurance training improves enzyme activity. Resistance training or muscle necrosis stimulates the incorporation of SATuniF6BA ELLITE CELLS into existing muscle fibres 105,106 . It is postu- lated that for sporadic mutations, resistance training might lead to an overall reduction in the proportion of mutated mtDNA versus wild-type, as satellite cells con- tain a low or negligible amount of mutated mtDNA 106,107 . Endurance training might therefore improve function by increasing wild-type mtDNA levels. However, there are concerns that mutated mtDNA might be preferen- tially amplified, and that this increase might become clinically relevant after deconditioning 105,108 . Studies are currently underway to address both the improvements and the concerns arising from these earlier reports. New avenues for treatment of mitochondrial diseases. The polyploid nature of the mitochondrial genome, the phenomenon of heteroplasmy, the inability to deliver potentially therapeutic nucleic acids to the organelle through mitochondrial transfection, and the slow realization of appropriate animal models of mtDNA disease are the main problems for the develop- ment of gene-based treatments. Despite these, several strategies that seek to rescue mitochondrial function through complementation of the genetic defect or direct manipulation of the levels of mutated mtDNA have been considered. One way of complementing a dysfunctional mitochondrial protein is through ALLOTOPIC EXPRESuniF6BA SION. Using this approach, it was possible to express the wild-type ATPase 6 protein allotopically from nucleus-transfected constructs in transmitochondrial cybrid cells that were homoplasmic for the 8993T>G MTATP6 (subunit 6 of mitochondrial ATP synthase) mutation (which causes neurogenic weakness, ataxia and retinitis pigmentosa (NARP) syndrome) and demonstrate a partial rescue of the biochemical defect 109 . A similar strategy has been used to express a modified NADH dehydrogenase subunit 4 (ND4) gene to complement the 11778G>A mutation that causes LHON 110 . To complement mitochondrial tRNA defects, an alternative approach has been pro- posed. Yeast cytosolic tRNA LysCUU (tK1) and similar derivatives can be imported into isolated human mitochondria if the tRNA is amino-acylated and sup- plied with soluble factors, including lysl-tRNA syn- thetases 111 . Using transmitochondrial cybrid cells and primary human fibroblasts that carry the 8344A>G TRNK (a tRNA gene) mutation, which causes myo- clonic epilepsy and ragged-red fibres (MERRF) syn- drome, it has recently been shown that the imported tRNA Lys is correctly amino-acylated and able to par- ticipate in mitochondrial translation, partially rescu- ing mitochondrial function 112 . Because the recessive nature of many pathogenic mutations dictates that a small percentage of the wild- type genotype is protective, a minor adjustment to the balance of mutated to wild-type genomes in favour of the wild type could improve both biochemical func- tion and resolve the clinical problem. The selective inhibition of mutated mtDNA replication by antige- nomic agents has been applied to manipulate mtDNA heteroplasmy 113 . Another strategy involves targeting the organelle with restriction endonucleases that, by differentiating between mtDNA genotypes, can cause the preferential elimination of the mutated geno- type and propagation of the wild-type genotype 114,115 . Pharmacological approaches to shift mtDNA het- eroplasmy are also being investigated. Oligomycin, an irreversible inhibitor of mitochondrial ATP synthase, has been used to increase the fraction of wild-type molecules in cells that harbour the 8933T>G MTATP6 mutation under culture conditions (using galactose as the sole carbon source) that specifically select for the wild-type molecule 116 . This culture medium could also selectively kill cells that are homoplasmic for a common (4,977-bp deletion) mtDNA rearrangement ? the molecular abnormality in many patients with KSS. In this case, a ketogenic medium has been used to shift the heteroplasmy of cells that contain a mixture of wild-type and partially deleted mtDNAs 117 . Finally, the revelation that overexpressing mitochondrial Tu translation elongation factor, TUFM, in yeast mutant counterparts of TRNL1 (a tRNA gene) MELAS (mito- chondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) mutations facilitates the res- cue of the respiratory defect might have implications for human disease-related mutations 118 . Prevention of transmission of mtDNA disease Improving genetic advice. The question often asked by both families and clinicians is whether there are any prospects for providing either better guidance for fam- ilies or even preventing the transmission of mtDNA disease. AMNIOCENTESIS and CHORIONIC VILLUS BIOPSY are widely used to diagnose autosomal and chromosomal abnormalities, and might be of value in heteroplasmic mtDNA disorders. Chorionic villus biopsy has been used in some families that have heteroplasmic mtDNA disorders, where the results have influenced the clinical management 119?121 . In many heteroplasmic mtDNA 64,122 disorders there are marked tissue-specific differences in the level of heteroplasmy, and therefore a reason- able concern is whether a prenatal sample will reflect the likely outcome for the fetus. The present evidence indicates that this should not be a problem 94,123 . Pre- implantation genetic diagnosis (PGD) allows either analysis of mtDNA from the POLAR BODY of unfertilized oocytes, or from one to two single cells that are taken from 8-cell embryos, followed by the implantation of healthy embryos. The potential value of this technique is obvious, but there is little reported information of its application in patients with mtDNA mutations. NATURE REVIEWS | GENETICS VOLUME 6 | MAY 2005 | 397 REVIEWS � 2005 Nature Publishing Group Pronuclei removed Pronucleus Germinal vesicle removed a b Mutant mitochondrion Normal mitochondrion GERMINAL VESICLE A stage during oocyte maturation in which the oocyte nucleus is located close to the surface of the egg cell and is clearly visible. The high number of mitochondrial genomes within oocytes 124 indicates that PGD should be feasible for mtDNA disease. Experiments in heteroplasmic mice have shown the levels of heteroplasmy to be virtually identical between the ooplasm and polar body of the mature oocyte and also between blastomeres of each of the 2-, 4-, and 6?8-cell embryo 125 . Alternative strategies associated with oocyte manipu- lation. Development of new strategies to prevent the transmission of mtDNA is a very important aspect of disease management. An obvious method to prevent the transmission of an mtDNA mutation to the offspring is oocyte donation. However, although the oocyte would be fertilized using the father?s sperm, the child would have the nuclear genotype of the donor female, a less- than-ideal option for many mothers. Cytoplasmic transfer involves the transfer of normal mitochondria into the abnormal oocyte to dilute the effect of any mtDNA defect 126 . Cytoplasmic transfer between human oocytes has been carried out to improve the outcome of assisted reproduction methods 127 . The effective- ness of this procedure is uncertain 128 , but some of the children born were heteroplasmic with low levels of mtDNA from the donor oocyte 129 . However, despite the observation that changes in heteroplasmy can occur, it is likely that this technique will have limited value for patients with mtDNA disease, as mouse experiments indicate that the amount of mtDNA that can be trans- ferred is relatively small 130 . Alternatively, nuclear chromosomes from an oocyte that contains mutated mtDNA could be transferred to an enucleated oocyte from a normal female 131 . This method has obvious advantages: the offspring will be genetically related to both par- ents and the chances of achieving very low levels of mutated mtDNA in the oocyte are high. However, important technical and ethical or legal issues make progress in this area difficult, principally because the technique involves the genetic manipulation of fer- tilized oocytes. To overcome these issues, the trans- fer could be carried out at different stages of oocyte maturation, although transfer from mature unferti- lized oocytes is difficult because of the serious risk of aneuploidy that is due to chromosome loss during transfer or due to missegregation following the dis- ruption of the meiotic spindle. Karyoplast transfer of the GERMINAL VESICLE between oocytes might be pos- sible before in vitro maturation and subsequent fer- tilization (FIG. 4). However, although maturation and fertilization of normal germinal-vesicle-stage oocytes has been achieved with both mouse 132?134 and human oocytes 135,136 , these methods are very inefficient. Pronuclear-stage transfer is possibly more feasible because human pronuclear oocytes can be manipu- lated to generate normal offspring 137 and because this technique is feasible in the mouse 138,139 . Acquired mtDNA mutations in ageing and cancer Mitochondrial theory of ageing. The mitochondrial theory of ageing proposes that progressive accumula- tion of somatic mutations in mtDNA during a lifetime leads to an inevitable decline in mitochondrial func- tion 140 . The most important biochemical factors in this process are considered to be reactive oxygen species (ROS), which are generated at very low levels during the normal function of the mitochondrial respiratory chain. It has been proposed that ROS can cause somatic mutations in mtDNA 140 . These mutations then result in impaired function of the respiratory chain, lead- ing to increased ROS production and the subsequent accumulation of more mutations. There is evidence that some pathogenic mutations, such as at 8993T>G, lead to increased ROS production 141 ; interestingly, for 8993T>G, free radical-mediated inhibition of OXPHOS can be restored by antioxidants 142 . The ROS vicious cycle is believed to account for an exponential increase in oxidative damage during ageing, which results in the eventual loss of cellular and tissue functions through a combination of energy insufficiency, signalling defects, apoptosis and replicative senescence. Studies in human and mouse models. Although the mitochondrial theory of ageing was first sug- gested in the 1950s, the first convincing evidence of a Figure 4 | Nuclear transfer techniques. The prevention of mutated maternal mtDNA transmission could be achieved by nuclear transfer techniques. In theory, this could be carried out at the germinal vesicle stage (a), when clearly identifiable structures are visible. The germinal vesicle contains nuclear chromosomes and could be transferred to an oocyte that contains normal mtDNA (green). Alternatively, pronuclear transfer can be carried out at the fertilized oocyte stage (b); this involves removal of the male and female pronuclei from the oocyte that contains mutated mtDNA (red) and their subsequent transfer to an oocyte that has normal mtDNA (green). 398 | MAY 2005 | VOLUME 6 www.nature.com/reviews/genetics REVIEWS � 2005 Nature Publishing Group GENETIC DRIFT Changes in the frequency of a genetic variant in a population owing to chance alone. biochemical defect in humans was the detection of COX-deficient cells in ageing post-mitotic tissues 143 (FIG. 2). These cells had identical characteristics to those found in patients with inherited mitochondrial disease 37 . Surprisingly, these cells show the clonal expansion of an individual mutation, which is often an mtDNA rear- rangement, rather than many individual mtDNA muta- tions 144,145 . In addition, studies from patients showed that, in the presence of many mtDNA genomes within a cell, the mutation is functionally recessive and a threshold effect is observed 37 . More recently, mtDNA mutations were found to accumulate to high levels in dividing cells and presumably stem cells 39,146 , and cause an observable biochemical defect 39 . How a mutation in an individual genome can clonally expand to high levels in individual cells is a crucial question. So far, several theories have been proposed, including the selective replication of mutated mtDNA, the longevity of mitochondria that contain mutated mtDNA and the ?sick mitochondrion? hypoth- esis (with preferential replication of mitochondria that have impaired OXPHOS activities and higher mutant load) 147?149 . However, mathematical-modelling stud- ies have also indicated that this process might occur by GENETIC DRIFT 150 . Studies from ageing humans and animal models showed good correlation between ageing and the pres- ence of mtDNA mutations. To test the mitochondrial ageing hypothesis, knock-in mice expressing a proof- reading-deficient version of PolgA were developed 10 . These mice acquire mtDNA mutations at a higher rate than is normal because the introduction of a point mutation into the exonuclease region of the PolgA gene drastically reduces its proof-reading exonuclease activity. The phenotype of the PolgA mutator mouse has many hallmarks of premature ageing, including a characteristic hunched appearance that is indicative of osteoporosis, reduced activity, severe weight loss with virtually no remaining adipose tissue and a marked reduction in muscle mass. Although this study pro- vides strong evidence that somatic mtDNA mutations contribute to ageing, further experiments are required to confirm that somatic mtDNA mutations are causal in the ageing process. Mitochondrial DNA mutations and cancer. In a landmark paper in 1998, Vogelstein and colleagues reported that mtDNA mutations were present in 7 out of 10 colorectal cancer cell lines that were studied 151 . Although these mtDNA mutations were somatic and absent in other patient tissues, many were homoplas- mic in the tumour tissue. Since then, numerous other studies have documented the presence of somatic mtDNA mutations in both solid tumours and leukae- mias 152?156 . How these mutations accumulate to high levels in individual tumours is still unclear, although both modelling and molecular studies indicate that they possibly accumulate without selection 38,39 . In addi- tion, there is no evidence of whether mtDNA muta- tions themselves contribute to the development of the tumour. Original work on colorectal tumours showed that many of the mutations detected were not associ- ated with a mitochondrial defect. Moreover, some of the mutations seen in other tumours are recognized polymorphic variants 151 . Therefore, although a direct link between the presence of the mtDNA mutation and the development of the tumour has not been made, the presence of a mtDNA mutation might prove significant in the detection of tumour recurrence 152 and possibly in the detection of genotoxic damage 157 . Conclusions and future prospects It is nearly 25 years since Sanger and colleagues sequenced the human mitochondrial genome, and sig- nificant progress in the mitochondrial field continues to be made. Our understanding of the pathogenesis of mtDNA disease will greatly improve by studying the basic processes involved in replication, transcription and translation of the mitochondrial genome. Unequivocally, mtDNA mutations are an impor- tant cause of genetic disease. The clinical variability of these disorders makes the recognition of patients with mtDNA disease a real challenge. 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Maternally inherited hearing loss in a large kindred with a novel T7511C mutation in the mitochondrial DNA tRNA Ser(UCN) gene. Neurology 52, 1905?1908 (1999). 171. Strachan, T. & Read, A. P. Human Molecular Genetics 2nd edn (John Wiley and Sons, New York, 1999). Acknowledgements The authors work is supported by the Wellcome Trust, Medical Research Centre, Alzheimer?s Research Trust, Muscular Dystrophy Campaign, Newcastle upon Tyne Hospitals NHS Trust and the European Union (FP6, EUmitocombat). We thank R.N. Lightowlers, N. Howell, R. McFarland and M. Herbert for valuable input, and L. Craven and M. Barron for help with the figures. Competing interests statement The authors declare no competing financial interests. Online links DATABASES The following terms in this article are linked online to: Entrez: http://www.ncbi.nih.gov/Entrez GFM1 | MRPS16 | ND2 | ND4 | POLRMT | RNR1 | TFAM | TFB1M | TFB2M | TRNK | TRNL1 OMIM: http://www.ncbi.nlm.nih.gov/Omim Alzheimer disease | diabetes | Kearns?Sayre syndrome | Leber hereditary optic neuropathy | Leigh syndrome | progressive external ophthalmoplegia | Parkinson disease | rhabdomyolysis FURTHER INFORMATION EUmitocombat web site: http://www.eumitocombat.org MITOMAP ? a Human Mitochondrial Genome Database: http://www.mitomap.org mtDB ? Human Mitochondrial Genome Database: http:// www.genpat.uu.se/mtDB Access to this interactive links box is free online. 402 | MAY 2005 | VOLUME 6 www.nature.com/reviews/genetics REVIEWS "
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