Mitochondrial DNA and disease

Abstract

In addition to the 3 billion bp of nuclear DNA, each human cell contains multiple copies of a small (16·5 kb) loop of double-stranded (ds) DNA within each mitochondrion—the mitochondrial genome (mtDNA). Although mtDNA contributes less than 1% to the total cellular nucleic-acid content, it is fundamentally important for the function of every human tissue1Lightowlers RN Chinnery PF Turnbull DM Howell N Mammalian mitochondrial genetics: heredity, heteroplasmy and disease.Trends Genet. 1997; 13: 450-455Summary Full Text PDF PubMed Scopus (348) Google Scholar Recent studies have also shown the importance of nuclear gene mutations as a cause of mitochondrial dysfunction,2Dahl HH Getting to the nucleus of mitochondrial disorders: identification of respiratory chain-enzyme genes causing Leigh syndrome.Am J Hum Genet. 1998; 63: 1594-1597Summary Full Text Full Text PDF PubMed Scopus (78) Google Scholar and the crucial role of the mitochondrion in the pathophysiology of well-established autosomal diseases3DiMauro S Schon EA Nuclear power and mitochondrial disease.Nat Genet. 1998; 19: 214-215Crossref PubMed Scopus (49) Google Scholar (panel 1). The role of acquired somatic mtDNA deletions in ageing and neurodegenerative disease is still being evaluated.4Wallace DC Mitochondrial diseases in mouse and man.Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2567) Google Scholar These disorders are outside the scope of this review, which is restricted to primary disorders of the mitochondrial genome itself.Panel 1Human mitochondrial disordersMitochondrial DNA defectsRearrangements(deletions and duplications, more than 100 identified)Chronic progressive external ophthalmoplegia (CPEO)Kearns-Sayre syndromeDiabetes and deafnessPoint mutations* (currently over 50 identified)Protein-encoding genes Leber's hereditary optic neuropathy (G11778A, T14484C, G3460A)Leber's hereditary optic neuropathy/dystonia (G14459A, T14569A)Neurogenic weakness, ataxia, and retinitis pigmentosa (T8993G/C)Leigh's syndrome (T8993G/C)tRNA genes Mitochondrial encephalopathy with lactic acidosis and stroke-tike episodes (A3243G, T3271C, A3251G)Myoclonic epilepsy with ragged-red fibres (A8344G, T8356C)CPEO (A3243G, T4274C)Myopathy (T14709C, A12320G)Cardiomyopathy (A3243G, A4269G)Diabetes and deafness (A3243G, C12258A)Encephalomyopathy (G1606A, T10010C)Leigh's syndrome (G1644T)rRNA genes Noh-syndromic sensorineural deafness (A7445G)Aminoglycoside-induced non-syndromic deafness (A1555G)Nuclear DNA defectsNuclear genetic disorders with a mitochondrial basisFriedreich's ataxia (frataxin)Autosomal-recessive hereditary spastic paraplegia (paraplegin) Wilson's disease(P-type ATPase)Nuclear genetic disorders of the mitochohdrial respiratory chainLeigh's syndrome (complex i deficiency-mutations in AQDQ subunit on chromosome 5)Optic atrophy and ataxia (complex It deficiency—mutations in Fp subunit of SDH on chromosome 3)Leigh's syndrome (complex IV deficiency—mutations in SURF I gene on chromosome 9ql)Nuclear genetic disorders associated with multiple mtDNA deletionsAutosomal dominant external ophthalmoplegia (chromosome 10q23·3-q24,3; 3pl4·1-21·2)Mitochondrial neuro-gastrointestinal encephalomyopathy (thymidine phosphorylase deficiency)—mutations in thymidine phosphorylase gene on chromosome 22ql3·32-qter*mtDNA nucleotide positions are on the L-chain. Mitochondrial DNA defects Rearrangements(deletions and duplications, more than 100 identified) Chronic progressive external ophthalmoplegia (CPEO) Kearns-Sayre syndrome Diabetes and deafness Point mutations* (currently over 50 identified) Protein-encoding genes Leber's hereditary optic neuropathy (G11778A, T14484C, G3460A)Leber's hereditary optic neuropathy/dystonia (G14459A, T14569A)Neurogenic weakness, ataxia, and retinitis pigmentosa (T8993G/C)Leigh's syndrome (T8993G/C) tRNA genes Mitochondrial encephalopathy with lactic acidosis and stroke-tike episodes (A3243G, T3271C, A3251G)Myoclonic epilepsy with ragged-red fibres (A8344G, T8356C)CPEO (A3243G, T4274C)Myopathy (T14709C, A12320G)Cardiomyopathy (A3243G, A4269G)Diabetes and deafness (A3243G, C12258A)Encephalomyopathy (G1606A, T10010C)Leigh's syndrome (G1644T) rRNA genes Noh-syndromic sensorineural deafness (A7445G)Aminoglycoside-induced non-syndromic deafness (A1555G) Nuclear DNA defects Nuclear genetic disorders with a mitochondrial basis Friedreich's ataxia (frataxin) Autosomal-recessive hereditary spastic paraplegia (paraplegin) Wilson's disease(P-type ATPase) Nuclear genetic disorders of the mitochohdrial respiratory chain Leigh's syndrome (complex i deficiency-mutations in AQDQ subunit on chromosome 5) Optic atrophy and ataxia (complex It deficiency—mutations in Fp subunit of SDH on chromosome 3) Leigh's syndrome (complex IV deficiency—mutations in SURF I gene on chromosome 9ql) Nuclear genetic disorders associated with multiple mtDNA deletions Autosomal dominant external ophthalmoplegia (chromosome 10q23·3-q24,3; 3pl4·1-21·2) Mitochondrial neuro-gastrointestinal encephalomyopathy (thymidine phosphorylase deficiency)—mutations in thymidine phosphorylase gene on chromosome 22ql3·32-qter *mtDNA nucleotide positions are on the L-chain. The first complete human mtDNA sequence was published in 1981.5Anderson S Bankier AT Barrell BG et al.Sequence and organization of the human mitochondrial genome.Nature. 1981; 290: 457-465Crossref PubMed Scopus (7448) Google Scholar Less than a decade later, the first pathogenetic mtDNA mutations were identified in human beings. Since that time, over 100 different rearrangements and 50 different point mutations have been associated with human disease (panel 1 and see also http://www.gen.emory.edu/mitomap.html).6Servidei S Mitochondrial encephalomyopathies: gene mutation.Neuromuscul Disord. 1999; 9: 15-22Google Scholar Patients with mtDNA defects present with a wide variety of phenotypes to physicians in almost any specialty (figure and panel 2).7Chinnery PF Howell N Andrews RA Turnbull DM Clinical mitochondrial genetics.J Med Genet. 1999; 36: 425-436Crossref PubMed Scopus (76) Google Scholar As a result, mitochondrial medicine is a rapidly expanding discipline that will undoubtedly gain further importance as we enter the new millenium.Panel 2Common Clinical syndromes due to mtDNA defects Tabled 1DisorderPrimary featuresAdditional featuresChronic progressive external ophthalmopiegia (CPEO)External ophtha Irnoplegia and bilateral ptosisMlid proximal myopathyKeams-Sayre syndrome (KSS)PEO onsent before age 20 with pigmentary retinopathy plus one of the following: CSF protein greter than 1 g/L, cerebellar ataxia, heart blockBllateral deafnessMyopathyDyphagiaDiabetes mellitusHypoparathyroidism DementiaPearson's syndromeSideroblastic anaemia of childhoodRenal tubular defectsPancytopaeniaExocrine pancreatic failureMitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS)Stroke-like episodes before age 40Diabetes mellitusSeizures and/or dementiaCardiomyopathy (hypertrophic leading to dilated)Ragged-red fibres and/or lactic acidosisBilateral deafnessPigmentary retinopathyCerebellar ataxiaMyoclonic epilepsy with ragged-red fibres (MERRF)MyoclonusDementiaSeizuresOptic atrophyCerebellar ataxiaBilateral deafnessMyopathyPeripheral neuropathySpasticityMultiple lipomataLeber's hereditary optic neuropathy (LHON)Subacute bilateral visual failureDystoniaMen:women about 4:1Cardiac pre-excitation syndromesMedian age of onset 24 years Open table in a new tab Tabled 1DisorderPrimary featuresAdditional featuresChronic progressive external ophthalmopiegia (CPEO)External ophtha Irnoplegia and bilateral ptosisMlid proximal myopathyKeams-Sayre syndrome (KSS)PEO onsent before age 20 with pigmentary retinopathy plus one of the following: CSF protein greter than 1 g/L, cerebellar ataxia, heart blockBllateral deafnessMyopathyDyphagiaDiabetes mellitusHypoparathyroidism DementiaPearson's syndromeSideroblastic anaemia of childhoodRenal tubular defectsPancytopaeniaExocrine pancreatic failureMitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS)Stroke-like episodes before age 40Diabetes mellitusSeizures and/or dementiaCardiomyopathy (hypertrophic leading to dilated)Ragged-red fibres and/or lactic acidosisBilateral deafnessPigmentary retinopathyCerebellar ataxiaMyoclonic epilepsy with ragged-red fibres (MERRF)MyoclonusDementiaSeizuresOptic atrophyCerebellar ataxiaBilateral deafnessMyopathyPeripheral neuropathySpasticityMultiple lipomataLeber's hereditary optic neuropathy (LHON)Subacute bilateral visual failureDystoniaMen:women about 4:1Cardiac pre-excitation syndromesMedian age of onset 24 years Open table in a new tab A basic understanding of human mitochondrial genetics is of great practical benefit to the clinician caring for patients with suspected or proven mtDNA disease: it provides an explanation for the pathophysiology, highlights the potential difficulties with investigation and diagnosis, and has important implications for prognostic and genetic counselling. Finally, since there is no effective treatment for most mitochondrial disorders, we must use our knowledge of mitochondrial genetics to devise new therapies. Each mtDNA molecule contains 13 polypeptide-encoding genes, and the 24 RNA genes that allow intramitochondrial protein synthesis (figure). Transcription and translation of mtDNA is controlled by the nucleus through the only non-coding region of the mitochondrial genome (the 1 kb D-loop). The polypeptides synthesised from the 13 mtDNA genes interact with more than 60 nuclear-encoded polypeptides to form the mitochondrial respiratory chain, which is essential for aerobic cellular metabolism. Mitochondrial function is, therefore, dependent on the interaction of many nuclear and mitochondrial genes, and abnormalities of either genome may cause mitochondrial disease.8Larsson NG Clayton DA Molecular genetic aspects of human mitochondrial disorders.Annu Rev Genet. 1995; 29: 151-178Crossref PubMed Scopus (400) Google Scholar, 9Howell N Human mitochondrial diseases: answering questions and questioning answers.Int Rev Cytol. 1999; 186: 49-116Crossref PubMed Google Scholar There are two fundamental differences between nuclear DNA and mtDNA that are important for the expression and transmission of mitochondrial genetic disease. Human cells contain at least 1000 copies of mtDNA. In normal individuals, all copies of the mtDNA are identical within the coding region. Individuals with mtDNA disease often harbour a mixture of mutated and wild-type (normal) mtDNA—this feature is called heteroplasmy.1 Within single cells, the proportion of mutated mtDNA must exceed a critical threshold before the cell expresses a mitochondrial respiratory-chain defect,8Larsson NG Clayton DA Molecular genetic aspects of human mitochondrial disorders.Annu Rev Genet. 1995; 29: 151-178Crossref PubMed Scopus (400) Google Scholar but the relation between the proportion of mutated mtDNA and the clinical phenotype of the whole organism is less clear. Different organs, and even adjacent cells within the same organ, may contain different amounts of mutated mtDNA. This variability, coupled with tissue-specific differences in the threshold and the dependence of different organs on oxidative metabolism, goes some way to explain why certain tissues are preferentially affected in patients with mtDNA disease.10Wallace DC 1994 William Allan Award Address.in: Mitochondrial DNA variation in human evolution, degenerative disease, and aging. 7th edn. Am J Hum Genet. 57. 1995: 201-223Google Scholar In general, postmitotic (non-dividing) tissues such as neurons, skeletal and cardiac muscle, and endocrine organs harbour high levels of mutated mtDNA and are often clinically involved. By comparison, rapidly dividing tissues such as the bone marrow are only rarely clinically affected,11Shoffner JM Maternal inheritance and the evaluation of oxidative phosphorylation diseases.Lancet. 1996; 348: 1283-1288Summary Full Text Full Text PDF PubMed Scopus (71) Google Scholar Differences in the proportions of mutated mtDNA between and within family members is one explanation for the extreme clinical variability that is characteristic of mtDNA disease. As a result, the same genetic defect can cause diabetes and deafness in one individual, and a severe encephalopathy with seizures and dementia in another.12Schon EA Bonilla E DiMauro S Mitochondrial DNA mutations and pathogenesis.J Bioenerg Biomembr. 1997; 29: 131-149Crossref PubMed Scopus (381) Google Scholar When large numbers of patients are studied, there does, however, appear to be a relation between mutation load and phenotype. For two of the most common point mutations (A3243G and A8344G), the frequencies of the major neurological clinical features are related to the level of mutated mtDNA in skeletal muscle,13Chinnery PF Howell N Lightowelrs RN Turnbull D Molecular pathology of MELAS and MERRF: the relationship between mutation load and clinical phenotypes.Brain. 1997; 120: 1713-1721Crossref PubMed Scopus (318) Google Scholar For example, stroke-like episodes, epilepsy and dementia are related to the level of the A3243G mutation in muscle, and cerebellar ataxia and myoclonus are related to the level of the A843G mutation in muscle. For the T8993G/C mutation, the mutation load in blood is related to the severity of the clinical phenotype14White SL, Collins VR, Wolfe R, et al. Genetic counselling and prenatal diagnosis for the mitochondrial DNA mutations at nucleotide 8993. Am J Hum Genet (in press).Google Scholar and presumably reflects the proportion in the clinically relevant organs. Maternal inheritance and transmission of heteroplasmy After fertilisation of the oocyte, sperm mtDNA is actively degraded.15Shitara H Hayashi JI Takahama S Kaneda H Yonekawa H Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage.Genetics. 1998; 148: 851-857PubMed Google Scholar As a consequence, mtDNA is transmitted exclusively through the maternal line. Thus, affected men do not transmit the genetic defect. Deleted molecules are rarely, if ever, transmitted from clinically affected women to their children.16Chinnery PF Howell N Lightowlers RN Turnbull DM Genetic counseling and prenatal diagnosis of mtDNA disease.Am J Hum Genet. 1998; 63: 1908-1911Summary Full Text Full Text PDF PubMed Scopus (23) Google Scholar By contrast, a woman with a heteroplasmic mtDNA point mutation, or mtDNA duplications, may transmit a variable amount of mutated mtDNA to her children.1 Early during development of the female germ-line, the number of mtDNA molecules within each oocyte is reduced before being subsequently amplified to reach a final number of about 100 000 in each mature oocyte. This restriction and amplification (also called the mitochondrial “genetic bottleneck”) contributes to the variability between individual oocytes, and the different concentrations of mutant mtDNA seen in the children of one woman.17Poulton J Macaulay V Marchington DR Mitochondrial genetics '98 is the bottleneck cracked?.Am J Hum Genet. 1998; 2: 52-57Google Scholar Despite this variability, recent studies have shown that for the most common pathogenetic mtDNA point mutations, mothers with a higher concentration of mutated mtDNA are more likely to have clinically affected children than mothers with lower levels of mutated mtDNA.14White SL, Collins VR, Wolfe R, et al. Genetic counselling and prenatal diagnosis for the mitochondrial DNA mutations at nucleotide 8993. Am J Hum Genet (in press).Google Scholar, 18Chinnery PF Howell N Lightowlers RN Turnbull DM MELAS and MERRF: the relationship between maternal mutation load and the frequency of clinically affected offspring.Brain. 1998; 121: 1889-1894Crossref PubMed Scopus (96) Google Scholar At present, it would be unwise to use these data for counselling purposes—but they do suggest that more accurate predictions may be possible after the prospective accumulation of data from mothers and children. A number of well-defined clinical syndromes are caused by mutations of mtDNA (panel 2).7Chinnery PF Howell N Andrews RA Turnbull DM Clinical mitochondrial genetics.J Med Genet. 1999; 36: 425-436Crossref PubMed Scopus (76) Google Scholar Large-scale deletions can cause chronic progressive external ophthalmoplegia (CPEO) and bilateral ptosis19Moraes CT DiMauro S Zeviani M et al.Mitochondrial DNA deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome.N Engl J Med. 1989; 320: 1293-1299Crossref PubMed Scopus (889) Google Scholar Some of these patients have little disability and may have limited extramuscular involvement. By contrast, similar deletions may also cause CPEO with bilateral sensorineural deafness, cerebellar ataxia, pigmentary retinopathy, diabetes mellitus, and cardiac conduction defects leading to complete heart block. When this begins in teenage years and is associated with high concentrations of protein in the cerebrospinal fluid, it is called the Kearns-Sayre syndrome (KSS), which is a devastating progressive neurological disorder.19Moraes CT DiMauro S Zeviani M et al.Mitochondrial DNA deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome.N Engl J Med. 1989; 320: 1293-1299Crossref PubMed Scopus (889) Google Scholar Most cases of CPEO and KSS are caused by sporadic mutations.16Chinnery PF Howell N Lightowlers RN Turnbull DM Genetic counseling and prenatal diagnosis of mtDNA disease.Am J Hum Genet. 1998; 63: 1908-1911Summary Full Text Full Text PDF PubMed Scopus (23) Google Scholar The causative mtDNA mutation probably occurs within the germ line.20Chen X Prosser R Simonetti S Sadlock J Jagiello G Schon EA Rearranged mitochondrial genomes are present in human oocytes.Am J Hum Genet. 1995; 57: 239-247Crossref PubMed Scopus (5) Google Scholar It is not clear why similar genetic defects can cause KSS or CPEO, but these two syndromes are the extremes of a range of disease, and many individuals lie somewhere between the pure extra-ocular and severe central neurological phenotypes. Pathogenetic point mutations of mtDNA are more common than rearrangements, partly because deletions within mtDNA cause sporadic disease, whereas many mtDNA point mutations are transmitted down the maternal line. The A3243G mutation in the leucine (UUR) tRNA gene is probably the single most common mtDNA defect, and is present in 1 in 7000 of the Finnish population21Majamaa K Moilanen JS Uimonen S et al.Epidemiology of A3243G, the mutation for mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes: prevalence of the mutation in an adult population.Am J Hum Genet. 1998; 63: 447-454Summary Full Text Full Text PDF PubMed Scopus (302) Google Scholar This mutation was first described in a patient with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS).22Goto Y Nonaka I Horai S A mutation in the tRNA(Leu)(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies.Nature. 1990; 348: 651-653Crossref PubMed Scopus (1701) Google Scholar Different families harbouring the same genetic defect may have different phenotypes. For example, some families with A3243G have predominantly diabetes and deafness, some have CPEO, and some present with cardiomyopathy.12Schon EA Bonilla E DiMauro S Mitochondrial DNA mutations and pathogenesis.J Bioenerg Biomembr. 1997; 29: 131-149Crossref PubMed Scopus (381) Google Scholar Why this is the case is currently not known, but probably there are other nuclear genetic factors that have an important role in modifying the expression of the primary mtDNA defect.23Chinnery PF Turnbull DM Mitochondrial genotype and clinical phenotype.J Inher Metab Dis. 1998; 21: 321-325Crossref PubMed Scopus (6) Google Scholar The A3243G mutation causes substantial morbidity and mortality, and it is estimated that 0·5-1·5% of cases of diabetes mellitus in the general population may be due to the A3243G mutation.24Maassen J Kadowaki T Maternally inherited diabetes and deafness: a new diabetes subtype.Diabetologia. 1996; 39: 375-382Crossref PubMed Scopus (162) Google Scholar Many cases of visual loss in young men are also caused by mtDNA mutations. About 50% of all males who have one of three point mutations (Gl1778A, T14484C, G3460A) develop bilateral, sequential visual loss in the second or third decade–a disorder known as Leber's hereditary optic neuropathy (LHON).25Howell N Leber hereditary optic neuropathy: how do mitochondrial DNA mutations cause degeneration of the optic nerve?.J Bioenerg Biomembr. 1997; 29: 165-173Crossref PubMed Scopus (55) Google Scholar Most individuals with a LHON mutation are homoplasmic with only mutated mtDNA, and yet it is fascinating that only 10% of women with the same genetic defect develop visual loss26Harding AE Sweeney MG Govan GG Riordan-Eva P Pedigree analysis in Leber hereditary optic neuropathy families with a pathogenic mtDNA mutation.Am J Hum Genet. 1995; 57: 77-86PubMed Google Scholar The pathogenesis of LHON is complex. Environmental factors may be important, and as yet unknown nuclear genetic factors probably contribute.9Howell N Human mitochondrial diseases: answering questions and questioning answers.Int Rev Cytol. 1999; 186: 49-116Crossref PubMed Google Scholar Although these, and many other syndromes, are strongly suspected of having a mitochondrial aetiology (figure and panel 2), many patients do not present with a classic phenotype. A mitochondrial genetic diagnosis should be thought of in any patient who has a disease with multiple organ involvement—particularly if there are central neurological features such as seizures and dementia, myopathy, cardiac involvement, or endocrine abnormalities such as diabetes mellitus (figure). Finally, many families with isolated inherited non-syndromic deafness have a homoplasmic mutation at position 1555 of the mitochondrial genome.27Estivill X Govea N Barcelo A et al.Familial progressive sensorineural deafness is mainly due to the mtDNA A1555G mutation and is enhanced by treatment with aminoglycosides.Am J Hum Genet. 1998; 62: 27-35Summary Full Text Full Text PDF PubMed Scopus (428) Google Scholar This mutation is associated with congenital and late-onset deafness, and the penetrance is enhanced by aminoglycoside exposure. Identification of these families is important because it may be possible to prevent the heating loss by avoiding aminoglycoside antibiotics. Not all patients with mtDNA disease can be diagnosed by a simple molecular genetic blood test that looks for one of the more common mtDNA mutations: a negative blood test in an index case does not mean that an individual does not have mtDNA disease. There are many potential difficulties. The same clinical phenotype can be caused by many different mutations, and even if the phenotype is “classic” for a particular genetic defect, the proportion of mutated mtDNA in blood may be undetectable by the routine methods used in many molecular diagnostic laboratories.28Chinnery PF Turnbull DM Walls TJ Reading PJ Recurrent strokes in a 34-year-old man.Lancet. 1997; 350: 560Summary Full Text Full Text PDF PubMed Scopus (17) Google Scholar The investigation of patients with suspected mtDNA disease involves the careful assimilation of clinical and laboratory data,29Chinnery PF Turnbull DM Clinical features, investigation, and management of patients with defects of mitochondrial DNA.J Neurol Neurosurg Psychiatry. 1997; 63: 559-563Crossref PubMed Scopus (95) Google Scholar and in many cases it requires the analysis of skeletal muscle. The histochemical analysis of muscle may reveal features of mtDNA disease, such as the subsarcolemmal accumulation of mitochondria, the so-called ragged-red fibres, or a mosaic deficiency of cytochrome oxidase.30Johnson MA Barron MJ Muscle biopsy analysis.in: 7th edn. Handbook of muscle disease. Marcel Dekker, New York1996Google Scholar Furthermore, for some mtDNA defects the abnormality is not detectable in leucocyte DNA, and the analysis of DNA extracted from muscle is essential to establish the diagnosis.7Chinnery PF Howell N Andrews RA Turnbull DM Clinical mitochondrial genetics.J Med Genet. 1999; 36: 425-436Crossref PubMed Scopus (76) Google Scholar Many patients with mitochondrial disease have a previously unrecognised mtDNA defect and direct sequencing of the mitochondrial genome is necessary.11Shoffner JM Maternal inheritance and the evaluation of oxidative phosphorylation diseases.Lancet. 1996; 348: 1283-1288Summary Full Text Full Text PDF PubMed Scopus (71) Google Scholar Automated sequencing itself is relatively straightforward with the proviso that more than 30% of the DNA sample is mutated mtDNA—hence the need for a muscle DNA sample—but the interpretation of the sequence data can be extremely difficult, mtDNA is highly polymorphic, and any two normal individuals may differ by up to 60 bp. In the strictest sense, a mutation can only be deemed to be pathogenetic if it has arisen many times in the population, it is not seen in controls, and it is associated with a potential disease mechanism. These stringent criteria depend on a good knowledge of polymorphic sites in the background population. If a novel base change is heteroplasmic, this finding suggests that it is of relatively recent onset. Family, tissue segregation, and single-cell studies may show that higher concentrations of the mutation are associated with mitochondrial dysfunction and disease, which strongly suggests that the mutation is causing the disease32 Thus, interpretion of sequence data is fraught with difficulties, and should not be undertaken lightly. The final molecular diagnosis may have important implications for future management (such as prenatal diagnosis), so there is no room for uncertainty. Management of patients with mtDNA disease After diagnosis, the management of mtDNA disease falls into four groups. Firstly, based upon retrospective studies of many patients, some guidance may be given about the future, and the chances of transmitting the genetic defect.13Chinnery PF Howell N Lightowelrs RN Turnbull D Molecular pathology of MELAS and MERRF: the relationship between mutation load and clinical phenotypes.Brain. 1997; 120: 1713-1721Crossref PubMed Scopus (318) Google Scholar, 18Chinnery PF Howell N Lightowlers RN Turnbull DM MELAS and MERRF: the relationship between maternal mutation load and the frequency of clinically affected offspring.Brain. 1998; 121: 1889-1894Crossref PubMed Scopus (96) Google Scholar Secondly, vigilant clinical monitoring over many years may prevent the known complications of mtDNA disease. Thirdly, intervention may be appropriate at some stage—either surgical (ptosis correction and cataract surgery), cardiac pacing, or a percutaneous gastrostomy—along with practical assistance with mechanical aids and social support. Finally, standard doses of vitamin C and K, thiamine, riboflavin, and ubiquinone (coenzyme Q10) may be of some benefit.31Taylor RW Chinnery PF Clark KM Lightowlers RN Turnbull DM Treatment of mitochondrial disease.J Bioenerg Biomembr. 1997; 29: 195-205Crossref PubMed Scopus (57) Google Scholar These treatments have no significant side-effects and are relatively inexpensive, but their efficacy is largely based on anecdotal reports. Hovel treatments are, however, under development. Exercise is important in patients with mtDNA disease, and isometric muscle contraction leads to an improvement in muscle strength,32Taivassalo T De Stefano N Matthews PM et al.Aerobic training benefits patients with mitochondrial myopathies more than other chronic myopathies.Neurology. 1997; 48: A214Crossref PubMed Scopus (78) Google Scholar and concentric exercise training may be accompanied by a decrease in the proportion of mutant mtDNA.33Taivassalo T Fu K Johns T Arnold D Karpati G Stourbridge EA Gene shifting: a novel therapy for mitochondrial myopathy.Hum Mol Genet. 1999; 8: 1047-1052Crossref PubMed Scopus (127) Google Scholar Drug-induced muscle necrosis followed by proliferation of myoblasts may also be important for the treatment of mitochondrial myopathy and ptosis34Fu K Hartlen R Johns T Genge A Karpati G Shoubridge EA A novel heteroplasmic tRNAleu(CUN) mtDNA point mutation in a sporadic patient with mitochondrial encephalomyopathy segregates rapidly in skeletal muscle and suggests an approach to therapy.Hum Mol Genet. 1996; 5: 1835-1840Crossref PubMed Scopus (142) Google Scholar, 35Clark KM Bindoff LA Lightowlers RN et al.Reversal of a mitochondrial DNA defect in human skeletal muscle.Nat Genet. 1997; 16: 222-224Crossref PubMed Scopus (125) Google Scholar A number of groups are designing methods to correct the underlying mtDNA defect. For example, an inhibitor of mitochondrial oxidation has been used in cultured cells to alter the ratio of mutant mtDNA to wild-type mtDNA36Manfredi G Gupta N Vazquez-Memije ME et al.Oligomycin induces a decrease in the cellular content of a pathogenic mutation in the human mitochondrial ATPase6 gene.J Biol Chem. 1999; 274: 9386-9391Crossref PubMed Scopus (93) Google Scholar In addition to the many difficulties that face nuclear gene therapy, there are a number of additional problems when trying to manipulate mitochondrial gene expression. Each cell contains multiple copies of mtDNA, many mtDNA mutations are heteroplasmic, and the therapeutic agent must be able to enter the mitochondria. At least two approaches are currently being explored. A self-replicating copy of a normal gene sequence has been successfully delivered into mitochondria in vitro,37Seibel P Trappe J Villani G Klopstock T Papa S Reichmann H Transfection of mitochondria: strategy towards a gene therapy of mitochondrial DNA diseases.Nucleic Acids Res. 1995; 23: 10-17Crossref PubMed Scopus (144) Google Scholar and an approach for heteroplasmic mtDNA disorders is to specifically inhibit replication of mutant mtDNA.38Taylor RW Chinnery PF Turnbull DM Lightowlers RN Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids.Nat Genet. 1997; 15: 212-225Crossref PubMed Scopus (207) Google Scholar Defects of the mitochondrial genome are a common cause of genetic disease. Many patients have sporadic disease due to deletions, but maternally inherited point mutations are common. Patients with pathogenetic mtDNA defects often have a mixture of mutated and wild-type mtDNA—heteroplasmy—which is “important for the expression and transmission of mtDNA disease. The investigation of patients with suspected mtDNA disease is a challenge, partly because of the complexities of the mitochondrial genetic system. Interpretation of the genetic tests is difficult, and it should only be done within the clinical context. An accurate diagnosis of mtDNA disease is important because it will have implications for the patient and for the family.