Mitochondrial DNA and disease

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1 Mitochondrial DNA and disease P F Chinnery, D M Tumbull 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 tissue? Recent studies have also shown the importance of nuclear gene mutations as a cause of mitochondrial dysfunction, 2 and the crucial role of the mitochondrion in the pathophysiology of well-established autosomal diseases 3 (panel 1). The role of acquired somatic mtdna deletions in ageing and neurodegenerative disease is still being evaluated. 4 These disorders are outside the scope of this review, which is restricted to primary disorders of the mitochondrial genome itself. The first complete human mtdna sequence was published in 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 6 Patients with mtdna defects present with a wide variety of phenotypes to physicians in almost any specialty (figure and panel 2). 7 As a result, mitochondrial medicine is a rapidly expanding discipline that will undoubtedly gain further importance as we enter the new millenium. 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. Basic mitochondrial genetics 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. 8,9 Lancet 1999; 354 (suppl I): Department of Neurology, University of Newcastle upon Tyne, NE2 4HH, UK (P F Chinnery MRCP, Prof D M Tumbull FRCP) Correspondence to: Prof DM Turnbull ( D.M.Turnbull@ncl.ac.uk) Differences between nuclear and mtdna There are two fundamental differences between nuclear DNA and mtdna that are important for the expression and transmission of mitochondrial genetic disease. Heteroplasmy and the threshold effect 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 wildtype (normal) mtdna--this feature is called heteroplasmy. 1 Within single cells, the proportion of Molecular medicine 354 July 1999 s117

2 THE LANCET Central nem Encephalol Stroke-like Seizures ar Psychosis: Ataxia Migraine Cardiac Hypertrophi Dilated can Heart block Pre-excitati ~pathy ~[ neuron meuraldeafne: Heteroplasmic mitochondrion / on Endocrine Diabetes m Hypoparath Hypothyrok Gonadal fai Renal Renal tubul~ Toni-Fancon D-LOOE 12SrRNA xq---"... ~ CYT ~.~,~"~ F T --~~, ~,u ~tem ~europathy ND1/~L(U'UR) M~_ "y n ~'" \ND5 ~ S (AGY) -~H. / ' ~ _.. W lid-type OL~ ~ ~S(UCN) ///D / N D 4 (normal) ~.. ~ ~ G ~ " ND4L mtdna molecule COX D ~ ~. ~ D 3 ' F - - ~ " N \COX III COX II ' ATPase6 ATPase8 ND2$,, W~ LA ~C ~ I Mitochondrial DNA and disease mutated m t D N A must exceed a critical threshold before the cell expresses a mitochondrial respiratory-chain defect, 8 but the relation between the proportion of mutated m t D N A 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 m t D N A. 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 m t D N A disease. 1 In general, postmitotic (non-dividing) tissues such as neurons, skeletal and cardiac muscle, and endocrine organs harbour high levels of mutated m t D N A and are often clinically involved. By comparison, rapidly dividing tissues such as the bone marrow are only rarely clinically affected, n Differences in the proportions of mutated m t D N A between and within family members is one explanation for the extreme clinical variability that is characteristic of m t D N A 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 another32 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 c o m m o n point mutations (A3243G and A8344G), the frequencies of the major neurological clinical features are related to the level of mutated m t D N A in skeletal muscle, x3 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 T G / C mutation, the mutation load in blood is related to the severity of the clinical phenotype ~4 and presumably reflects the proportion in the clinically relevant organs. $I 1 8 Matemal inheritance and transmission of heteroplasmy After fertilisation of the oocyte, sperm m t D N A is actively degraded, x~ As a consequence, m t D N A 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. 16 By contrast, a woman with a heteroplasmic m t D N A point mutation, or m t D N A duplications, may transmit a variable amount of mutated m t D N A to her children. 1 Early during development of the female germ-line, the number of m t D N A molecules within each oocyte is reduced before being subsequently amplified to reach a final number of about 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 m t D N A seen in the children of one woman. 17 Despite this variability, recent studies have shown that for the most common pathogenetic m t D N A point mutations, mothers with a higher concentration of mutated m t D N A are more likely to have clinically affected children than mothers with lower levels of mutated m t D N A. 14'~8At 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. Clinical range and prevalence disease of mtdna A number of well-defined clinical syndromes are caused by mutations of m t D N A (panel 2).7 Large-scale deletions can cause chronic progressive external ophthalmoplegia (CPEO) and bilateral ptosisj 9 Some of these patients have little disability and may have limited extramuscular involvement. By contrast, similar deletions may also cause C P E O with bilateral sensorineural deafness, cerebellar Molecular medicine 354 July 1999

3 THE LANCET 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 Keams-Sayre syndrome (KSS), which is a devastating progressive neurological disorder. '9 Most cases of CPEO and KSS are caused by sporadic mutations. 16 The causative mtdna mutation probably occurs within the germ line. s 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 extraocular 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 populationj I This mutation was first described in a patient with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS)J ~ 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. 1~ 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. 23 The A3243G mutation causes substantial morbidity and mortality, and it is estimated that % of cases of diabetes mellitus in the general population may be due to the A3243G mutation. 24 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). 25 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 lossj 6 The pathogenesis of LHON is complex. Environmental factors may be important, and as yet unknown nuclear genetic factors probably contribute. 9 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 meuitus (figure). Finally, many families with isolated inherited non-syndromic deafness have a homoplasmic mutation at position 1555 of the mitochondrial genomej 7 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. Genetic tests for mtdna disease 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 laboratoriesj s The investigation of patients with suspected Molecular medicine 354 July 1999 si19

4 mtdna disease involves the careful assimilation of clinical and laboratory data, 29 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 c oxidase2 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, v Many patients with mitochondrial disease have a previously unrecognised mtdna defect and direct sequencing of the mitochondrial genome is necessary. 11 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. 13'1s 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 benefit21 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 strengthfl 2 and concentric exercise training may be accompanied by a decrease in the proportion of mutant mtdna23 Drug-induced muscle necrosis followed by proliferation of myoblasts may also be important for the treatment of mitochondrial myopathy and ptosis24'3s 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 mtdna26 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, 3~ and an approach for heteroplasmic mtdna disorders is to specifically inhibit replication of mutant mtdna28 Conclusion 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. Mitochondrial genetics is at a very different stage of development from other areas of genetics. It is a tiny genome that can easily be sequenced in its entirety. Nevertheless, we understand relatively little when it comes to managing our patients. However, the next decade should be one in which we are able to answer the main clinical questions relating to mtdna disease---how to give good genetic counselling, how to advise on prognosis, and how to treat these disorders. Acknowledgements PFC is a Wellcome Trust Research Fellow and DMT is supported by a programme grant from the Wellcome Trust, by the MRC, and the Muscular Dystrophy Group of Great Britain. References 1 Lightowlers RN, Chinnery PF, Turnbun DM, Howell N. Mammalian mitochondrial genetics: heredity, heteroplasmy and disease. Trends Genet 1997; 13: Dahl HH. Getting to the nucleus of mitochondrial disorders: identification of respiratory chain-enzyme genes causing Leigh syndrome. Am ff Hum Genet 1998; 63: DiMauro S, Schon EA. 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