Summary Since the human mitochondrial genome was characterised and sequenced in 1981(l),it has been viewed as the likely site of genetic diseases showing a maternal inheritance pattern and associated with defects of the respiratory chain, such as the mitochondrial myopathies ( MMS)?(~). The properties that make it a candidate For the source of such conditions are that it encodes polypeptides involved in electron and that it is maternally inherited(5).However, several of the mtDNA diseases only fulfill one or other of these criteria: the first group OF mtDNA diseases showed only sporadic deletion~(~.~), and the first point mutation in Leber’s Hereditary Optic Neuropathy(*)(LHON) is not associated with a clear biochemical defect. Furthermore, it is now clear that both autosomal dominant(9)and probably recessive(lO)nuclear genes can cause abnormalities of mtDNA. Each of these major groups will be considered in turn. Introduction Until 1988, the human mitochondrial genome was the province solely of basic biological science. Since then, mitochondrial DNA has made frequent appearances in medical journals and even in the popular press. Discoveries of pathological mitochondria1 DNA (mtDNA) mutations have burgeoned, and have already reached double figures. A comprehensive review is therefore outside the scope of this article. It will introduce the various conditions rather than describe them in depth, in order to discuss some of the salient points of this field. Mitochondrial DNA Mitochondria are intracellular organelles which are key elements in oxidative phosphorylation. They contain their own DNA, which is the only source of extranuclear DNA in man (reviewed in full by Atlardi(’’ 1). Unlike nuclear DNA, there

are lhousands of copicc of this 16.569 base circular moleculc in every nucleate cell, each mitochondrion containing between two and ten copies. In normal individuals, the\e are virtually all identical(”), and each copy encodes thirteen proteins (subunits of the electron transport chain), 22 transfer RNAs (tRNAs) and two ribosomal RNAs (Fig. 1). Mitochondria require their own ribosomes and transfer RNAs (tRNAs) because they have thcir own version of the genetic code. The mitochondrial genome is extremely compact with most of the genes abutting, and only just over 1 kb of noncoding DNA (the so-called D-loop). This is an important control region, containing both the origins of replication of rntDNA (OH and OL from which synthesis of the daughter heavy and light ytrands start), and the promoters for RNA synthesis (LSP and HSP corresponding to light and heavy strand transcript\). MtDNA is almost entirely maternally inherited, presumably because the sperm contributes very little cytoplasm to the sygote(l3).

Major Re-arrangements of mtDNA: simple The first evidence that rearrangements of mtDNA might cause human disease was obtained by Holt et 0 1 . ‘ ~ )who used Southern hybridisation to demonstrate large deletions in mtDNA from muscle, which were not detectable in blood o f a group of patients with muscle disease (a subtype of the mitochondrial myopathies comprising Kearns-Sayre syndrome or KSS ‘and chronic progressive external ophthalmoplegia or CPEO). Both normal and mutant mtDNAs were present in these patients, as is usually the case in patients with mtDNA diseases. The existence of more than one mitochondrial type in the same cell, tissue or individual is known as heteroplasmy. Although circumstantial evidence is accumulating that these deletions are causative, the pathophysiological mechanism is still obscure. I will first describe the deletions which have been observed in sporadic cases. then discuss the experimental data with respect to some key ques-

a 9,

MTATP6 MTATPB

DEILJLD

NORMRL

and ragged red fibres; LHON. Leber’s Hcreditary Optic Neuropa-

Fig. 1. The common deletion alongside thc normal mitochondrial genome &awn to scale. Boxed region reprcsents the deleted region. Circles represent genes for transfer RNAs, Otl: Origin of replication of the heavy strand. OL: Origin of replication of the light slrand. MTATP6 and 8: subunits 6 and 8 of ATP synthase,MTCOXI to 111: subunits I to I11 oP cytochrome oxidase, MTCYB: cytochrome b, MTNDl to 6 and 4L: subunits 1 to 6 and 4L of NADH dehydrogc-

thy.

nase.

fmtDNA, Mitochondrial DNA; MM. Mitochondrial myopathy; MELAS; Mitochondrial encephalomyopathy, lactic acidosis and stroke4 ke episodes; MERKF, Mitochondrial enccphaloinyopathy

tions, including the relationship of the deletions to the phenotype.

The %ommon deletion’ All the deletions described so far(6.14)include one or more tRNA genes and part or all of one or more protein reading frames. The deletion found most frequent1y(l5),the so-called ‘common deletion’ (Fig. 1) is about 4.9kb in length and comprises approximately 50% in most series of Part or all of seven protein reading frames arc deleted (Fig. 1). Does the sequence data give us any clues as to how these re-arrangements occurred? Recombination does not appear to be a common feature of mtDNA in higher organisms. Extensive population surveys have not identified healthy individuals in whom major re-arrangements are present at high levels. Sequence analysis(’s.‘7) reveals that there are exact homologies at either end of the deleted segment, 13bp in length. Furthermore, i t appears that this particular stretch may be a recombination ‘hot spot’ (or more strictly ‘warmer spot’ than other sites) as a number of deletions share one end or the other. Sequence data from an increasingly large number of deletions shows that many have similar direct repeats flanking the deleted segment. Replication slippage may be a feature of the recombination For instance, during replication the heavy strand might be particularly vulnerable as it remains single stranded while the replication fork proceeds 2/3 of the way round the genome(3). The single stranded heavy strand might mispair with any single stranded regions of the light strand (for instance by means of a bubble in a region rich in As and Ts). 1s the distribution of mutant and wild type mtDNA uniform within an individual? Deleted mtDNAs may be found not only in muscle but in other tissues such as brain, liver, ludney and b l o ~ d ( ~ The ~ , ~proportion ~). of mtDNAs which are mutant appears to depend on the tissue sampled and the patient’s age. The same patient inay progress from Pearson’s syndrome (a multisystem disease with high levels of rearranged mtDNAs in all tissues) to KSS(23),,where deleted mtDNAs acculmulate in muscle but are present at much lower levels in blood (for example, mutants might comprise 60% of mtDNA in muscle and only 5% in blood). Cell lines which have been cloned from these patients suggest that individual cells within the same tissue may contain differing ratios of mutant to wild type mtDNA(20). Do individual mitochondria contain mixtures of mtDNA types, or only one type‘?If the latter, is there free communication between mitochondria, so that wild type gcnomes may ‘complement’ the mutants? There is some evidence that the latter may occur. Firstly, the classical view of the sausage shaped mitochondrion containing 2-10 copies of the genome has been challenged by morphological studies which put forward an alternative network-like structure (Shoubridge E, personal communication j. Secondly, Wallace and co-workers studied interactions between chloramphenicol (cap)-sensitive and chloramphenicol-resistant mitochondria in a hybrid cell line(21).They showed that the cap-sensitive mitochondria were able to synthesize polypeptides in the presence of cap-resistant mitochondria at cap levels which would

normally be lethal. They concluded that this could only occur if there were cooperation between thc two types of mitochondria. For example, mRNA from the cap-sensitive mitochondria must have been translated using rRNA from the capresistant mitochondria. Thirdly, it is probable that an RNA component of the mitochondrial R N A processing complex is transported into the mitochondrion, so it is possible that this pathway might be able to transport components such as mitochondrial tRNAs. HOWdo these rearrangements match up to the clinical fealures? Surprisingly, Moraes et u Z . ( ~ ) found no clear rclationship of the phenotype either to the region of the mitochondrial genome deleted, the length of mtDNA deleted (1 to 7kb) or the percentage of mitochondrial DNAs which are deleted (which may range from 20 to 80% in muscle). The identical deletion may give rise to varicd phenotype~(~,~’,, mild or severe. Where a number ofbiopsies have been taken from the same individual the proportion of deleted gcnomes increases with time(23), consistent with clinical deterioration. (And where multiple biopsies have been taken from different sites in the same individual, they are not significantly different). The factors affecting this ratio may have parallels in the phenomenon of suppressivity in yeast. Here there appears to be a race between deleted and wild type mtDNA where shorter genomes replicate faster, particularly if they have several origins of Some investigators have reported that deleted genomes are confined to muscle in patients with CPEO(2s),but may be distributed more widely in KSS, consistent with their clinical involvement. Thus, there may be a dosage effect: clinical severity increases with increasing proportion of deleted genomes within an individual or tissue. However, it is likely that the distribution of the mutant mtDNAs relative to wild type is of crucial importance in determining the phenotype. Studies of segments of single muscle fibres have thrown new light on this aspect of the pathogenesis of the deletions. Shoubridge et al.(26)did in situ hybridisation and microdissection and PCR (polymerase chain of single fibre from muscle from patients with known deletions. They confirmed Mita et d ’ s results localising deleted mtDNAs to muscle fibres whose cytochrome oxidase activity was clearly reduced(28). They found that the wild-type mtDNA was present in these muscle fibres at near normal levels. They concluded that the deletions must be ‘functionally dominant’. In other words, the dcleted genomes are not merely harmful because they are non-functional -if so the wild type genomes should function nornially - but are actively harmful. They may impair the wild-type genomes either by removal of some limiting mitochondrial or nuclear product (such as tRNAs or an enzyme involved in replication of mtDNA as discussed above) or by producing some toxic product. The latter would only be possible if there were some interaction of mutant and wild-type genome products. Thus, any of these three alternatives were supported by Shoubridge’s Subsequently, Harding and co-workers have found single fibres with reduced levels of both wildtype and deletcd mtDNA(29)which had reduced cytochrome oxidase activity (CONDM fibres). In these fibres. the cytochrome oxidase deficiency is unlikely to be caused by a

limiting nuclear factor. Very recently, Hayashi ei investigated the consequences of mtDNA deletions by fusing cybrids containing high levels of deleted mtDNA with a mtDNA-free cell line. They identified abnormal translation products corresponding to the junction region at low levcls of mutant. However, at high levels, no translation products were detectable, presumably bccauqe some factor such as tRNAs from the deleted region became limiting. It may be that cytochrome oxidase deficiency can be caused by different mechanisms in the different fibre types.

Duplications of mitochondrial DNA Duplications of nitDNA have been described by two groups(3’.32).To summarise, two sporadic patients with KSS and diabctes mellitus had duplications which were approximately 8 kb in length. A third family had a duplication of intDNA about lOkb in length, detectable by Southern hybridisation in two sibs, and by PCR in the mother. The phenotype in these three families was similar to that found in deletion patients, and once again, the pathophysiology is uncertain. How could a duplication cause a disease? It is possible that an imbalance of normal products from the duplicated regions could be harmful, either, for instance, a relative deficiency of tRNAs from the non-duplicated region or an excess of products from the duplicated region? Alternatively, abnormal gene products of the junction region between duplicated segments could be harmful. For inslance, either abnorinal transcripts or polypeptides might impair mitochondrial function by competing with the normal gene products. Therc is a precedent for this in cytoplasmic male sterility (non-functional pollen(33))in plants. Thc fact that the phenotype of plan@) and lizards(35)does not appear to be arfected by some mitochondrial genome duplications makes the latter slightly more likely. On the other hand, the rearrangements are different in each of the three cases. so any fusion molecules from the junction region would be dissiinilar, yet the phenotypes are obscrvcd to bc similar. Preliminary data also suggest that two further re-arrangcd molecules are present in all These comprise monomers and probably dimers of the duplicated region which would be equivalent to delcted molecules. At least two recombination events and probably a dimerisation must have occurred to generate this family of molecules. Sequential muscle biopsy and cell culture studies suggest that the duplication is only present transiently in muscle, fibroblasts and lymphocytes. It is tempting to speculate that the first recombination generated a duplication, and some of these recombined to a wildtype deleted mtDNA by homologous recombination. The duplicated molecule would thus be an intermediate in the lormation of deletions. There may be parallels between the familks of re-arrangements found in plants, and thc two distinct recombinant molecules described here. It is possible that there are other suhgcnomic intermediates at low levels in patients with both duplications and deletions, caused by a common underlying defect. At the very least the evidence suggests that both the origins and effects of the duplications could be similar to deletions.

Point Mutations of Mitochondria1DNA Point mutations of mtDNA have been descibed in Leber’s Hereditary Optic Neuropathy (LHON)(sJ, two mitochondrial myopathies mercifully abbreviated ‘MELAS’t”) and ‘MERlZF’(”) (‘mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes’ and ‘mitochondrial encephalomyopathy and ragged red fibres’), Leigh’s en~ephalopathy(~~1 and a c a r d i ~ m y o p a l h y ( ~~ While ) . the association of mutation and phenotype is excellent in most of these, therc are a numbcr of other mutations for which the correlations have rather less Bccausc mtDN A is highly polymorphic one would expect that there would be many differenccs from thc reference genome in any complete mtDNA sequenced. After excluding those which do not give risc to an amino acid substitution. there would be several candidates as the causative mutation. I n the abscncc of a clcar biochemical defect, their significance is extremely hard to investigate. Three examples of point mutaions will be discussed briefly with these problems in mind.

Leber’s Hereditary Optic Neuropathy (L HON) LHON causes bilateral blindness in adolescents and young adults. Although strictly maternally inherited, the incidence is much higher in males than females, suggesting that nuclear gene products must also be involved in the phenotype. Two point mutations have now been closely associated with thc phenotype, one in ND4(S)(about 70%(“3)) and the other in NDl(44,45)(15%).Indirect evidence suggests that these are causative but no definite mechanism has yet been identificd. In both cases, this results in a conserved amino acid change which would be expected to alter protein structure and function. Both mutations are present in LHON families and not in controls(8.“) and comparison of mtDNA typing suggests that the ND4 (NADH dehyrogenase subunit 4) mutation occurred independently at least twice in the past, 40-80,000 years ago(“). While some families appear to be homoplasmic for this defect (i.c. having a single population of mtDNA in each individual), others are h e t e r ~p l a s m i c ((two ~ ~ ) or more populations of mtDNA in the same individual), the severity correlating with dosage of abnormal mtDNAs(“) in some cases. However, heteroplasmy i b unable to explain the sex difference, and there is recent data suggesting that an X linked gene may be involved(49).Secondly, although there is a clear defect in complex 1 activity@’) in the NDl mutation, this has been hard to demonstrate in the ND4. Thus, subtleties such as interactions with isoforms specific to the eye must be invoked. Very recently. a number of groups have implicated several other point mutations. There is another NDl mutation found in a single atypical and a number of probable polyniorphisms which occur morc frequently in these patients than in controls(51).Investigators have suggested that and that the final pheiiotype may depend on a complex interaction between several gene products. Alternativly, one of the mutations could be causative, and the others simply polymorphisms which were present on the ancestral mtDNA in which the mutation took place. The absencc of a clear biochemical defect associated with the ND4 mutation will make this problem particularly hard to investigate.

mtDNA in MERRF A point mutacion in tRNA lysine has been found in the majority of patients with MERRFi”) (Myoclonic Epilepsy and ragged-red fibre disease). It is located in the TqC loop of this tRNA, and would be expected to ‘bend’ the tRNA and might result in faulty lysine incorporation. Translation products with the highest number of lysine codons are reduced suggesting that this hypothesis is correct(”). mtDNA in MELAS h4ELAS is associated with a point mutation in tRNA Unlike MERRF, pulse labelling of translation products does not clearly implicate faulty leucine incorporation. As well as possibly influencing tRNA function, the mutation lies within the binding site for the so-called termination factor which is involved in controlling the proportions of two alternativc transcripts(54).Zti vitro, the point mutation reduces binding of the termination factor and results in a lower level of the shorter of two alternate transcripts. However. this is not reflected by different levels in ~ i v and o it is not clear whether either or both of these factors is significant. It seems likely that some of these mutations arc located in elements that regulate functions which have yet to be characterised, such as RNA processing.

Nuclear Genes and Other Factors in Mitochondrial Disease Mutations in many of the nuclear genes encoding proteins involved in mitochondrial biogenesis arc able to cause respi1-atorydefects in yeast(”).Thus, in the early literature on mitochondrial diseases, some investigators asserted that nuclear rather than mitochondrial mutations were likely to be causative as in yeast. These were thought to be responsible for those defects which had tissue specificity or were developmentally regulated: tissue or age specific isoforms were implicated. Since then, the pendulum of opinion has swung away from this view. Heteroplasmy with variation in the ratio of normal to mutant mtDNA in different tissues probably explains the tissue specificity in some of the MMs. Similarly, alterations in the tissue distribution of the proportion of mutant intDNAs with the passage of time explains some of the agc dependency. Mutations of mtDNA have been proposed as candidates underlying a wide range of neurodegenerative disorders including Parkinson’s disease(55),Huntingon’s, Alzheiineds6), Retts syndrome(s7), maternally inherited deafness and even aging(58)as well as all of the mitochondrial myopathies. In practice, the truth is probably somewhere between. The role of the mtDNA mutations in disease may be central or epiphenomenal. For example, it may well be that in some diseases, mtDNA mutations arc secondary to some primary defect e.g. an environmental toxin in Parkinson’s, or to ischaemia in myocardial infarction, or to mutations in the many nuclear genes whose products regulate mtDNA replication, transcription etc. However, it is probable that some of these phenotypes involve interactions between mutations of mtDNA and nuclear genes. The following three sections will discuss two con-

ditions where nuclear genes appear to be causativc, and where the phenotype appcars to depend on the mtDNA abnormality.

Major Re-arrangements of mtDNA: complex There appear to be at least two distinct syndromes where patients are heteroplasmic for several different mtDNA mutations. The mutations appear to arise de n o w in each individual, and rollow an autosomal dominant pattern of inheritance. Zeviani el described families with multiple deletions in tnuscle mitochondria which were not detectable in blood. These aberrations were not dependent on mtDNA type, as they demonstrated that two affected individuals wilhin a pedigree had arisen from distinct mitochondrial lineages. The end points of the deletions were variable, but in all cases the D-loop or control region was conserved. They have recently cloned the gene for a protein which may correspond to the single stranded binding protein (SSB) which has been described in prokaryotes. A mutation in this protein might result in inappropriate unwinding of DNA, and hence its availability for recombination. It would thus be a candidate gene for the presumed nuclear mutation in these cases. Munnich’s group have described a family in which each individual had variable deletions detectable in all tissues investigatcd, but thc predominant mutant varied in different tissues. Mutations were also present in assymptomatic individuals. The variation in tissue distribution suggests that these are somatic mutations which occur early in embryogenesis and become distributed by random segregation. It is possible that there are additional factors such as different kinds of selection are operating in different tissues, as has been postulated to explain the tissue distribution in a patient in whom the common deletion was probably present in the germline(5”. Depletion of mtDNA in Infantile Cytochrome Oxidase Deficiency Profound cytochrome oxidase deficiency is commonly found in patients presenting with MM in infancy. This clinical syndrome may affect the central nervous system, liver and/or ludney in addition to muscle. Indeed, musculo-skeletal symptoms may be a minor feature. Very recently, Moraes et uZ.(In) have demonstrated that in a high proportion of these patients, the affected tissue is depleted in mtDNA using both Southern hybridisation and immunohistochemistry. The remaining nitDNA appears to be structurally normal. In the rare familial cases(10,60), inheritance appears to be autosoinal recessive. This may be cauwd by a defect in mtDNA replication. Similarly, mtDNA is reduced as a sidc elTect of AZT (idovudine) in the treatment of patient5 with AIDS (acquired immunodeficiency syndrome), as this inhibits the gamma polymerase used in mtDNA replication(61). Abnormality of Chaperonins in Mitochondrial Disease After import of nuclear encoded mitochondrial components

into the mahix space, the leader sequence is cleaved off and the molecule is re-folded and assembled. Deficiency of the chaperonin, HSP-60, has been shown to prevent assembly of multimeric components of the respiratory chain in yeast mutants(62).Agsteribbe el u2.(63)have obtained preliminary results suggesting that this may occur in Leigh’s syndrome. Although mRNA for the HSP-60 gene product was present, the protein was not detectable in one patienl with a global dcficit of several respiratory complexes. This suggests an assembly defect caused by th HSP-60 deficiency. It is also likely that other autosomally inherited diseases will be found to cause their effects via the mitochondrial genome. Similarly, there is evidence that non-specific mtDNA damage may occur in Parkinson’s disease and in normal ageing. However, it is not clear whether this is directly involved in producing the phenotype, or is an irrelevant secondary effect.

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