Neur.mtacular Otsordcrs. Vol. I. No. 3. pp. 165-172. 19(11 Printed in Great Bnta|n

0960-8966i91 $3.00 * 0.00 C 1991 Pergamon Press pie

REVIEW ARTICLE NEUROLOGICAL

DISORDERS

DUE TO MUTATIONS

MITOCHONDRIAL

OF THE

GENOME

MASSIMO ZEVIANI a n d STEFANO DIDONATO* *Divisione di Biochimica e Genetica, Istituto Nazionale Neurologico C Besta. 20133 Milano. Italy

(Received 28 March 1991; accepted 6 June 1991)

Abstract--The rapidly expanding list of human diseases due to lesions of mitochondrial DNA includes myopathies, encephalopathies, eardiomyopathies, or various combinations of the latter, leading to multisystemdisorders, which can also affect visceral organs. Five maternally inherited diseases, mainly affecting muscle and brain, are due to point mutations of mitochondrial genes encoding either respiratory chain polypeptides or transfer RNAs. On the other hand, three sporadic entities, Chronic Progressive External Opthalmoplegia, KearnsSayre syndrome, and Pearson's pancreas-bone marrow syndrome, are due to single large-scale deletions of mitochondrial DNA. In addition, multiple deletions are the molecular hallmark of familial encephalomyopathies,inherited as either autosomal dominant or autosomal recessive traits. Finally, tissue-specific depletion of mitochondrial DNA was found in an autosomal recessive disease affecting either muscle, liver, kidney, or a combination of the three. Point mutations and slipped mispairingduring, or impairment of, mitochondrial replication are likely mechanisms involved in the pathogenesisof these lesions. Key words: Mitochondrial DNA, maternal inheritance, mtDNA disease, mtDNA deletions, mtDNA point mutations.

INTRODUCTION

Human cells possess two different genomes: the nuclear DNA, a 3 x 109 base pair (bp) long genome, present in two copies per cell, and the mitoehondrial DNA (mtDNA), a 16,569 bp long genome present in 2-10 copies per mitochondrion and, therefore, in several hundred copies per cell. The sequence and organization of the human mtDNA has long been elucidated. The mtDNA is a circular chromosome formed by two complementary strands: one filament is rich in Guanine nucleotide residues, while the other is rich in Cytosine residues. They are conventionally called Heavy (H) and Light (L) strands, respectively. Mitochondrial DNA encodes for 2 ribosomal RNAs (12 and 16 S rRNA), 22 transfer RNAs (tRNA), and 13 messenger RNAs (mRNA) specifying as many polypeptides of the respiratory chain (7 subunits of complex I, 3 subunits of COX, complex IV, subunits 6 and 8 *Author to whom correspondence should be addressed.

of ATPase, complex V, and apocytochome b of complex III). The gene organization is highly compact: all of the coding sequences are contiguous with each other with no introns. The only non-coding stretch of mtDNA is the displacement-loop (D-loop), a region of about 1000 bp which contains the origin of replication of the Hstrand, and the promoters for L- and H-strand transcription. This region is an important area of interaction of mtDNA with nuclear-encoded proteins and riboproteins regulating mtDNA housekeeping functions [I]. Two additional features characterize the mitochondrial genome. Firstly, its genetic code differs from the universal code, making the nuclear DNA and the mtDNA reciprocally untranslatable. Triplets UGA and AUA specify tryptophan and methionine, instead of termination and isoleucine, respectively; AGA and AGG are termination codons, instead of arginine-specific codons. Secondly, the entire mitochondrial genotype of each individual, either male or female, is exclusively inherited from the mother. This type of non-Mendelian, 165

166

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"maternal" inheritance is due to the fact that, during egg fertilization, the sperm cell contributes only its nuclear DNA to the zygote [I, 2]. More than 50 neurological disorders have been mapped to the human nuclear genome in the last 10 yr [3]. On the other hand, evidence of the contribution of the smaller and simpler mtDNA to human pathology has long remained circumstantial, based on the identification of maternally-inherited clinical syndromes. In 1988, a breakthrough in the field of mtDNA-related disorders was made by a study reporting the association of sporadic human encephalomyopathies with large-scale deletions of mtDNA [4]. Shortly thereafter, a point mutation was linked to Leber's Hereditary Optic Neuroretinopathy (LHON), a maternally-inherited disease [5]. Since then, knowledge of this area of human pathology has so rapidly expanded, that an ever increasing number of commentaries have been dedicated to mtDNA-associated diseases in the major scientific journals [6-9]. Table I reports an updated classification of these disorders, based on clinical, genetic and molecular features.

MATERNALLY INIIERITED mtDNA DISEASES:

POINT MUTATIONS

Inherited optic atrophy with cardiac dysrhythmia and pcripapillary microangiopathy (Leber's Hereditary Optic Neuroretinopathy, LHON) is a rare disease of young adults. Paternal transmission was never reported, suggesting maternal inheritance. Wallace and co-workers [5] proved, by sequencing most of the mtDNA, that a functionally relevant G-to-A transition was present at nucleotide 11778 (G--*A (Ij77x)) of mtDNA in patients and matrilinear relatives of families with LHON. The mutation replaced a highly conserved arginine with histidine at position 340 in subunit 4 of complex I (ND4). Diagnosis of the mutation, based on restriction analysis of the Polymerase Chain Reaction (PCR)-amplified mtDNA region, was made easier by the fact that the G--,A "j77K' transition knocks off a restriction site for the enzyme Sfa NI, present in the wild-type template. Biochemically, the mutation was expected to impair the activity of mitochondrial NADH-dehydrogenase (complex I). Only one paper reported a defect of this enzyme in one family, but the clinical diagnosis of LHON was controversial, and

no molecular-genetic studies were performed [10]. Two questions were still to be explained: firstly, all of the maternal members of LHON families were homoplasmic for the mutation: in other terms, the mitochondrial genotype of both the patients and the healthy maternal relatives was composed by a single, mutated mtDNA species. This finding was difficult to reconcile with the variable penetrance of LHON. Secondly, in-spite of sharing an identical mutation, males develop LHON 20 times more frequently than females. Partial answers to these questions came from two recent studies: one reported that. in several English families, the G---,A q~77X~mutation was heteroplasmic (i.e. mutant and wild-type were co-existing) [I 1]. The other reported that, in families with the G--,A "s77~) mutation, susceptibility to develop LHON was linked to a still unknown gene in locus DXS7 of the X chromosome [12]. Finally, not all of the LHON families showed the G--,A (I1778)mutation, supporting the idea that the disease may be genetically heterogeneous [I I]. This concept is further supported by the discovery of alternative mutations in complex I genes in LHON families that did not harbour the 11778 mutation. However, the pathogenetic significance of these observations requires further confirmation, because the alternative mutations, although more frequently observed in LHON patients, arc also present in a small percentage of the general population, and do not affect highly conserved amino acids [I 3]. Another rare maternally-inherited disease associated with a mtDNA point mutation is a neuromuscular disorder characterized by the combination of retinitis pigmentosa and blindness, seizures, ataxia, proximal neurogenic muscle weakness, sensory neuropathy and dementia [14]. Four females were affected in the maternal lineage of one family. In the symptomatic and asymptomatic family members of matrilinear descendance a mutation was present in position 8993 of mtDNA. The mutation converted a highly conserved leucine to arginine in subunit 6 of mitochondrial ATPase [14]. The mtDNA from muscle biopsies and peripheral blood cells was heteroplasmic for the mutation. A threshold effect was demonstrated by the fact that, in the maternal relatives, overt clinical signs were only present when mutant mtDNA_was over 80% of the total [14]. The molecular basis of a third, devastating maternally-inherited mitochondrial disorder, Myoclonus Epilepsy with Ragged-Red Fibres (MERRF), has been recently elucidated. Clinical

Review Article

features of M E R R F include intention myoclonus, epilepsy, progressive ataxia, muscle weakness and wasting, deafness and dementia. Wallace and co-workers found a positive correlation between severity of the neurological symptoms, reduced activities of respiratory complexes I and IV, and defective aerobic metabolism of individuals, in the maternal members of a M E R R F family [15]. The individual variability of the clinical phenotype was attributed to the uneven segregation of a yet undiscovered, heteroplasmic mutation. Later, the same group identified the MERRF mutation in the mtDNA of a large American pedigree: the mutation was an A-to-G transition at nucleotide 8344 (A---,G~"~) in the pseudouridyl loop of the tRNA ~" gene [16]. This finding was later confirmed in European families, by rapid methods based on the creation of diagnostic restriction sites during PCR-amplification of the mtDNA fragment containing the mutation [17, 18]. Variability of the clinical phenotype in M E R R F [15, 16] seems to be dependent on both the amount and the tissue distribution of mutant mtDNA in each individual. Unequal replicative segregation of heteroplasmic mitochondrial genomes could occur either in the ovum ofduring embryonic organogenesis, and, in mitotically active tissues, throughout the life of the organism. The outbreak of the clinical syndrome will depend on whether the degree of heteroplasmy overcomes a critical threshold, which is dependent on the intrinsic metabolic needs of each tissue. Accordingly, the nervous system, and the skeletal and cardiac muscles are usually the most affected structures, because they strongly depend on mitochondrial respiration for ATP synthesis. Furthermore, these stable tissues are relatively unable to eliminate deleterious mtDNA mutations by negative selection of the most affected cells. Mutations can therefore accumulate and eventually become symptomatic. Since there is a physiological decline of mitochondrial respiration with age, symptoms of brain and muscle dysfunction may not appear at birth, but later in life [2, 15]. The tRNA ~" mutation is present in most of the M E R R F families described, suggesting an etiological relationship between the mutation and the clinical phenotype. The possibility of a common ancestor, leading to a genetic ~'founder'" effect, is unlikely because of the widespread ethnic origins of the M E R R F families investigated. Further evidence for a linkage between specific point mutations and discrete clinical phenotypes

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came from the recent report describing an A-toG transition at nucleotide 3243 in 26 out of 31 unrelated Japanese patients with Mitochondrial Encephalomyopathy, Lactic Acidosis and Strokelike episodes [19] (MELAS). This mutation, which affects a nucleotide position in the dihydrouridine loop of the tRNA ~'"~UUR'mtDNA gene, was considered the cause of the disease because of the high evolutionary conservation of the A ~Jz~3j.By Southern blot analysis, 50-92% of the patients" mtDNA was mutated. Maternal transmission was documented in one family [19]. Two other Japanese patients with M E L A S , reported elsewhere [20], had the same mutation. In addition, another Japanese infant with a MELAS-iike syndrome and fatal cardiomyopathy had a different mutation: an Ato-G transition at conserved position 4317 in the pseudouridine loop of the tRNA ~` gene [21]. The association of different tRNA mutations [16, 19, 21] with different clinical syndromes seems to be rather specific. This is surprising if one considers that these mutations act on the same mitochondrial function, i.e. the ability of mitochondria to translate their own genes. Moreover, the three described tRNA mutations have virtually indistinguishable consequences at the biochemical level, usually producing multiple partial defects of the mtDNA-dependent respiratory complexes. The fll vitro detailed demonstration of the specific effects of each mutation on the mtDNA translation will probably help elucidate the consequences of these lesions on the mitochondrial biogenesis.

SPORADIC mtDNA DELETIONS

In a seminal study on mtDNA-related human pathology, Holt and co-workers [4] analysed the mtDNA of different encephalomyopathies associated with various respiratory enzyme defects. In 9 out of 25 patients, they found two populations o f m t D N A in muscle mitochondria; one population was composed of wild-type mtDNA; the other was missing a large region of the molecule. Single deletions were found in each individual, but among different individuals they varied both by size and localization. Later studies correlated mtDNA deletions with clinical phenotypes. Either large-scale deletions [22-25] or duplications [26] were found in about 50% of the cases of sporadic, adult-onset Chronic Progressive Ophthalmoplegia (CPEO) with raggedred fibres, and in nearly 100% of the cases of

168

Review Article Table I. Molecular m t D N A pathology

Inheritance

Disease

Type of lesion

Proposed mechanism of lesion

Maternal

LHON

mtDNA germ line

[5]

mtDNA germ line

[14]

mtDNA germ line

[16]

mtDNA germ line

[19]

? Trans-acting nucleuscoded factor Trans-acting nucleuscoded factor Trans-acting nucleuscoded factor Slipped-mispairing Slipped-mispairing Slipped-mispairing Slipped-mispairing

[341 [431

Maternal? Autosomal dominant

PEO ~ PEO +

Point mutation at ND 4 gene Point mutation at APTase 6 gene Point mutation at tRNA ~*. Point mutation at t R N A ~ t t R, Deletions Multiple deletions

Autosomal recessive

PEO +

Multiple deletions

Retinopathy and ataxia MERRF MELAS

Fatal lactic acidosis and PEO mtDNA depletion Sporadic

PEO 4Kearns-Sayre s. Pearson's anaemia Parkinson's dis.?

Single deletion Single deletion Single deletion Single deletion

Kearns-Sayre (KS) syndrome. The latter is a multisystem disorder characterized by short stature, CPEO, muscle weakness, pigmentary retinopathy, heart block, cerebellar ataxia, hearing loss, dementia and increased CSF proteins [23]. Onset is in the infantile or juvenile age. In a study performed on 123 patients, Moraes and coworkers [27] showed that mtDNA deletions specifically segregated with CPEO or KS syndromes: no other mitochondrial encephalomyopathy had deletions. Moreover, the activities of complexes I and IV, two respiratory enzymes that contain-mtDNA-encoded polypeptides, were decreased in muscle of CPEO and KS patients with mtDNA deletions, but were normal in "non-deleted" cases of CPEO. Molecular work [27, 28] established that: (I) the percentage of deleted mtDNA was similar in muscles of both CPEO and KS patients; (2) deletions were widely distributed in extramuscular tissues of the multisystem disease (i.e. KS syndrome), and restricted to skeletal muscle in pure CPEO; (3) the proportion of deleted vs wild-type mtDNA in different tissues roughly correlated with the severity of clinical involvement; (4) only one type of deletion was found in each patient and, in the case of multisystemie involvement, the same deletion was present in different tissues; (5) some unrelated patients had an identical deletion; (6) all patients were heteroplasmic; (7) all deletions spanned more than one gene, including mRNA and tRNA genes [27]; (8) almost all of the reported deletions, ranging from 1.3 to 7.6 kb, were localized between the end of the D-loop and the origin of L-strand replication: in only four cases were deletions found in the minor region between the origins of replication [4, 29, 301. Deletions

Ref.

[461 [48] [27] [23] [3 I] [54]

were also found in a non-neurological disease, characterized by pancytopenia and pancreatic dysfunction [31] (Pearson's syndrome). Interestingly, one infant with a mtDNA deletion found in blood cells and muscle, was first diagnosed as Pearson's syndrome, and later developed the typical signs of KS syndrome [32]. The evolution from a tissue-specific (e.g. Pearson's syndrome) to a multisystem disorder (e.g. Kearns-Sayre syndrome) could be explained by the observation that the mutated mitochondrial DNA fraction increases in an agedependent fashion [33]. Most of the cases of KS syndrome, CPEO and Pearson's syndrome associated with single mtDNA deletions were sporadic. Maternal inheritance of the trait was proposed by Ozawa et al. in two familial cases (see Table 1). Single deletions were found in skeletal muscle of a mother and her daughter, both affected by CPEO [34]. However, direct "maternal" transmission of the lesions was made unlikely by the fact that deletions were not identical in the two patients, differing in both size and localization. In a later paper, the same authors described a Japanese woman with CPEO: her mother and grandmother had ptosis also, but they could not be further analysed [35]. In the proband the presence of deleted mtDNA, was not detectable by Southern blot, but was suggested by results of PCR analysis, which indicated the presence of differently deleted mtDNA populations in skeletal muscle (multiple heteroplasmy or pleioplasmy). The authors suggested that also in this family deletions were maternally inherited [35], but the possibility of an autosomal dominant transmission of the trait (see below) was not ruled out.

Revie~v Article

The molecular pathogenesis of mtDNA deletions has been investigated by sequence analysis of the junctional regions in "'deleted" mtDNAs. These studies have demonstrated that most deletions are flanked by direct repeats of variable length [36--39], suggesting that rearrangements of the molecule may ensue by either non-replicative homologous recombination or intrareplicative slipped mispairing through direct repeats [39]. The slipped-mispairing model requires that at some points both direct repeats be present as complementary single strands, thus allowing pairing and recombination to occur. Incidentally, this model could explain the rarity of deletions occurring within the minor region between the origins of replication (see above): this region is not subjected to displacement of single-stranded DNA during synthesis of daughter strand, thus remaining in a permanent double-stranded configuration throughout the entire replication cycle [30]. A particularly frequent deletion, called the "common" deletion after Schon and co-workers [36], occurred across two 13-bp long perfect direct repeats, 4.9 kb apart from each other. The presence of a single mtDNA deletion in each individual can be explained by the clonal amplification of a single mutational event. Furthermore, the fact that most of the observed cases were sporadic indicates that the molecular rearrangement probably occurs as a somatic mutation after egg fertilization. The rapid cell proliferation during embryonic organogenesis will promote mitotic segregation, "loss" of mutant mtDNAs in the extra-foetal structures, and negative selection of respiratory-deficient cells in the rapid-turnover tissues. These phenomena can determine the observed variability in the quantitative distribution of the heteroplasmic mtDNAs in different clinical phenotypes. It is not yet understood, however, why in multisystem disorders, such as KS, in which deleted mtDNAs are virtually ubiquitous, mutations are not transmitted through female gametes to the progeny. One possibility is that the germinal cells containing deleted genomes are not viable for gametogenesis and/or fertilization. The correlation between deletions and clinical and biochemical phenotypes has been investigated by in situ hybridization of muscle mtDNA. Mita and co-workers showed that the ragged-red, COX-depleted fibres of muscle biopsy specimens from KS patients were exclusively composed of deleted mitochondrial genomes [40]. Shoubridge et al. confirmed that

169

deleted genomes were largely predominant in ragged-red fibres, but found virtually normal levels of wild-type mtDNA and mRNAs [41]. In spite of these partially different results, both these studies, as well as in vitro experiments in cultured fibroblasts containing deleted mtDNA [42], proved that ragged-red fibres were competent for mitochondrial transcription but not for translation. These findings suggest that a stalling of mtDNA translation could ensue by either an absolute (Mita et al.) or a relative (Shoubridge et aL) deprivation of the tRNA genes encompassed by deletions. NUCLEUS-DRIVEN MUTATIONS OF mtDNA

A specific disruption of the nucleusmitochondria complementation, which controls mtDNA replication, has been reported in Italian families with autosomal dominant inheritance and mitochondrial disease [43--45]. Patients had CPEO, proximal weakness and wasting, sensorineural hypoacusia, cataract, peripheral neuropathy and, in some cases, were victims of precocious death. Examination of muscle biopsies showed ragged-red fibres, decreased histochemical reaction to COX and neurogenic changes. Biochemically, the activities of respiratory complexes I and IV were decreased to about 50%. Pleioplasmy due to multiple deletions ofmtDNA was detected in the patients' muscles by Southern blot. Most deletions spanned a mtDNA region of several kilobases, between the end of the D-loop and the region encompassing genes for ATPase 8 and 6, and COX I. Sequence analysis of PCR fragments flanking the deletions showed that these occurred across short direct repeats [43], suggesting homologous recombination. Extensive analysis in several individuals, from three unrelated families, demonstrated that the origins of both H-strand and L-strand replication were consistently conserved in deleted mtDNAs, a prerequisite for their propagation [44]. The existence of an autosomal dominant human disease implied a mutation in a nucleusencoded gene. The abnormal product of this gene is supposed to interact, most likely during mtDNA replication, with the mitochondrial genome to cause the accumulation of multiple lesions of the molecule. A mitochondrial disorder, also associated with mtDNA pleioplasmy, was reported independently by Yuzaki and co-workers [46] in a

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Japanese family. Deletions, flanked by direct the activity of complex I in parkinsonian repeats, were localized randomly in the mtDNA patients, and found that the activity of this with a relative sparing of the D-loop region. enzyme was specifically reduced in the substantia Phenotypically, the disease was nearly identical nigra of affected individuals. Subsequent work to that described by Zeviani and co-workers [43] from these [52] and other investigators [53], except that autosomal recessive, instead ofauto- confirmed the enzyme defect, and showed, by somal dominant, transmission was suggested by immunoblotting techniques, that some of the the fact that the two affected brothers were sons mtDNA-encoded subunits of complex I were of unaffected consanguineous parents [47]. decreased in the nigrostriatal region of brains Finally, a new autosomally inherited disease of from parkinsonian patients [53]. Southern blot mtDNA was recently reported by DiMauro and analysis failed to reveal major deletions of co-workers [48]. Two infant sisters had fatal mtDNA in the substantia nigra [52]. However, a mitochondrial myopathy, CPEO and lactic semi-quantitative PCR-amplification o f m t D N A acidosis, and their second cousin died of hepatic regions commonly involved in deletions showed failure associated with mitochondrial abnor- that affected striatai specimens contained more malities in liver. Transmission was autosomal than 5% deleted mtDNA [54]. Only 0-0.5% of recessive. Quantitative Southern analysis mtDNA were deleted in aged- and anatomicalrevealed severe depletion of mtDNA in the matched control samples. The authors suggested available affected tissues from one sister and her that Parkinson's disease might ensue when cousin. Depletion of mtDNA was also found in deleted mitochondrial genomes reach a certain two unrelated patients, one affected by isolated threshold. This conclusion is difficult to reconcile infantile myopathy and the other by myopathy with the observation that the threshold for and nephropathy. The disorder is reminiscent of phenotypic expression of a mtDNA-related the rho ° phenotype, selected as spontaneous disease is usually reached when the mutant mutants in yeast and by prolonged exposure to species largely exceeds 50% of the total mitoethidium bromide [49] in cultured human fibro- chondrial genotype [14, 16, 19]. It is possible, blasts. A mutation in any ofthe proteins involved however, that, in Parkinson's disease, mtDNA either in mtDNA replication or in the control of deletions are concentrated in a specific neuronal mtDNA copy number could result in such a subtype of the striatum. In situ hybridization depletion-mutant phenotype [48]. techniques could help verify this hypothesis. Interestingly, the same group has recently It has been recently suggested that degenreported [50] a "rho-minus" human phenotype, erative diseases of adulthood or senescence may observed in nine AIDS patients under long-term be due to mtDNA mutations accumulating treatment with Zidovudine (AZT), a powerful throughout the life of the individuals [55]. inhibitor of the mitochondrion-specific, gamma- Arguments supporting this hypothesis are the DNA polymerase. Patients developed a des- following: (I) mitochondrial mutations occur at tructive mitochondrial myopathy with ragged- a rate much higher than nuclear mutations; (2) red fibres and proliferation of abnormal mito- unlike nuclear DNA, mtDNA lacks a repair chondria. Amounts of mtDNA were severely mechanism; (3) heteroplasmic segregation of reduced in muscle biopsy specimens of the mtDNA occurs in somatic cells. In agreement patients (up to 78% reduction vs normal adult with the hypothesis are also two additional controls). Depletion of mtDNA was reverted in observations: the age-dependent decline of one patient after discontinuation of the therapy. muscle aerobic metabolism in humans [56] and the reduction of mtDNA transcription in senescent rats [57]. It is not yet clear, however, whether P A R K I N S O N ' S DISEASE AND AGEING the (rare) mutational events observed in senescence can be a cause or rather an effect, with An intracellular metabolite of I-methyl-4- no functional consequences, of ageing. phenylpyridinum, MPP" is a specific toxin of the substantia nigra which causes parkinsonism in A,'knowh,dgem,'nts--The authors are gratefully indebted to primates. MPP ~ is also a strong inhibitor of A R I N , Associazione Italiana per le Ricerche Neurologiche. NADH-Coenzyme Q reductase (complex I). To Milano, Italy, for the generous support of their work. The verify whether a defect of mitochondrial res- skilful work of Cinzia Gellera, Marco Rimoldi, Patrizia Amati, Valeria Tiranti and Diego Lorenzetti (Divisione di piration could be correlated with Parkinson's Biochimica e Genetica. Istituto Nazionale Neurologico) is disease, Shapira and co-workers [51] measured also acknowledged.

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