MHREP$0206

Molecular Human Reproduction vol.3 no.2 pp. 133–148, 1997

Mitochondrial disorders

M.Zeviani1,3,4 and C.Antozzi2 1Department

of Biochemistry and Genetics, 2Neuromuscular Research Department, Istituto Nazionale Neurologico ‘Carlo Besta’, Milan and 3Division of Molecular Medicine, Children’s Hospital Bambino Gesu`, Rome, Italy

4To whom correspondence should be addressed at: Istituto Nazionale Neurologico ‘Carlo Besta’, Via Celoria 11, 20133 Milan, Italy

Mitochondria, the organelles devoted to energy production, have unique genetic features. They possess their own genome encoding several subunits of the respiratory chain, the majority of which are encoded by nuclear DNA, as well as factors involved in replication, transcription and translation of mitochondrial DNA. In the past few years, molecular lesions of mitochondrial DNA have been reported with increasing frequency as a source of human disorders. Several mutations of mitochondrial DNA, either as sporadic large scale rearrangements (deletions, duplications) or maternally-inherited point mutations, have been associated with well defined clinical syndromes. Furthermore, because of the nuclear DNA contribution to the synthesis of respiratory chain enzymes, phenotypes transmitted as Mendelian traits have also been identified and associated with qualitative (multiple deletions) and quantitative (depletion) lesions of the mitochondrial genome. The clinical manifestations of mitochondrial DNA mutations are extremely heterogeneous, ranging from myopathies, encephalomyopathies, cardiopathies, to complex multisystem syndromes. Clinical, morphological, biochemical and molecular genetic data are necessary for diagnosis. The recent advances in genetic studies provide both diagnostic tools and new pathogenetic insights into this rapidly expanding area of human pathology. Key words: human disorders/mitochondrial DNA/respiratory chain

Introduction Mitochondria are double-membrane organelles devoted to energy production. Their unique genetic features have been extensively investigated in recent years and several human diseases have been attributed to pathogenic mutations of mitochondrial DNA (mtDNA). The study of mtDNA as a source of human pathology has remarkably changed the approach to mitochondrial encephalomyopathies (ME), providing pathogenic insights as well as powerful diagnostic tools for the identification and classification of several syndromes. To understand better the biological complexity, and hence the clinical heterogeneity of ME, we shall present first an overview of the molecular features of the mitochondrial genome, of its gene products and their function and of the complex interrelationships between mitochondrial and nuclear DNA (nDNA).

The mitochondrial respiratory chain Mitochondrial respiration is carried out by sequentially ordinated redox reactions that utilize reducing equivalents derived from the oxidative degradation of carbon substrates to convert molecular oxygen to water. These reactions are catalysed by four multiheteromeric enzymes, the respiratory chain complexes I, II, III and IV, embedded in the inner mitochondrial membrane, in close contact with each other and with two small ‘shuttle’ molecules, co-enzyme Q and cytochrome c. © European Society for Human Reproduction and Embryology

The energy liberated in the redox reactions is partially stored as a transmembrane proton gradient, generated by active extrusion of protons from the inner mitochondrial compartment, and ultimately utilized by complex V, the ATP-synthase, to phosphorylate ADP to ATP. The entire process, known as oxidative phosphorylation (OXPHOS), provides most of the ATP in the cell (Tzagoloff, 1982) (Figure 1). The mitochondrial respiratory chain is under a dual genetic control: the nuclear genome and the mitochondrial genome. Most of the mitochondrial proteins, including most of the subunits of the respiratory complexes, are encoded by nDNA genes, and synthesized by cytoplasmic ribosomes, usually as precursors containing an N-terminal extension. The latter serves as a leader peptide that precisely addresses the protein to mitochondria. Import into mitochondria is carried out by a complex, ATP-dependent transport system, and is followed by a series of post-translational modifications, including the cleavage of the leader peptide, that eventually produce a mature, functional protein (Hartl and Neupert, 1990; Neupert et al., 1990). On the other hand, mtDNA is a small but essential structure that retains the genetic information to encode a few crucial components of the respiratory chain, as well as the RNA apparatus that carries out their autochthonous translation (Anderson et al., 1981). The increased dependency of eukaryotic cells upon OXPHOS as the most efficient energy supply and the divergence between the universal and the mitochondrial genetic codes, that presumably blocked the 133

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Figure 1. Schematic representation of the mitochondrial respiratory chain.

massive transfer of genetic information from the proto-mitochondrial genome to the nucleus, are thought to have determined the perdurance of both mitochondria and mitochondrial DNA as stable components of modern eukaryotes (Margulis, 1970).

Sequence and organization of human mitochondrial DNA The human mitochondrial genome is a 16 569 base pair (bp) long circular chromosome composed of double-stranded DNA (Figure 2). Its nucleotide sequence and gene organization have been fully elucidated (Anderson et al., 1981). A strand bias in the G 1 T content produces the separation of the two strands of mtDNA into a heavy (H) and light (L) strand by centrifugation through a density gradient. As shown in Figure 2, human mtDNA contains 37 genes which encode the RNA components of the mitochondrial translational apparatus, i.e. 22 transfer RNA (tRNAs) genes and the 12S and 16S ribosomal RNA (rRNA) genes, as well as 13 polypeptide-encoding genes (mRNAs). All 13 polypeptides are essential components of four of the five complexes that form the mitochondrial OXPHOS pathway. Seven polypeptides, ND1, ND2, ND3, ND4, ND4L, ND5, ND6, are subunits of complex I (NADH-dehydrogenaseubiquinone reductase); cytochrome b is part of complex III (ubiquinol-cytochrome c reductase); COI, COII and COIII are the catalytic subunits of complex IV (cytochrome c oxidase), and ATPase 6 and 8 are subunits of the lipophilic portion of complex V. All the subunits of complex II (succinate dehydrogenase-ubiquinone reductase) are encoded by nDNA (Figure 1) (Attardi, 1986). In order to produce functionally active complexes, the mtDNA-encoded subunits of each complex must interact with several subunits which are encoded by nDNA and then imported into mitochondria from the cytoplasm. The mtDNA has an extraordinarily compact gene organiza134

tion (Figure 2). There are no introns; all of the coding sequences are contiguous with each other, or are separated by only a few nucleotides, and lack significant untranslated flanking regions. There are only two non-coding stretches in mtDNA of functional significance. One is the displacement-loop (D-loop), a region of about 1 kb which contains the origin of replication of the H-strand (OH), and the promoters for L- and H-strand transcription. The other important non-coding sequence is a 30 nucleotide (nt)-long region, positioned at 2/3 of the mtDNA length from the OH, in the leading strand replication sense. This region, which is surrounded by a cluster of five tRNA genes, is able to form a stable hairpin structure, and serves as the origin of replication of the L-strand (OL). The two rRNA genes and most of the polypeptide-encoding genes are flanked by regularly interspersed tRNA genes. The punctuation of the genome with tRNAs is thought to allow the generation of mature RNA species through the action of RNA-processing enzymes at the 59 and 39 ends of the tRNAs (Ojala et al., 1980, 1981; Montoya et al., 1981).

Replication of mtDNA Replication of mtDNA occurs in the mitochondrial matrix, independently from the cell cycle phase and from replication of nDNA. The synthesis of each mtDNA strand proceeds asynchronously from the two widely separated origins of replication. Synthesis of the H-strand, the so-called ‘leading strand’, initiates first, from the origin of replication in the Dloop region (Clayton, 1982). Here, the two complementary parental strands are unwound, and RNA primers are generated by the cleavage of an L-strand promoter transcript. It has been proposed that the cleavage enzyme for primer RNA processing may be a site-specific endoribonuclease RNase [mitochondrial RNA processing (MRP) protein] consisting of an RNA species and a protein component, both encoded by nuclear genes (Chang and Clayton, 1987). Once the RNA priming is

Mitochondrial disorders

Figure 2. Map of human mitochondrial DNA. Outer and inner circles represent the heavy (H) and light (L) strand respectively; HSP 5 H-strand promoter; LSP 5 L-strand promoter; OH 5 origin of H strand replication; OL 5 origin of L strand replication; 12S, 16S 5 mitochondrial ribosomal RNAs; ND1-6 5 complex I subunits; CO I–III 5 complex IV subunits; Cyt. b 5 cytochrome b; single capital letters 5 amino acid-specific tRNA genes.

completed, elongation of the primer RNA is carried out by the mitochondrion-specific gamma-DNA polymerase, to produce a 7S DNA replication-initiation sequence synthesized using the L-strand as template. Most of the 7S DNA molecules are removed and re-synthesized at a much higher rate than replication rate. The functional role of this redundant phenomenonis unknown. When the H-strand has been replicated by 2/3 of its length, the replication origin of the L-strand is exposed as a single-stranded template, allowing the formation of the hairpin structure that is recognized by a specific mtDNA primase. RNA priming on the light-strand origin is followed by DNA elongation of the daughter L-strand around the displaced parental H-strand, until the circle is completed. Completion of the replicative process implies the removal of RNA primers, gap-filling and closure of each circular strand, followed by separation of the catenated pair of circles, and introduction of superhelical turns (Clayton, 1982).

Transcription and cessing of mtDNA

post-transcriptional

(Chang and Clayton, 1984; Clayton, 1984). Once the synthesis of the giant polycistronic transcripts has been completed, release of the functional RNA species (tRNAs, rRNAs and mRNAs) is carried out post-transcriptionally by cleavage of each polycistronic RNA. The strategic distribution of tRNAs or tRNA-like structures along the polycistronic transcripts allows recognition of cleavage sites at the 59 and 39 ends of tRNAs by specific RNA processing enzymes (Ojala et al., 1980). After the tRNAs have been precisely cleaved out of the primary transcript, mRNAs from those reading frames with no termination codon would be left with either U or UA at their 39 ends, in phase with the reading frame. Post-transcriptional polyadenylation of the mRNAs by a poly-A RNA polymerase creates an in-phase UAA termination codon. Shorter poly-A stretches are also added to the 39 end of rRNAs. Finally, the 39 terminal CCA of tRNAs is not encoded in mtDNA, but added post-transcriptionally by a nucleotidyl-transferase activity (Deutscher, 1982).

pro-

Nucleus-encoded genes are usually transcribed individually, through the activation of specific promoters residing in the upstream non-coding regions of each gene. By contrast, both mtDNA strands are transcribed as whole, genome-length polycistronic RNAs, from their own, spatially, and possibly functionally, independent promoters in the D-loop region

Translation of mtDNA The genetic code directing translation of mtDNA differs from the universal genetic code (Anderson et al., 1981). In mammals, UGA encodes tryptophan instead of being a termination codon, AUA encodes methionine instead of isoleucine, and AGA and AGG are termination codons, instead of encoding arginine. The main consequences of this phenomenon have been the 135

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interruption of the gene transfer from the primordial protomitochondrial genome to the nucleus, and the evolution of an in-situ translational apparatus that includes unique rRNA and tRNA encoding genes. Only 22 tRNAs are sufficient for translation of the protein coding sequences of the human mitochondrial genome, due to a more simplified codon– anticodon pairing than that required to read the universal genetic code. In humans eight mitochondrial tRNAs recognize eight codon families with four-fold degeneracy, and 14 recognize the remaining codon pairs. A single tRNAMet occurs in human mtDNA, specifying both methionine and N-formyl methionine. As in prokaryotes, the latter replaces methionine as the initial aminoacid. Moreover, AUA or AUU are sometimes used as initiation codons instead of AUG (Anderson et al., 1981). While the RNA components of the translation apparatus are mtDNA-encoded, the genes encoding the protein factors involved in translation are encoded in the nucleus. These include the aminoacyl-tRNA synthetases, the ribosomal proteins, elongation and termination factors etc. None of these protein gene products has been identified to date.

As mentioned above, the essential first step for mitochondrial replication is the generation of primers for heavy-strand replication deriving from transcripts initiating at the lightstrand promoter. Therefore, by activating mtDNA transcription, mtTFA provides a crucial control point for the maintenance of mtDNA as well. A second important transcriptional control mechanism is carried out by a 34 kDa nDNA gene product, the mitochondrial termination factor, mTERF (Kruse et al., 1989), which causes early H-strand transcription termination by binding to a promoter-independent bidirectional termination site at the 16S rRNA-tRNALeu gene boundary. As few as 13 bp, located entirely within the gene for tRNALeu(UUR), are sufficient to direct termination (Christianson and Clayton, 1988). When mTERF is not bound to the termination site, H-strand transcription can proceed completely, from the tRNAPhe gene 59-end to the tRNAThr gene 39-end, as a giant RNA species. This mechanism provides a 25-fold increase in the synthesis rate of the short H-strand transcript, relative to the full-length Hstrand transcript, thus allowing the production of sufficient amounts of rRNAs for protein translation.

Control mechanisms In order to carry out their function, many nucleus-encoded factors must interact with cis-elements distributed along the mtDNA sequence or the mitochondrial RNA transcripts. A few binding sites in mtDNA are already known, such as those identified in the D-loop, OL and elsewhere. However, given the compactness of mammalian mtDNA, regulatory functions are likely to converge on mtDNA sequences that also retain a more obvious role, being contained, for instance, within the coding region of a gene. An example is the case of the tRNA gene transcripts, serving as cleavage points for transcription processing, or the transcription termination site in the tRNALeu(UUR) gene. Other, as yet unsuspected, mtDNA regions are likely to behave as ‘cryptic’ regulatory elements. Their identification would be important also from a clinical standpoint, because it could perhaps contribute to an explanation of some of the specific pathogenic effects of different mtDNA mutations. In any case, the recent discovery of some of the factors involved in mtDNA replication and transcription have begun to shed light on the detailed mechanisms by which this control is performed.

Transcriptional control Transcription is carried out by a mitochondrial RNA polymerase that has been partially purified and characterized in HeLa cells (Walberg and Clayton, 1983; Shuey and Attardi, 1985). However, the mitochondrial RNA polymerase is not able to recognize selectively the transcription initiation sites. Promoter selection, as well as activation, is conferred on RNA polymerase by a second nDNA gene product, the mitochondrial transcription factor A, mtTFA (Fisher and Clayton, 1985; Parisi and Clayton, 1991). This factor binds to sequences upstream of both Heavy Strand and Light Strand Promoters (HSP, LSP), spanning positions –12 to –39 of the respective transcription initiation sites. 136

Translational control Control mechanisms may occur during translation as well, and may explain the wide variation observed in the translational efficiency of different mtDNA-encoded polypeptides. One possibility is that translation of individual mitochondrial mRNAs is limited by the variable abundance of nuclearencoded subunits belonging to the same respiratory complex. Alternatively, similar to the situation in yeast mitochondria, specific nuclear-encoded factors may modulate the synthesis rate of individual mRNAs of mammalian mitochondria, according to the specific metabolic needs and developmental conditions of differentiated tissues.

Higher control mechanisms Finally, since mtDNA-encoded polypeptides are an integral part of the respiratory chain, their expression must be coordinated with that of nDNA-encoded subunits and with the rate of synthesis of cytochromes and other prosthetic components of the OXPHOS system. Higher control mechanisms must then provide a link between the expression of mtDNA- or nDNAencoded OXPHOS genes to the metabolic needs of different cell types and tissues of the organism. Two important components of this regulatory chain are the nuclear respiratory factors, NRF1 and NRF-2. Human NRF-1 consists of a single polypeptide of 68 kDa. NRF-2 consists of five polypeptides, only one of which has intrinsic DNA-binding ability. The other peptides are likely to provide specific binding properties to the heteromeric complex (Virbasius et al., 1993a,b, 1994). NRF-1 and NRF-2 are powerful activators of a number of OXPHOS-related genes. NRF-1 and NRF-2 specific binding sites are present in the promoter regions of genes encoding cytochrome c, several nDNA-encoded subunits of respiratory complexes III, IV and V, and 5-aminolevulinate synthase, the rate-limiting enzyme in the biosynthesis of heme for respiratory

Mitochondrial disorders

Figure 3. Mitotic segregation: variable distribution of wild-type and mutated mtDNA.

cytochromes. However, NRF-1 and NRF-2 are also transcriptional activators of mtTFA and MRP-RNA genes. Therefore, NRF-1 and NRF-2 provide an important control point linking the expression of several OXPHOS genes with the nuclear gene set that provides the perpetuation and gene expression of the mitochondrial genome. Finally, NRF-1 specific binding sites have been identified in the promoter regions of genes not directly related to OXPHOS, such as eukaryotic initiation factor 2a and tyrosine aminotransferase, suggesting an engagement in the coordination of the respiratory metabolism with other biosynthetic and degradative pathways.

Genetic features of mtDNA The genetics of mtDNA differ from that of nDNA in the following unique properties: (i) the mitochondrial genome is maternally inherited. The mother transmits her oocyte mtDNA to all of her offsprings, and her daughters transmit their mtDNA to the next generation. This is due to the fact that during fertilization, the few mitochondria from the sperm that enter the egg are rapidly eliminated through an unknown mechanism; (ii) mitochondria are polyploid. Each human cell has hundreds of mitochondria, each containing 2–10 mtDNA molecules. At cell division, mitochondria and their genomes are randomly distributed to daughter cells; (iii) the mitochondrial genome has a much faster evolution rate than the nuclear genome. The average number of bp differences between two human mitochondrial genomes is estimated to be from 9.5 to 66. This is explained by the fact that, although the mitochondrial gamma-DNA polymerase may retain a proofreading activity, and certain types of repair enzymes have been identified in mitochondrial fractions, mitochondria lack an efficient DNA repair system, based, for instance, on homologous recombination or removal of pyrimidine dimers. In addition, the mitochondrial genome lacks protective proteins like histones, and is physically associated with the inner mitochondrial membrane,

where highly mutagenic oxygen radicals are generated as by-products of OXPHOS; (iv) normally, the mitochondrial genotype of an individual is composed of a single mtDNA species, a condition known as homoplasmy. However, the intrinsic propensity of mtDNA to mutate randomly can occasionally determine a transitory condition known as heteroplasmy, where the wild-type and the mutant genomes co-exist intracellularly. Because of mitochondrial polyploidy, during mitosis the two mtDNA species are stochastically distributed to subsequent cell generations. Eventually, an intracellular genetic drift known as mitotic segregation determines the separation of the two mitochondrial genomes into two distinct cell lineages, each one containing only one mtDNA type. To explain the rapid segregation observed in vertebrate mitochondrial DNA, despite its high copy number and mutational rate, a model based on a ‘bottleneck’ effect has been proposed to occur during oogenesis and early embryogenesis. During the follicular phase of oogenesis the number of mitochondria is enormously amplified; however, the mtDNA copy number increases to a lesser extent, resulting in a decrease of the mtDNA/mitochondria ratio to near 1. Mitochondria with a reduced mtDNA copy number will then segregate into the dividing cells of the embryo. As a consequence of this mitochondrial partitioning, a very limited number of mtDNA molecules serve to define the cytoplasmic genotype from one generation to the next. High mutation rate, maternal inheritance, mitotic segregation and absence of recombination cooperate to make mutations become fixed, after a transient period of heteroplasmy, as homoplasmic changes in a given maternal lineage. Eventually, a distinct human mitochondrial haplogroup may occur, characterized by the presence of a particular set of mitochondrial polymorphisms. By contrast, deleterious mutations may arise frequently, but are rapidly eliminated by negative selection: therefore, they do not become fixed in any specific mitochondrial haplogroup, but are rather found in different haplogroups, and are often heteroplasmic. Hetero137

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Figure 4. Principal morphological findings of muscle tissue in mitochondrial disorders: (A) ragged red fibres (increased subsarcolemmal staining, modified Gomori trichrome stain); (B) increased subsarcolemmal reactivity (succinate dehydrogenase staining); (C) ultrastructural appearance of abnormal mitochondria with paracrystalline inclusions; (D) COX negative fibres (cytochrome oxidase staining). (Courtesy of Dr Marina Mora.)

plasmy, distribution among different human populations and, of course, specific segregation with the disease, are distinctive features of pathogenic mutations; (v) because of mitotic mtDNA segregation and polyploidy, a threshold effect dictates the phenotypic expression of a mtDNA-associated character (Figure 3). For a given heteroplasmic mutation, only when mutated gene copies accumulate over a certain threshold, the deleterious effects of the mutation will no longer be complemented by the co-existing wild-type mtDNA, and will be expressed phenotypically as a cellular dysfunction leading to disease. In addition, phenotypic expression will depend upon the nature of the mutation, i.e. its intrinsic pathogenicity, its tissue distribution, and the relative reliance of each organ system on the mitochondrial energy supply. In general, the visual and auditory systems, the central and peripheral nervous systems, the heart, muscle, endocrine pancreas, kidney and liver are, in that order, the organs most sensitive to OXPHOS failure. The influence of nuclear genes, the age and sex of the individual, and environmental factors may also play an important, albeit poorly understood, role in the phenotypic expression of mtDNA mutations.

Clinical considerations A wide range of clinical phenotypes has been classified under the heading of mitochondrial encephalomyopathies (DiMauro 138

and Moraes, 1993). Until recently, the diagnosis relied upon clinical features and the morphological finding of the typical ‘ragged red fibres’ (RRF) on light microscopy examination of muscle specimens stained with the modified Gomori trichrome stain (Figure 4). The transformation of muscle fibres into RRF is due to the accumulation of mitochondria, abnormal in number and size, under the sarcolemmal membrane, as demonstrated by ultrastructural studies (Figure 4). A second frequently associated finding is the presence of scattered muscle fibres that appear non-reactive with the cytochrome oxidase histochemical stain (Figure 4). Morphological investigations were followed by biochemical studies that identified several specific defects of the mitochondrial respiratory chain. However, it soon became evident that neither RRF nor biochemical data alone were able to provide a systematic classification of ME. Moreover, the general but still frequently used term ‘mitochondrial myopathy’ was too restrictive in the light of the extreme clinical heterogeneity of these diseases. Since tissues with the highest aerobic demand, such as skeletal muscle, brain, heart, and less frequently liver and kidney, are exquisitely affected, the corresponding phenotypes range from pure myopathies, to encephalomyopathies, cardiomyopathies, and complex multisystem syndromes. The main clinical features observed in patients with ME are reported in Table I. The identification of mutations of mtDNA, first reported in

Mitochondrial disorders

Table I. Clinical features of mitochondrial disorders Neurological manifestations ophthalmoplegia exercise intolerance, fatigue myopathy ataxia seizures myoclonus stroke optic neuropathy sensorineural hearing loss dementia peripheral neuropathy headache dystonia myelopathy Systemic manifestations

cardiomyopathy cardiac conduction defects short stature pigmentary retinopathy cataract metabolic acidosis nausea and vomiting hepatopathy nephropathy intestinal pseudo-obstruction pancytopenia sideroblastic anaemia diabetes mellitus exocrine pancreatic dysfunction hypoparathyroidism

1988 (Holt et al., 1988; Wallace et al., 1988a), has provided the basis for the modern, molecular genetic classification of mitochondrial disorders. On the other hand, patients in whom extensive investigation of mtDNA is non-informative are still classified according to biochemical criteria (DiMauro and Moraes, 1993). Accordingly, mitochondrial disorders can be divided into two main groups: (i) genetically defined defects of mtDNA; and (ii) biochemically-defined defects of the respiratory chain.

Genetically-defined defects of mtDNA According to the molecular and genetic features of the mutation of mtDNA, this group of defects includes clinical syndromes due to: (i) large scale rearrangements of mtDNA; (ii) point mutations of mtDNA; and (iii) Mendelian traits associated with mtDNA lesions.

Large-scale rearrangements of mtDNA These can be either single mtDNA deletions (mtDNAD1) or, more rarely, duplications. Both mutations are heteroplasmic since they coexist with variable amounts of wild-type mtDNA (mtDNAwt) and are most frequently found in patients with Kearns–Sayre syndrome (KSS) and sporadic chronic progressive external ophthalmoplegia (CPEO) (Holt et al., 1988; Zeviani et al., 1988; Moraes et al., 1989; Poulton, 1992). KSS is a sporadic disorder characterized by the invariant triad of: (i) CPEO; (ii) pigmentary retinopathy; and (iii) onset before the age of 20 years, and at least one or more of the following: cerebellar syndrome, heart block and increased

cerebrospinal fluid protein content. The prognosis of the disease is poor and most patients die within the fourth decade of life, even after placement of a pacemaker. Sporadic CPEO is characterized by bilateral ptosis of the eyelid and ophthalmoplegia, frequently associated with variable degrees of proximal muscle weakness and wasting and exercise intolerance. Detailed clinical and molecular genetic studies are necessary to differentiate the disease from autosomal dominant CPEO and CPEO associated with the MELAS mutation (see below). mtDNAD1 have also been reported in Pearson’s bone marrow–pancreas syndrome, a rare disorder of infancy characterized by sideroblastic anaemia with pancytopenia and exocrine pancreatic insufficiency. Interestingly, infants surviving into adolescence may develop the clinical features of KSS (Ro¨tig et al., 1990; McShane et al., 1991). Therefore, the three phenotypes described may represent different expressions of the same molecular defect. mtDNA large scale rearrangements have been described in patients with different phenotypes such as the DIDMOAD syndrome (diabetes insipidus, diabetes mellitus, optic atrophy and deafness) (Ro¨tig et al., 1993; Barrientos et al., 1996), diffuse leukodystrophy (Nakai et al., 1994), and infantile chronic diarrhoea (Cormier-Daire et al., 1994). Although rearranged mtDNA species have been detected in human oocytes, the clinical syndromes associated with large scale mtDNA rearrangements are usually sporadic (Chen et al., 1995). In rare cases, however, maternal transmission of heteroplasmic partial duplications of mtDNA, but not of deletions, has been reported, in association with clinical syndromes such as familial diabetes mellitus and deafness (Ballinger et al., 1992; Dunbar et al., 1993), or proximal tubulopathy, diabetes mellitus and cerebellar ataxia (Ro¨tig et al., 1992). A list of phenotypes associated with large scale rearrangements of mtDNA is reported in Table II.

Molecular features of mtDNA rearrangements Single mtDNAD1 are detected by Southern blot analysis of mtDNA in at least 50% of patients with sporadic CPEO and in nearly 100% of patients with KSS (Zeviani et al., 1988; Moraes et al., 1989). The occurrence of a single mtDNA deletion (or duplication) in each patient suggests that they are produced through the clonal amplification of a single mutational event. As for other mtDNA mutations, mitotic segregation can increase the variability of the tissue distribution of rearranged mtDNAs, hence influencing the clinical phenotype. For instance, mtDNAD1 are found in muscle, but not in rapidturnover tissues of CPEO patients; by contrast, they are usually detected in several extramuscular tissues, including blood cells and fibroblasts, in the more severe KSS (Shanske et al., 1990; Zeviani et al., 1990b), and Pearson’s syndrome. Most of the mtDNA deletions range from 1.3–7.6 kb in length, are localized between the end of the D-loop region and the origin of the Lstrand replication, and all span more than one gene, including both mRNA and tRNA genes (Holt et al., 1989; Moraes et al., 1989). Sequence analysis of mtDNAD1 showed that deletions are flanked by direct repeats of variable length (Schon et al., 139

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Table II. Large scale rearrangements of mtDNA and associated phenotypes Phenotype

mtDNA mutation

Inheritance

Reference

KSS

Large scale deletion Large scale duplication Large scale deletion Large scale deletion Deletion-duplication Large scale duplication Large scale deletion Large scale duplication Large scale deletion Deletion-duplication 260 bp duplication Multiple deletions Multiple deletions Multiple deletions Multiple deletions Multiple deletions Multiple deletions Multiple deletions Multiple deletions Severe depletion Severe depletion Severe depletion Partial depletion

Sporadic

Zeviani et al., 1988 Poulton et al., 1992 Holt et al., 1988 Ro¨tig et al., 1990 Superti-Furga et al., 1993 Dunbar et al., 1993 Ro¨tig et al., 1993 Ro¨tig et al., 1992 Nakai et al., 1994 Cormier-Daire et al., 1994 Manfredi et al., 1995 Zeviani et al., 1989 Ohno et al., 1991 Yuzaki et al., 1991 Casademont et al., 1994 Cormier et al., 1991 Suomalainen et al., 1992 Hirano et al., 1994 Takei et al., 1995 Moraes et al., 1991 Moraes et al., 1991 Moraes et al., 1991 Tritschler et al., 1992

CPEO Pearson’s syndrome Diabetes, deafness Diabetes, deafness, optic atrophy Tubulopathy, diabetes, ataxia Ataxia, leukodystrophy Chronic diarrhoea, villous atrophy Myopathy Familial CPEO Familial recurrent myoglobinuria Myopathy Sideroblastic anaemia, myopathy Encephalomyopathy Familial cardiomyopathy MNGIE Hypertrophic cardiomyopathy Fatal infantile myopathy Fatal infantile hepatopathy Myopathy, nephropathy Myopathy of childhood

Sporadic Sporadic Sporadic Maternal Sporadic Maternal Sporadic Sporadic Sporadic AD AR AR AR AD AD AR Sporadic AR AR AR AR

KSS 5 Kearns–Sayre syndrome; CPEO 5 sporadic chronic progressive external ophthalmoplegia; MNGIE 5 mitochondrial neurogastrointestinal encephalomyopathy syndrome; AR 5 autosomal recessive; AD 5 autosomal dominant.

1989). The latter feature suggests that mtDNA rearrangements can occur through a mechanism of slipped-mispairing of the single mtDNA strands during replication (Shoffner et al., 1989). From a functional point of view, the loss of tRNA genes contained in the deletion makes the mtDNAD1 species translationally incompetent. Therefore, translation of these genomes can take place only through the complementation by wild-type mtDNA (mtDNAwt) contained in the same organelle. The relative distribution of mtDNAD1 versus mtDNAwt genomes can thus influence dramatically the functional consequence of the mutation. For instance, studies performed in cybrids obtained by introducing variable amounts of mtDNAwt and mtDNAD1 into p° cells (i.e. cells deprived of mtDNA) demonstrated that mtDNA translation is abolished when the relative proportion of mtDNAD1 increases above 60% (Hayashi et al., 1991). Experiments of in-situ hybridization and immunostaining in muscle have demonstrated that RRF contain an overwhelming proportion of mtDNAD1 molecules and that mtDNA translation of mtDNA does not occur (Mita et al., 1989; Nakase et al., 1990; Shoubridge et al., 1990).

Point mutations of mtDNA Point mutations of mtDNA are usually maternally inherited, and can occur in mRNA, tRNA or rRNA genes. Since mtDNA has a very high mutational rate, pathogenic mutations should fulfil the following criteria: (i) high phylogenetic conservation of the mutated nucleotide (or amino acid); (ii) segregation of the mutation with the clinical phenotype; and (iii) correlation between the severity of the clinical and biochemical phenotype and the degree of mtDNA heteroplasmy (if present). An updated list of point mutations of mtDNA and associated phenotypes is reported in Table III. We will discuss here the most frequently observed syndromes. 140

Clinical syndromes associated with point mutations of mtDNA Myoclonus epilepsy with ragged-red fibres (MERRF) is a devastating, maternally-inherited neuromuscular disorder characterized by myoclonus or myoclonus epilepsy, muscle weakness and wasting, cerebellar ataxia, deafness and dementia (Wallace et al., 1988b). The most commonly observed mutation of mtDNA associated with MERRF is an A→G transition at nt 8344 in the tRNALys gene (Shoffner et al., 1990). Extensive clinical, biochemical and molecular investigation of large pedigrees showed a positive correlation between the severity of the disease, age at onset, mtDNA heteroplasmy and reduced activity of respiratory chain complexes I and IV in skeletal muscle. The great majority of patients has been associated with the 8344 transition but genetic heterogeneity occurs in MERRF syndrome. A second mutation has been reported in the same gene, at position 8356 (Silvestri et al., 1992; Zeviani et al., 1993) and, recently, a T→C mutation at position 7512 involving the tRNASer(UCN) has been observed in a family with a MELAS/MERRF overlap syndrome (Nakamura et al., 1995). A significant phenotypic heterogeneity has been observed within families with MERRF in which signs of adult onset-myopathy can be variably combined with a myoclonic encephalopathy. Even though the genotype-phenotype correlation between MERRF syndrome and the 8344 mutation is tighter than that of other mutations, the A8344G transition has also been reported in phenotypes as different as Leigh’s syndrome, myoclonus or myopathy with truncal lipomas and myopathy alone (Hammans et al., 1993; Silvestri et al., 1993). In any case, molecular investigation of mtDNA can be of considerable help in the differential diagnosis of MERRF from other progressive myoclonus epilepsies (such as the Unverricht–Lundborg disease and the Ramsay–Hunt syndrome)

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Table III. Point mutations of mtDNA and associated phenotypes Phenotype

mtDNA mutation

Gene location

Reference

CPEO–CPEO Plus

nt 3243 A→G nt 3256 C→T nt 5692 A→G nt 5703 A→G nt 12311 T→C nt 3251 A→G nt 8344 A→G nt 8356 T→C nt 8356 T→C nt 7512 T→C nt 7472 insertion nt 3243 A→G nt3252 A→G nt 3271 T→C nt 3291 T→C nt 9957 T→C nt 3260 A→G nt 8993 T→G nt 8993 T→G nt 8993 T→C

tRNALeu(UUR) tRNALeu(UUR) tRNAAsn tRNAAsn tRNALeu(CUN) tRNALeu(UUR) tRNALys tRNALys tRNALys tRNASer(UCN) tRNASer(UCN) tRNALeu(UUR) tRNALeu(UUR) tRNALeu(UUR) tRNALeu(UUR) COX subunit III tRNALeu(UUR) ATPase 6 ATPase 6 ATPase 6

Hammans et al., 1991 Moraes et al., 1993 Seibel et al., 1994 Moraes et al., 1993 Hattori et al., 1994 Sweeney et al., 1993 Shoffner et al., 1990 Silvestri et al., 1992 Zeviani et al., 1993 Nakamura et al., 1995 Tiranti et al., 1995a Goto et al., 1990 Morten et al., 1993 Goto et al., 1991 Goto et al., 1994 Manfredi et al., 1995 Zeviani et al., 1991 Holt et al., 1990 Tatuch et al., 1992 de Vries et al., 1993

nt 3460 G→A nt 11778 G→A nt 14484 T→C nt 3394 T→C nt 4160 T→C nt 4216 T→C nt 4917 A→G nt 5244 G→A nt 7444 G→A nt 9101 T→C nt 9438 G→A nt 9804 G→A nt 13708 G→A nt 15257 G→A nt 15812 G→A nt 14459 G→A nt 11696 A→G nt 14596 T→A nt 3250 T→C nt 3302 A→G nt 15590 C→T nt 3303 C→T nt 9997 T→C nt 4300 A→G nt 4269 A→G nt 4320 (–) T 15 bp microdeletion nt 15923 A→G nt 9176 T→C nt 5549 G→A nt 3243 A→G nt 14709 T→C nt 7445 T→C nt 1555 A→G single nt pair deletion

ND1 ND4 ND6 ND1 ND1 ND1 ND2 ND2 COX I ATPase COX III COX III ND5 Cytochrome b Cytochrome b ND6 ND4 ND6 tRNALeu(UUR) tRNALeu(UUR) tRNAPro tRNALeu(UUR) tRNAGly tRNAIle tRNAIle tRNAIle COX III tRNAThr ATPase 6 tRNATry tRNALeu(UUR) tRNAGlu tRNASer(UCN) 12S rRNA tRNALeu(UUR)

Huoponen et al. 1991 Wallace et al., 1988 Johns et al., 1992 Harding and Sweeney, 1994

CPEO, sudden death MERRF MERRF–MELAS Myoclonus, ataxia, hearing loss MELAS

MIMyCa NARP NARP–MILS LHON Primary mutations Secondary mutations

LHON, dystonia LHON, hereditary spastic dystonia Myopathy Cardiomyopathy

Recurrent myoglobinuria Fatal multisystem syndrome Bilateral striatal necrosis Dementia, chorea Diabetes, deafness Diabetes, myopathy Sensorineural deafness Aminoglycoside-induced deafness Encephalomyopathy

Jun et al., 1994 De Vries et al., 1996 Goto et al., 1992 Bindoff et al., 1993 Moraes et al., 1993 Silvestri et al., 1994 Merante et al., 1994 Casali et al., 1995 Taniike et al., 1992 Santorelli et al., 1995 Keightley et al., 1996 Yoon et al., 1991 Thyagarajan et al., 1995 Nelson et al., 1995 Van den Ouweland et al., 1992 Hao et al., 1995 Reid et al., 1994 Prezant et al., 1993 Shoffner et al., 1995

CPEO 5 sporadic chronic progressive external ophthalmoplegia; MERRF 5 myoclonus epilepsy with ragged-red fibres; MELAS 5 mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; MIMyCa 5 maternally-inherited myopathy and cardiomyopathy; NARP 5 neuropathy, ataxia and retinitis pigmentosa; MILS 5 maternally-inherited Leigh’s syndrome; LHON 5 Leber’s hereditary optic neuroretinopathy.

that can be clinically indistinguishable from MERRF at least at the earliest stages of the disease when myoclonic epilepsy and ataxia are the prominent features (Berkovic et al., 1993; Franceschetti et al., 1993). Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is defined by the following obligatory criteria: (i) stroke-like episodes [confirmed by focal brain lesions by computerized tomography (CT) or magnetic

resonance imaging (MRI) and more often localized in the parieto-occipital lobes]; (ii) lactic acidosis and/or presence of RRF in the muscle biopsy. Elevated lactate concentrations have also been reported by proton magnetic resonance spectroscopy (Barkovich et al., 1993; Matthews et al., 1993; Castillo et al., 1995). Other signs of central nervous system involvement occur frequently in MELAS, including dementia, recurrent headache and vomiting, focal or generalized seizures, and 141

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deafness (Hirano et al., 1992). The pathogenesis of the strokelike episodes is uncertain but a mitochondrial dysfunction in blood vessels has been postulated. The walls of intramuscular arteries show large granular deposits with strong succinate dehydrogenase (SDH) activity (Hasegawa et al., 1991) due to marked proliferation of predominantly mutant mitochondria (Tokunaga et al., 1994). Morphological signs of abnormal mitochondrial proliferation were also noted in the endothelium of endomyocardial biopsy specimens from MELAS patients with cardiomyopathy (Sato et al., 1994b). MELAS was first associated with a heteroplasmic point mutation in the tRNALeu(UUR), an A→G transition at position 3243 (Goto et al., 1990). As in MERRF syndrome, genetic heterogenity occurs and other point mutations involving positions 3271, 3252, 3256 and 3291 in the tRNALeu(UUR) gene (Goto et al., 1991, 1994; Morten et al., 1993; Sato et al., 1994a) and position 9957 in cytochrome oxidase III gene have been reported (Manfredi et al., 1995a). The genotype– phenotype correlation of the 3243 mutation is rather loose since the observed clinical manifestations are not limited to the full-blown MELAS syndrome. For instance, the A3243G mutation has been detected in some patients with CPEO, myopathy alone, and in pedigrees with maternally-inherited diabetes mellitus and deafness (Ciafaloni et al., 1992; Reardon et al., 1992; Moraes et al., 1993a,b; Mosewich et al., 1993; Mariotti et al., 1994; Hammans et al., 1995). Neuropathy, ataxia and retinitis pigmentosa (NARP) is a rare syndrome characterized by maternal inheritance, developmental delay, sensory neuropathy, proximal neurogenic muscle weakness, retinitis pigmentosa, ataxia, seizures and dementia. RRF fibres are absent in the muscle biopsy. A heteroplasmic T→G transition at position 8993 in the ATPase 6 subunit gene was reported (Holt et al., 1990). The degree of heteroplasmy was correlated with the severity of the disease. Further studies identified the same mutation in patients showing the clinical, neuroradiological and neuropathological findings of Leigh’s syndrome (hence called MILS, maternally-inherited Leigh’s syndrome) when the percentage of mutant mtDNA is .95% (Tatuch et al., 1992). Interestingly, the two phenotypes of NARP and Leigh’s syndrome may coexist in the same family. A second transition (T8993C) has also been described (de Vries et al., 1993). These findings imply that molecular studies of mtDNA should always be carried out in patients with Leigh’s syndrome not associated with cytochrome c oxidase deficiency (see under defects of the respiratory chain). An impairment of ATP synthesis has been reported in a patient harbouring the T8993G mutation causing instability and altered assembly of the enzyme complex (Houstek et al., 1995). Leber’s hereditary optic neuroretinopathy (LHON) was the first human disorder linked to point mutations of mtDNA (Wallace et al., 1988a; Newman et al., 1991). Clinically, the disease is characterized by bilateral, acute or subacute loss of central vision due to optic atrophy. The typical funduscopic finding in the acute stage of the disease is a peripapillary microangiopathy. The visual defect is usually the only clinical feature. However, it can occasionally be associated with cardiac conduction abnormalities (pre-excitation syndrome), peripheral neuropathy and/or ataxia. Penetrance of LHON is variable, 142

with male predominance. The onset is usually in the second and third decade. LHON has been linked to several point mutations of mtDNA. Three mutations are considered pathogenic (hence called primary mutations), as they have never been found in non-LHON families. They are located at positions 11778 (subunit I of complex I, ND1), 3460 (ND1), and 14484 (subunit 6 of complex I, ND6), respectively (Harding and Sweeney, 1994). Detailed clinical and genetic studies of LHON patients harbouring primary mutations report a poor prognosis for vision (Harding et al., 1995; Riordan-Eva et al., 1995). The 14484 mutation seems to be linked to a relatively mild course, compared to the 3460 or 11778 mutations, the latter being associated with the worst visual outcome. A positive correlation between partial visual recovery and onset before the age of 20 years has also been observed. Interestingly, a multiple sclerosis-like disease has been described in some female patients with the 11778 mutation (Harding et al., 1992; KellarWood et al., 1994). However, whether the presence of the LHON mutations can trigger or worsen the course of multiple sclerosis remains an interesting but as yet unproven hypothesis. Primary mutations have not been detected in normal controls while several other point mutations were reported in a small percentage of the general population (hence called secondary mutations). The pathogenetic role of the latter mutations is unclear. Several aspects of LHON remain unexplained. The observed mutations are usually homoplasmic in all the maternal relatives of LHON pedigrees, regardless of their clinical status. Moreover, unequivocal data on the biochemical consequences of the reported mutations are still lacking. Therefore, other factors are likely to contribute to the variable expressivity of the disease, including the later onset in females, the male predominance, and the selective involvement of the optic nerve. The influence of an X-linked nuclear factor on the expression of an mtDNA mutation has been postulated (Bu and Rotter, 1991; Vilkki et al., 1991), but not confirmed (Sweeney et al., 1991, 1992). LHON has been reported in association with dystonia. Symmetrical basal ganglia abnormalities were found by brain imaging (Novotny et al., 1986). The presence of a pathogenic G→A transition at nt 14459 in the ND6 gene (complex I) has been reported in three independent LHON-dystonia pedigrees (Jun et al., 1994; Shoffner et al., 1995b). Moreover, a heteroplasmic A→G transition at nt 11696 in the ND4 gene and a homoplasmic T→A transition at nt 14596 in the ND6 gene were associated with low complex I activity in a large Dutch family with LHON complicated by hereditary spastic dystonia (de Vries et al., 1996). Several other point mutations of mtDNA have been detected in single patients or in pedigrees affected with different phenotypes (Table III). For instance, maternally-inherited myopathy and cardiomyopathy (MIMyCa) was first described in an Italian pedigree affected by exercise intolerance, proximal muscle weakness, increased blood lactate production at rest and during exercise, and impaired cardiac ejection fraction. The most severely affected patient had a severe myopathy, hypertrophic cardiomyopathy and the Wolff–Parkinson–White syndrome (Zeviani et al., 1991). Genetic studies revealed the

Mitochondrial disorders

presence of a heteroplasmic A→G transition at nt 3260 in the tRNALeu(UUR) gene. A positive correlation was found between the degree of mtDNA heteroplasmy, oxygen consumption in vivo, cardiac performance during exercise, and the activities of the respiratory chain enzymes in vitro. A second pedigree has been reported by Sweeney et al. (1993a). Interestingly, the mutations leading to MIMyCa and MELAS, as well as several other mutations associated with myopathies or cardiomyopathies are concentrated on the same tRNALeu(UUR) gene. The reasons for the high frequency of pathogenic mutations in this hot spot are unclear (Moraes et al., 1993b). Finally, a microdeletion of 15 bp in the mtDNA-encoded cytochrome c oxidase (COX) subunit III gene has been detected in a patient with recurrent myoglobinuria and severe isolated COX deficiency in skeletal muscle. This heteroplasmic mutation is the first genetic defect documented in a component of COX, and it is suspected to interfere with the assembly and stability of the enzyme complex (Keightley et al., 1996).

Cellular and molecular pathogenesis of point mutations of mtDNA The relationship between the clinical presentation, the underlying biochemical defect and molecular genetic findings is still unclear. Point mutations involving structural genes of mtDNA (i.e. LHON and NARP mutations) are not associated with RRF. They should impair the specific activity of the corresponding enzyme complex but evidence of this is still limited. Interesting data are available from studies on muscle tissue from patients affected with point mutations involving tRNA genes. These patients usually have RRF and biochemical studies in muscle show multiple defects of the respiratory chain, most frequently complexes I and IV. Point mutations involving tRNA genes cause a reduced availability of functional tRNAs that impairs the overall mitochondrial protein synthesis. The effect of point mutations has been investigated in vitro using human cell lines deprived of mtDNA (p° cells) and repopulated with mutant mtDNA to obtain cybrids. Studies performed in cybrids containing mtDNA with the MELAS, MERRF and MIMyCa mutations showed that mitochondrial protein synthesis and respiration are markedly reduced above a threshold of 80– 90% of mutant mtDNA (Chomyn et al., 1991, 1992; King et al., 1992; Yoneda et al., 1992, 1994; Mariotti et al., 1994). Identical abnormalities of mitochondrial protein synthesis were shown in cybrids harbouring either the A8344G or the T8356C ‘MERRF’ mutations, suggesting that different mutations within the same gene, associated with the same phenotype, are able to determine similar defects at the cellular level (Masucci et al., 1995). Moreover, a specific functional defect has been persuasively demonstrated in the case of the A8344G MERRF mutation, where aminoacylation of tRNALys is impaired, leading to premature translation termination at lysine codons (Enriquez et al., 1995). Mendelian traits associated with mtDNA lesions The presence of mtDNA abnormalities inherited as Mendelian traits indicates the existence of mutations in nuclear genes ultimately affecting the structural integrity of the mitochondrial genome. An attractive hypothesis is that the responsible genes

are involved in the nuclear control of mitochondrial biogenesis or maintenance. Multiple deletions of mtDNA were first reported (Zeviani et al., 1989, 1990a,b) in several Italian pedigrees affected with autosomal dominant chronic progressive external ophthalmoplegia (AD-CPEO). The main features of the disease are adultonset CPEO, proximal muscle weakness and wasting, vestibular areflexia, cataracts, ataxia, and sensory-motor peripheral neuropathy. Muscle morphology shows RRF and COX-depleted fibres together with neurogenic changes. The activities of mtDNA-encoded respiratory chain complexes are reduced in muscle. Southern blot analysis of mtDNA from muscle shows the presence of multiple heteroplasmic deletions of mtDNA, that are individually similar to the single deletions responsible for sporadic KSS or CPEO. Linkage studies have recently demonstrated the genetic heterogeneity of the AD-CPEO trait: one disease locus, specific to a large Finnish family, has been assigned to chromosome 10q 23.3–24.3 (Suomalainen et al., 1995), while linkage on chromosome 3p 14.1–21.2 has been found for three Italian pedigrees (Kaukonen et al., 1996), and evidence of at least a third locus has been provided by the absence of linkage to both loci in yet other AD-CPEO families. Multiple deletions of mtDNA have also been reported in other familial or sporadic phenotypes including sporadic CPEO cases, isolated recurrent myoglobinuria (Ohno et al., 1991), MNGIE syndrome (mitochondrial neurogastrointestinal encephalomyopathy) (Hirano et al., 1994), sideroblastic anaemia and mitochondrial myopathy (Casademont et al., 1994), periodic paralysis (Prelle et al., 1993), mitochondrial myopathy and hypertrophic cardiomyopathy (Takei et al., 1995) and inclusion body myositis (Oldfors et al., 1993). Depletion of mtDNA is an autosomal recessive tissue specific disorder whose clinical manifestations fall into three groups: (i) a fatal infantile congenital myopathy with or without a DeToni–Fanconi renal syndrome; (ii) a fatal infantile hepatopathy leading to rapidly progressive liver failure; and (iii) a late infantile or childhood myopathy, with onset after 1 year of age, characterized by a progressive myopathy causing respiratory failure and death by 3 years of age. The presence of affected siblings born from healthy parents suggested an autosomal recessive mode of inheritance, possibly affecting a nuclear gene involved in the control of the mtDNA copy number. Southern blot analysis is diagnostic, demonstrating the severe reduction of mtDNA in affected tissues (up to 98% in the most severe forms) (Moraes et al., 1991; Tritschler et al., 1992). Mutations in candidate genes such as mitochondrial transcription factor A (mtTFA) and mitochondrial singlestranded DNA binding protein (mtSSB) genes were ruled out in one patient (Mariotti et al., 1995b). An acquired form of mtDNA depletion has been reported in patients with AIDS treated with zidovudine, probably due to drug-induced inhibition of the mitochondrial gamma-DNA polymerase (Dalakas et al., 1990; Herzberg et al., 1992).

Biochemically-defined defects of the respiratory chain The observation of familial cases with Mendelian inheritance and severe isolated defects of the respiratory-chain complexes, not associated with mtDNA lesions, suggests the presence of 143

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mutations of nuclear genes as a source of respiratory chain deficiencies. However, no mutations in nuclear genes related to the respiratory chain have been reported to date. The only exception was a point mutation in the nuclear-encoded flavoprotein subunit gene of succinate dehydrogenase that was recently found in two siblings affected with Leigh syndrome associated with deficiency of complex II (Bourgeron et al., 1995). Therefore, for those mitochondrial disorders that still lack a molecular genetic definition, the classification is based on biochemical criteria only (DiMauro and Moraes, 1993). The clinical presentation of defects of the respiratory chain is heterogeneous, with onset ranging from neonatal to adult life. The clinical features include fatal infantile multisystem syndromes, encephalomyopathies, or isolated myopathies sometimes associated with cardiopathies. In paediatric patients the most frequent clinical features are severe psychomotor delay, generalized hypotonia, lactic acidosis and signs of cardiorespiratory failure. Patients with later onset usually show signs of myopathy associated with variable involvement of the central nervous system (CNS, e.g. ataxia, hearing loss, seizures, polyneuropathy, pigmentary retinopathy, and rarely movement disorders). Other patients complain only of muscle weakness and/or wasting with exercise intolerance. Defects of complex IV (cytochrome c oxidase) are the best known and probably the most frequent. Two clinical presentations have been described, the myopathic and the encephalomyopathic forms. Fatal infantile myopathy is characterized by severe muscle weakness, lactic acidosis and ventilatory insufficiency causing death before 1 year of age, associated with DeToni–Fanconi syndrome in some cases. This form must be differentiated from a reversible (benign) form, initially indistinguishable from the former, characterized by a progressive spontaneous improvement with remission by the age of 3 years (Zeviani et al., 1987; Tritschler et al., 1991). The encephalomyopathic form (Leigh’s syndrome or subacute necrotizing encephalomyopathy) is characterized by the predominant involvement of CNS. Affected infants show severe psychomotor delay, cerebellar and pyramidal signs, dystonia, seizures, respiratory abnormalities, incoordination of ocular movements and recurrent vomiting. Focal symmetrical lesions have been found in the brainstem, thalamus and posterior columns of the spinal chord, as clearly demonstrated by MRI (Van Coster et al., 1991). Morphological studies do not show RRF while the absence of reactivity for cytochrome c oxidase is found in most cases. Biochemically, the majority of patients present a severe defect of complex IV in muscle. Less frequently, a defect of pyruvate dehydrogenase or complex I of the respiratory chain has been found (Rahman et al., 1996). In some children with Leigh’s syndrome without cytochrome c oxidase or pyruvate dehydrogenase deficiency, the disease has been associated with a point mutation at nt 8993 of mtDNA (see above under NARP). The nuclear gene origin of the COX defect in Leigh’s syndrome has recently been demonstrated by the re-expression of the biochemical phenotype in cybrids composed of a nuclear gene complement derived from a patient and a mitochondrial gene complement derived from control cells (Tiranti et al., 1995b). 144

Systemic manifestations of mtDNA defects Clinical studies on mitochondrial disorders have drawn considerable attention to their neurological manifestations. However, systemic manifestations of mtDNA disorders must not be overlooked since in some cases they represent the main clinical findings and can drive towards the correct diagnosis. The main systemic manifestations observed in ME are listed in Table I. Cardiac manifestations are relatively common and include conduction defects (such as heart block and the Wolff– Parkinson–White syndrome) and hypertrophic or dilated cardiomyopathies. Signs of cardiomyopathy have been frequently reported in patients with the 3243 MELAS mutation, in which cardiac findings may be the presenting feature (Hiruta et al., 1995). A heteroplasmic T→C transition at nt 9997 in the mitochondrial tRNA(gly) has been detected in a pedigree with maternally-inherited hypertrophic cardiomyopathy (Merante et al., 1994). Endocrine manifestations have also been reported, including diabetes mellitus (DM), hypoparathyroidism and hypogonadism. Families in which maternally-inherited DM was the sole or the predominant clinical feature have been described in association with the A3243G MELAS mutation (van den Ouweland et al., 1992; Reardon et al., 1992), and mtDNA duplications (Ballinger et al., 1992; Dunbar et al., 1993). In general, DM is a frequent complication of mitochondrial encephalomyopathies, and it is probably due to failure of the energy-dependent process controlling insulin secretion in the β-cells of pancreatic islets. Finally, a strong association has been observed between the A8344G ‘MERRF’ mutation and the presence of multiple, symmetric lipomas, and in some families multiple lipomas may be the sole manifestation of this mutation (Holme et al., 1993). Mitochondria and the ageing process Free radicals, as normal by-products of OXPHOS, can be harmful compounds when accumulated in excessive amounts, leading to damage of membranes, proteins and DNA. The consequences of oxidant leakage are usually limited by scavenging enzymes and other molecules with antioxidant activity. The ageing process is associated with a gradual decline of tissue function, and a progressive reduction of respiratory chain activities, particularly of complex I, has been documented to occur with age (Trounce et al., 1989; Luft, 1994; Shapira, 1994). mtDNA is particularly susceptible to the toxic effects of free radicals since it lacks an efficient repair mechanism and is not protected by histones. A pathogenic role has been attributed to oxygen radicals in several neurodegenerative disorders and ageing. In this regard, it has been hypothesized that the accumulation of mtDNA lesions may lead to a reduced OXPHOS activity, increasing free radical production and hence oxidative stress. Indeed, accumulation of mtDNA deletions with age has been observed in several tissues by PCR amplification (Cortopassi et al., 1990, 1992; Hattori et al., 1991; Cooper et al., 1992), but the amount of mutated mtDNA is very small compared with normal mtDNA. Therefore, the low levels of mutated mtDNA alone are unlikely to be the cause of reduced respiratory function of ageing tissues.

Mitochondrial disorders

Mitochondria and the reproductive system Recently, mitochondrial dysfunction has been considered as a factor possibly implicated in infertility (Cummins et al., 1994). Mitochondrial ATP is thought to be essential for sperm motility. Ultrastructural abnormalities of mitochondria have been observed in asthenozoospermic subjects compared to controls, suggesting a link between reduced energy production and poor sperm function (Mundy et al., 1995). Indeed, sperm motility was reduced and mitochondria were ultrastructurally abnormal in a patient with a mitochondrial disease associated with respiratory chain deficiency (Folgero et al., 1993). A relationship between ATP content and embryonic viability has been found in human oocytes, and in mouse oocytes treated with uncouplers of mitochondrial oxidative phosphorylation to investigate preimplantation development. A lower ATP content, indicating a reduced mitochondrial metabolism, has been associated with reduced embryonic viability. In this regard, the possible role of mitochondria in maternal age-related decline in fertility should be investigated (Van Blerkom et al., 1995). A high incidence of pre-eclampsia and eclampsia has been reported in a large pedigree affected with a mitochondrial disorder (Torbergsen et al., 1989), and a decrease in cytochrome c oxidase activity and cytochrome oxidase subunit I mRNA levels was found in placental tissue from women with preeclampsia in comparison with controls (Furui et al., 1994). However, the contribution of mitochondria to the pathophysiological mechanisms underlying pre-eclampsia still remains to be elucidated.

Acknowledgements Telethon Italy (grant no. 767 to M.Z.) and ARIN (Associazione Italiana per la Promozione delle Ricerche Neurologiche) are gratefully acknowledged.

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