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Human Reproduction, Vol. 15, (Suppl. 2), pp. 28-43, 2000 Genetic defects causing mitochondrial respiratory chain disorders and disease John Christodoulou1 Western Sydney Genetics Program, Royal Alexandra Hospital for Children, Westmead, and Department of Paediatrics and Child Health, University of Sydney, Sydney, Australia 'To whom correspondence should be addressed at: Western Sydney Genetics Program, Royal Alexandra Hospital for Children, Westmead, NSW 2145, Australia. E-mail: [email protected] Genetic defects of the mitochondrial respiratory chain show marked phenotypic variability. Laboratory diagnosis is complicated and includes biochemical screening tests, tissue histopathology, functional enzyme studies, and molecular tests where available. Normal respiratory chain function necessitates the co-ordinated expression of over 100 different gene loci, and the interaction of two genetic systems, the nuclear and mitochondrial genomes. Thus genetic counselling for the mitochondrial disorders is extremely challenging. In this review, the classes of mitochondrial and nuclear defects that give rise to functional abnormalities of the mitochondrial respiratory chain are discussed, with specific instructive examples described in some detail. Keywords: genetic s/mitochondria/mitochondrial disease/mitochondrial DNA/respiratory chain Clinical spectrum, diagnosis, and treatment of respiratory chain disorders The mitochondrial respiratory chain plays a critical part in the biosynthesis of adenosine triphosphate (ATP) by the process of oxidative 28 phosphorylation, and is composed of five enzyme complexes (Figure 1), each of which is made up of multiple polypeptide subunits (Shoffner and Wallace, 1995). A constant supply of this newly formed ATP is critical for the normal function of many key organs. Clinically, defects of the mitochondrial respiratory chain show marked phenotypic variability, both between and within families, and can interfere with the function of single organs (including skeletal muscle, the central nervous system, kidneys, endocrine organs and the gastrointestinal system) or result in life-threatening multisystem disease (DiMauro et ai, 1998). The clinical onset may be at any age, and the rate of progression of symptoms can range from a rapid, fulminant lactic acidosis in the neonatal period to a slow or even static condition at a later age, albeit usually with impaired survival. Some mitochondrial disorders are associated with a characteristic set of clinical and pathological features, for which they have been given eponymous names (for a review see Shoffner and Wallace, 1995). Diagnosis is a staged process, with initial screening tests that include measuring plasma and cerebrospinal fluid (CSF) concentrations of lactate and pyruvate, and imaging the brain (Munnich et al., 1996). The next phase of investigations involves biopsy and laboratory © European Society of Human Reproduction & Embryology Mitochondria! respiratory chain disorders glucose pyruvate intermembrane spac matrix atty acids carnitine outer membrane membrane respiratory chain Figure 1. Schematic representation of the mitochondrial energy production pathways. The five complexes that make up the respiratory chain are physically located at the inner mitochondrial membrane. Also shown are the tricarboxylic acid (TCA) cycle and the fatty acid (3-oxidation spiral, both of which are the major suppliers of reduced equivalents as electron donors. Note also the multiple copies of circular mitochondrial DNA within the mitochondrial matrix. analysis of skeletal muscle and/or liver, using histochemistry, electron microscopy, and functional studies, which can include polarography or direct enzyme assays (Trounce et al, 1996). Enzymatic studies of cultured skin fibroblasts, isolated leukocytes, or transformed lymphoblasts can also be valuable (Rustin et al, 1994). However, as some defects are not expressed in all tissues, including cultured cells, selective functional assays can be problematical (Chretien et al, 1994). Molecular analysis of specific mitochondrial (mt) DNA defects is available in many cases, but it is important to note that certain mtDNA molecular defects are found in some tissues but not others, or are found at different levels in different tissues (heteroplasmy) (Shoffner and Wallace, 1995). While 'common' mutations have been identified in some syndromic mitochondrial disorders (see below), the majority of patients (including most children), still turn out to have nuclear or mtDNA mutations that are not identified (Rahman et al, 1996). The development of the rho-0 cell, an immortal human cell line that has been rendered mtDNA-free (King and Attardi, 1989) (though still with mitochondrial organelles), provides an extremely valuable experimental tool not only for evaluating the biochemical and molecular pathogenicity of putative mtDNA mutations (Attardi et al, 1995), but for distinguishing disorders with Mendelian (nuclear) inheritance from those due to defects of the mitochondrial genome (Tiranti et al, 1995). There have been many attempts at treating the effects of mitochondrial disease, including avoidance of agents that can precipitate lactic acidosis, dietary modifications, treatment of acute lactic acidosis with slow bicarbonate infusions, administration of artificial electron acceptors, vitamin cofactors, and other pharmacological agents (Przyrembel, 1987). Although occasional patients show some response to therapeutic interventions of this type (Ogle et al, 1997; Mowat et al, 1999), therapeutic efforts in most individuals have generally been disappointing (Walker and Byrne, 1995). Moreover, systematic evaluation 29 J.Christodoulou of efficacy of these treatments is hampered by clinical, biochemical and genetic heterogeneity, difficulty in accessing some therapeutic agents, and difficulty assessing the response to treatment of conditions that in any case can show considerable fluctuation in clinical features from week to week. It is widely felt that the development and use of a standardized, objective means of assessing symptoms and response to treatment would be a step forward. The key, meanwhile, to developing effective treatments will lie in a better understanding of the genetic bases of the respiratory chain defects. Special aspects of mitochondrial genetics The five enzyme complexes of the respiratory chain are each composed of multiple polypeptides, totalling over 80 different gene products. In addition, a number of essential proteins are involved in regulation of mtDNA transcription and subunit assembly. Most of these genes (up to 80%) are encoded by the nuclear genome, but some 37 (including specific transfer and ribosomal RNAs) are encoded by the mitochondrial genome. Normal mitochondrial respiratory chain function for efficient oxidative phosphorylation thus necessitates the co-ordinated expression of >100 different gene loci from two very different genomes (Figure 2), with some of these showing tissuespecific expression. Thus genetic counselling for the mitochondrial disorders is particularly difficult and challenging. There are a number of characteristics of the mitochondrial genome that render its genetic influences quite unlike that of the more familiar Mendelian inherited, and even polygenic nuclear genomic disorders. Maternal inheritance In humans, mtDNA is maternally inherited, transmitted by a female to all of her offspring 30 Human Chromosomes 100's - L000' o autosomes sex chromosomes 1-22 X or Y 50-100,000 genes 2-10 copies 37 genes proteins regulating mtDNA replication Figure 2. The relationship between the mitochondrial and nuclear genomes. The protein subunits of the respiratory chain are encoded by both nuclear and mitochondrial genes. In addition, the mitochondrial genome encodes a full complement of tRNAs and rRNAs, providing all the necessary machinery to permit intramitochondrial transcription and translation. Other (nuclear encoded) factors are essential for the correct targeting of cytoplasmically synthesized proteins to the mitochondria, import into the mitochondria, and their correct assembly there. There are also essential nuclear genes that play a role in the replication, transcription, and translation of the mitochondrial genome. through the ovum's cytoplasm (Giles et al., 1980). Clinical clues to maternal inheritance comprise involvement of multiple generations through a common female ancestor, absence of transmission of the defect by a male to his offspring, and potential prevalences among offspring higher than that expected for autosomal dominant nuclear inheritance (albeit with marked clinical variability between members of the same family, see below). Replicative segregation Mitochondria (and the group of mtDNA circles they contain) are believed to behave as discrete units within the cell (Wallace, 1995), although there is evidence to suggest that there may be functional intermitochondrial interactions (Takai etal., 1997). Accordingly, during cellular replication mitochondria are randomly segregated to daughter cells. In the case of the female germ cells of a woman with a mitochondrially inherited disorder, the result- Mitochondrial respiratory chain disorders progenitor cell normal fully energy competent mitochondria mtDNA mutation develops mitochondria replicate independently mitochondria randomly distributed in daughter cells complete energy deficiency varying degrees of energy deficiency fully energy competent Figure 3. Replicative segregation and the threshold effect. Mitochondria (and the mtDNA within them) are generally considered to function as genetically discrete units and to replicate by a process of budding. During cell division, mitochondria behave stochastically, i.e. they segregate in a random way among daughter cells. Thus, where a progenitor cell harbours a pathogenic mtDNA mutation, individual daughter cells can have varying mutant loads. Once the mutant load reaches a certain threshold, which differs from one tissue to another, respiratory chain function collapses and clinical consequences appear. ant individual oocyte could bear mtDNA all of normal sequence (homoplasmic normal), all of abnormal sequence (homoplasmic for the mutant mtDNA, which is lethal in some instances but not in others, depending on a number of factors), or a mixture of normal (wild-type) and mutant mtDNA (heteroplasmy) (Wallace, 1991). Threshold effect During the early stages of embryogenesis there is no mitochondrial or mtDNA replication (Piko and Matsumoto, 1976), resulting in random mitochondrial segregation of the fertilized oocyte's mtDNA among daughter cells during cleavage, again producing a potential wide range of mtDNA genotypes from virtual homoplasmy for wild-type mtDNA to virtual homoplasmy for the mutant mtDNA (Wallace, 1986). At a certain mutant mtDNA load, a threshold is reached beyond which cellular dysfunction will become evident (Figure 3) (Shoffner et al., 1990). This threshold differs according to the particular mutation and the energetic needs and characteristics of individual tissues or organs, but there is generally a predictable hierarchy of vulnerability for a given mtDNA mutation. The organs most likely to be affected are the central nervous system (including the eye), type 1 skeletal muscle, cardiac muscle, pancreatic islet tissue, liver, and kidney (Wallace, 1992); and this is the general pattern of abnormalities most often encountered clinically. High mutation rate The mutation fixation rate of the mitochondrial genome is 10-20 times higher than that of the nuclear genome (Wallace, 1987). Factors responsible for this include the fact that free radicals generated within the mitochondrion are highly mutagenic (Richter et al., 1988) and that mitochondria contain limited DNA repair mechanisms (Tritschler and Medori, 31 J.Christodoulou 1993). Thus, with advancing age in postmitotic (differentiated) tissues, there is thought to be a potentially important level of accumulation of mitochondrial mutations that can exceed the threshold for clinical consequence. In summary, where the metabolic disorder is due to a defect of a mitochondrially encoded gene, the clinical consequences of that mtDNA mutation are a function of the mtDNA mutant load in a given tissue, the seriousness of the mtDNA mutation itself, and the energetic requirements and functional reserve of the organ or tissue in question. In addition, nuclear genes, age-related accumulation of somatic cell mtDNA mutations, and certain environmental insults (Shoffner and Wallace, 1995) can all modulate the clinical phenotype. Mutations causing mitochondrial disease In recent years there have been great advances in our understanding of the range of molecular defects that can give rise to functional abnormalities in the mitochondrial respiratory chain (Table I). Below is a description of a number of different types of mutations. The list is not exhaustive but is sufficient to highlight some of the important clinical features of mitochondrial diseases. Primary mtDNA defects mtDNA point mutations: protein reading frames In all, 13 mtDNA genes encode some of the respiratory chain subunits (seven subunits of complex I, one subunit of complex III, three subunits of complex IV, two subunits of complex V). Mutations have been described in some but not all of these genes. Clinical examples are described below. Leber hereditary optic neuropathy (LHON) This disorder usually has its onset in early adulthood, and causes a progressive retinal 32 Table I. Classes of genetic defects giving rise to respiratory chain disorders Defects Primary mtDNA defects mtDNA point mutations Protein subunits tRNAs rRNAs mtDNA rearrangements Large-scale deletions Large-scale duplications Small deletions Nuclear encoded defects Protein subunits Protein trafficking, unfolding, assembly mtDNA replication, transcription, translation Other defects a Examples8 LHON, NARP MEL AS, MERRF Sensorineural deafness KSS CPEO Recurrent myoglobinuria NDUFS4, NDUFS8 hsp60, SURF-1 Thymidine phosphorylase Frataxin, paraplegin For explanations of abbreviations, see text. microangiopathy and optic nerve degeneration, resulting in progressive blindness (Harding and Sweeney, 1994). Some individuals also develop cardiac arrhythmias, but lactic acidaemia (a consequence of widespread metabolic disruption) is very uncommon. For reasons that are unclear, males are 5-10 times more likely to be affected than females. LHON was the first elucidated mtDNA defect (Wallace et al., 1988), with >15 mtDNA nucleotide substitutions now identified (1999). A guanine to adenine mutation at nucleotide position 11778 (notation, G11778A), which results in the substitution of histidine for a highly conserved arginine in a subunit NADH dehydrogenase 4 (ND4) gene, accounts for 50-70% of cases. Unlike other mitochondrial diseases, homoplasmy for the mutation is common, even before the onset of symptoms. Neuropathy, ataxia and retinitis pigmentosa (NARP) syndrome This condition is characterized by a pigmentary retinal dystrophy and blindness from macular degeneration in association with neurological dysfunction, including neuro- Mitochondria! respiratory chain disorders genie muscle weakness and ataxia from olivopontocerebellar atrophy (Holt et al., 1990). It is caused by a thymidine to guanine mutation involving nucleotide 8993 (T8993G), which swaps an arginine for a highly conserved leucine in ATP synthase subunit 6, and thus places an additional positive charge group in the proton channel of ATP synthase. This seriously interferes with proton pumping into the intermembranal space, thus blocking ATP production and uncoupling oxidative phosphorylation. Death always occurs before homoplasmy can be reached. With high levels of inherited mutation, maternally inherited Leigh disease can manifest instead. Leigh disease This is a progressive neurodegenerative disorder notable for optic atrophy, ophthalmoplegia, nystagmus, respiratory abnormalities, ataxia, hypotonia, spasticity, and regression, with its onset in the neonatal period or the first few months of life, and survival spanning only a few years (van Erven et al., 1987). Neuropathologically, there is symmetric gliosis, demyelination, and necrosis predominantly involving the basal ganglia and brainstem, with relative sparing of cerebral cortex. Most cases are thought to be caused by autosomal recessive mutations affecting nuclear-coded subunits of the mitochondrial respiratory chain, but some cases are due to the T8993G (or T8993C) mutation also responsible for NARP. In addition, functional defects of pyruvate dehydrogenase, or complexes I and/or IV have been described with Mendelian (nuclear) inheritance patterns (Robinson, 1995), including a recently described mutation in the SURF-1 gene (resulting in isolated complex IV deficiency, see below). mtDNA point mutations: transfer RNA mutations Transfer RNAs are critical for translation of all the mitochondrially inherited subunits of the electron transport chain, so tRNA defects £re usually associated with multiple functional respiratory chain abnormalities. Muscle biopsies often show a massive accumulation of subsarcolemmal mitochondria, which when stained with modified Gomori trichrome give the so-called ragged red fibre picture. Two clinically recognized phenotypes are described below. Myoclonic epilepsy and ragged red fibres (MERRF) syndrome This disorder has its onset in early childhood through to adulthood, and is characterized by a mitochondrial myopathy, myoclonic epilepsy, and slowly progressive dementia, with hearing loss and ataxia being common findings (Moraes et al., 1989). On laboratory testing, patients typically show functional abnormalities of complexes I and IV, and sometimes complex III. Mutations have been identified in several tRNAs, with the tRNA for lysine (tRNALys) most often affected, particularly by the mutation G8344A (Shoffner et al., 1990). Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) Recurrent stroke-like episodes and a mitochondrial myopathy are characteristic of this progressive neurodegenerative disorder (Shoffner and Wallace, 1995). These 'metabolic strokes', which can have their onset between 5 and 15 years of age, do not follow a vascular distribution and are thought to be the result of transient cerebral respiratory chain dysfunction. Most patients to date have been found to have mutations in the tRNA for leucine (tRNALeu), with the mutation A3243G being most common (Goto, 1995). Migrainelike headaches are common in these families. Other combinations of problems can be seen, including cardiomyopathy, deafness, ophthalmoplegia, cardiac conduction defects, diabetes mellitus, easy fatiguability, dementia, and 33 J.Christodoulou renal tubular dysfunction, as well as lactic acidaemia (Schon, 1994). mtDNA point mutations: ribosomal RNA mutations There have been several families reported with maternally inherited childhood-onset sensorineural deafness, which in one family was apparently induced by aminoglycoside antimicrobial usage. A homoplasmic mutation of the mitochondrial genome was identified (A1555G), which is within the gene for 12S rRNA (Prezant et al, 1993). This mutation has also been associated with maternally inherited cardiomyopathy (Santorelli et al, 1999). In addition, other mutations in the 12S rRNA gene have been described in association with type 2 diabetes mellitus (Tawata et al, 1998). mtDNA rearrangements Large-scale deletions or duplications of mtDNA are associated with distinct phenotypes, which in almost cases do not appear to be maternally inherited but present as sporadic mutational events. mtDNA rearrangements: major deletions In these patients up to 50% (occasionally more) of the 16.6 kb mitochondrial genome can be deleted, which if homoplasmic in the embryo would almost certainly be lethal because of the profound impact this would have on respiratory chain function (Wallace, 1991; Schon, 1994). Therefore, all individuals are heteroplasmic for the deletion. Over 100 mtDNA deletions have been identified (MITOMAP, 1999). Most are flanked by direct repeats, suggesting that for at least some of these deletions slipped mispairing may be responsible for the development of the deletion (Schon et al, 1989; Shoffner et al, 1989). The deletions are generally in the range 1.334 7.6 kb, localized between the end of the D-loop and site of origin of light chain synthesis, thus spanning more than one gene (and including tRNA and rRNA genes as well as respiratory chain polypeptide genes). Examples of recognized clinical phenotypes are described below. Kearns-Sayre syndrome (KSS) This neurodegenerative disorder usually has its onset at <20 years, with affected individuals typically manifesting a progressive external ophthalmoplegia and partial ptosis, with retinitis pigmentosa (Berenberg et al, \911; Zeviani et al, 1988). Additional features not seen in all patients include cerebellar signs, cardiac conduction abnormalities, and a raised CSF protein. Less commonly seen problems include diabetes mellitus, hypoparathyroidism, deafness, peripheral neuropathy, seizures, dementia, renal tubular and glomerular dysfunction, cardiomyopathy, and lactic acidosis. Patients with an onset at >20 years usually have a less severe and more slowly progressive variant called chronic progressive external ophthalmoplegia (CPEO) (Shoffner and Wallace, 1995). Most KSS and CPEO patients have a heteroplasmic deletion, 4.9-9 kb in size (including the so-called 'common' deletion, AmtDNA4977), which can be readily identified by Southern-blot analysis or specifically designed polymerase chain reaction (PCR)based assays (Manfredi et al, 1997). In some patients no deletion is identified; some of these patients have a mtDNA point mutation, while others could harbour a nuclear DNA mutation (Shoffner and Wallace, 1995). Over 100 cases of KSS have been reported. The condition is usually sporadic: as explained above, oocyte transmission of the deletion generally fails and the large-scale rearrangement presumably occurs as a somatic mutation after embryogenesis has commenced (De Vivo, 1993). A reported maternally inherited case is thought to have involved transmission Mitochondrial respiratory chain disorders of a duplication (prone to subsequent deletion) (Rotig et al, 1992). The birth of normal children to affected women has occurred (De Vivo, 1993). The higher the mutational load is, the earlier the clinical presentation, with a preference for expression in mitotically active tissues (see below). With lower mutational loads respiratory function is not significantly compromised in mitotically active tissues and the disease does not express until an age-dependent rise in the mutational load occurs in post-mitotic tissues. Sometimes mitotic tissue presentations ameliorate spontaneously, as mitochondrial selection takes place, only to give way to postmitotic tissue involvement at a later age (as has been seen in Pearson syndrome; see below). Pearson syndrome This is also a progressive degenerative disorder caused by large deletions in the mitochondrial genome, but (unlike KSS) with onset in early infancy (Rotig et al, 1991). The clinical picture is characterized by aplastic or sideroblastic anaemia, with vacuolated myeloid and erythroid cell lines, and ringed sideroblasts in bone marrow, watery diarrhoea, pancreatic insufficiency, liver failure, proximal renal tubule dysfunction, and lactic acidosis (Pearson et al, 1979; Rotig et al., 1990). The deletion is initially most prominent in blood mtDNA (i.e. in mitotically active cell lines), but in children who survive beyond infancy the deletion disappears from blood cells, probably because of successful clonal advantage for non-affected cell lines (Shoffner and Wallace, 1994). There is, however, a progressive increase in the proportion of deleted mtDNA in muscle (unlike blood, a post-mitotic tissue), and as the surviving child grows older the clinical phenotype can evolve into KSS (McShane et al., 1991). Most cases appear to be due to de-novo deletions, although there is a report of maternal transmission of a deletion resulting in Pearson syndrome (Bernes et al., 1993). Villous atrophy and lactic acidosis This infantile-onset disorder has features that overlap with KSS and Pearson syndrome, including watery diarrhoea, lactic acidosis, growth retardation, sensorineural deafness, retinitis pigmentosa, cerebellar ataxia, diabetes mellitus, and renal failure, with death usually in the second decade (Cormier-Daire et al., 1994). Muscle samples often show ragged red fibres, with a mitochondrial genomic deletion 3.4^.2 kb in size. Diabetes mellitus and deafness Affected individuals usually manifest deafness in mid-childhood to late adolescence, with insulin-dependent diabetes mellitus developing in the third to fourth decade, and vascular strokes (Ballinger et al., 1992). Muscle analysis shows functional defects of complexes I, III, and IV, but not complex II (DiMauro and Moraes, 1993). (This laboratory pattern of respiratory chain abnormalities is highly suggestive of a mtDNA disorder, because all of the four complex II subunits are encoded by the nuclear genome.) Large heteroplasmic deletions, up to 10.4 kb, have been found in these families, whilst others are found to have the A3243G mutation also seen with MELAS (van den Ouweland et al., 1992). mtDNA rearrangements: major duplications Duplications of the mitochondrial genome are also heteroplasmic, and usually sporadic (occasional maternally inherited cases have been reported). Patients with a KSS or CPEO phenotype have been described who have a duplication of up to 10 kb in size (Poulton et al., 1989), as have some patients with Pearson syndrome (Rotig et al, 1991). Duplications and deletions can co-exist, and 35 J.Christodoulou recent studies suggest that in such individuals it is the deletion rather than the duplication which appears to be most pathogenic (Manfredi et al, 1997). mtDNA rearrangements: small deletions subunits (Adams et al., 1997; Jaksch et al., 1998; Lee et al, 1998), only a small number of such genetic defects have been found. The first to be discovered involved a subunit of succinate dehydrogenase (Bourgeron et al., 1995). More recently mutations of the NDUFS4 subunit (van den Heuvel et al., 1998), the NDUFS8 subunit (Loeffen et al, 1998) and NDUFV1 subunit (Schuelke et al, 1999) of complex I have been reported. There have been only a few reports of a very small deletion of the mitochondrial genome. Shoffner and colleagues (Shoffner et al, 1995) described a patient with a mitochondrial encephalomyopathy and cerebral calcifications, Defects involving protein trafficking, who was found to have a deletion of one of unfolding or assembly the three T:A base pairs (involving nucleotides 3271-3273) in the tRNALeu<UUR> gene. In Over the last few years there have been great another report, the affected individual had advances made in understanding the processes a transient neonatal lactic acidosis and that target cytoplasmically synthesized procardiac conduction defects, and subsequently teins to mitochondria, the mechanisms developed growth failure, spasticity, nystag- involved in their translocation through the mus and strabismus, and a further episode of mitochondrial membranes, and their sorting lactic acidosis in association with an intercur- to and assembly in the different mitochondrial rent illness (Seneca et al, 1996). This patient compartments (Lill et al, 1996; Neupert, was found to have an apparently homoplasmic 1997; Ryan et al, 1997). Defects of the 1 bp deletion in the ATPase 6 gene. Keightley transportation and processing machinery in and associates (Keightley et al., 1996) identi- some cases would be expected to result in fied a 15 bp deletion in the cytochrome c multiple mitochondrial protein defects, involoxidase (COX) III subunit in a young woman ving not just proteins of the respiratory chain with recurrent episodes myoglobinuria pro- but other key pathways. Such a disorder has voked by exercise or catabolic illnesses. De been described in two siblings, involving heat Coo et al. (1999) reported a patient with a shock protein 60 (hsp60) (Agsteribbe et al, childhood onset encephalopathy and a 1993). Other defects involving mitochondria] MELAS type phenotype who was found to protein trafficking and processing have also have a 4 bp deletion in the cytochrome c gene. been proposed (Schapira et al, 1990), but the All of these cases were sporadic (or de novo, molecular mechanisms have not been idenalthough muscle tissue was not available for tified. analysis in the probands' mothers). Most cases of Leigh disease (see above) show Mendelian (nuclear) inheritance, either autosomal recessive or X-linked, the latter Nuclear mutations affecting oxidative being the case in those patients who have phosphorylation been shown to have a defect in the El a subunit of the pyruvate dehydrogenase complex (Brown et al, 1989). Defects of protein subunits In the majority of patients with Leigh disDespite extensive attempts to identify ease an isolated defect involving COX is the mutations in nuclear encoded respiratory chain primary functional abnormality. One of the 36 Mitochondrial respiratory chain disorders somatic cell techniques which has been used in the genetic study of Leigh disease is complementation analysis, where cell strains from different patients exhibiting the same functional defect are fused. If the fused heterokaryon recovers the function under study, one can infer that the two cell strains have mutations in different genes (i.e. the two cell strains have complemented each other's genetic defects, and thus belong to different complementation groups). On the other hand, if there is no recovery of function, the two cell strains are likely to have mutations in the same gene, and would therefore be assigned to the same complementation group. Complementation analyses in COX-deficient Leigh disease have shown that most patients belong to the same complementation, suggesting they would have a defect in the same gene (Brown and Brown, 1996; Munaro et al, 1997). Using different approaches in a series of elegant experiments, two groups simultaneously identified mutations in a highly conserved gene called SURF-1 in patients belonging to this major complementation group (Tiranti et al, 1998; Zhu et al, 1998), thereby defining a new class of genes causing neurodegenerative diseases in humans. The function of the SURF-1 gene product is not well understood, but it is believed to play a role in assembly or maintenance of the COX complex. Thus, in some instances individual genes involved in these process may be specific for certain enzyme complexes. Defects affecting mtDNA replication, transcription, or translation Disorders with Mendelian inheritance in which the defect appears to involve genes responsible for the regulation of mtDNA replication, transcription, or translation have been reported. At the molecular level, these disorders are manifest by qualitative or quantitative abnormalities of mtDNA. Multiple mtDNA deletions Patients have been identified with a CPEO clinical phenotype, but with an autosomal dominant pattern of inheritance and additional clinical features, including peripheral neuropathy, ataxia, cataracts, muscle weakness and mild lactic acidosis, and muscle biopsies that reveal mitochondrial proliferation (Zeviani et al, 1989; Zeviani, 1992). Rather than a single deletion as seen in typical CPEO, affected family members show multiple deletions on Southern blots. Linkage studies (in two unrelated families) have mapped genes likely to be responsible to the 10q23.3-24.3 region (Suomalainen et al., 1995) and the 3pl4.121.2 region (Kaukonen et al., 1996). Another disorder, possibly autosomal recessive or Xlinked, and manifesting clinically with recurrent myoglobinuria in brothers, has been reported with the identification of multiple mtDNA deletions (Ohno et al., 1991). The mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome is a multisystem autosomal recessive disorder mapped to chromosome 22ql3.32-qter, and characterized by ptosis, progressive external ophthalmoplegia, skeletal myopathy, gastrointestinal dysmotility, poor weight gain, and leukoencephalopathy. Multiple mtDNA deletions have been observed in skeletal muscle (Hirano et al., 1998) and are presumably also present in other tissues. Recently diseasecausing mutations were identified in the thymidine phosphorylase gene, making this the first gene isolated which plays a role in intergenomic interactions, perhaps by regulating the availability of thymidine for DNA synthesis and mtDNA maintenance (Nishino et al., 1999). mtDNA depletion In this disorder mtDNA is qualitatively normal, but there is quantitative depletion of mtDNA circles in mitochondria (Moraes et al, 37 J.Christodoulou 1991; Tritschler et al, 1992). The neonatalonset form is fatal in early infancy and is characterized by a skeletal myopathy or liver failure, while the later-onset form (initial presentation at ~1 year of age) appears to involve only skeletal muscle. In both forms there are multiple functional defects of the respiratory chain. Mitochondrial proliferation occurs in affected tissues in an apparent attempt at compensation. The inheritance pattern is autosomal recessive. In the early-onset form, there is a reduction in mtDNA of up to 98% compared with normal controls, whilst in the late-onset form mtDNA depletion is less severe, at -70-90% (Tritschler et al, 1992). The molecular mechanism responsible for the mtDNA depletion has not yet been identified. Other nuclear defects affecting the respiratory chain Friedreich ataxia is an autosomal recessive disorder in which many of the clinical features are very reminiscent of a primary respiratory chain defect (progressive ataxia, dysarthria, peripheral neuropathy, diabetes mellitus and cardiomyopathy), so it should not be surprising that it was recently found that affected individuals have an unusual pattern of abnormalities, namely functional defects of complexes I, II and III (Rotig et al, 1997). The most common genetic defect is an expansion of a GAA triplet repeat in the gene for frataxin, and recent evidence suggests that the pathogenesis of this disorder is related to a disruption of iron-sulphur dependent enzymes, including the respiratory chain complexes listed above and the enzyme aconitase (Rotig et al, 1997). The hereditary spastic paraplegias are a group of disorders characterized by progressive weakness and spasticity, particularly involving the lower limbs, and peripheral neuropathy. Other features in this clinically and genetically heterogeneous condition 38 include retinitis pigmentosa, optic atrophy, deafness, and intellectual disability. Recently, the gene responsible for an autosomal recessive form (SPG7) has been characterized (De Michele et al, 1998). Called paraplegin, this novel protein is highly homologous with a family of yeast mitochondrial metalloproteinases. Recent work has found evidence suggesting that defects of the human protein might have similar consequences to yeast mutants, i.e. a deficiency in the assembly of functional respiratory and ATPase complexes (Casari et al, 1998). Some cases of KSS, CPEO and the mitochondrial disorder diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD) show autosomal dominant or recessive inheritance (Shoffner and Wallace, 1995), but the genetic defects are unknown. Genetic counselling for mitochondrial disorders Genetic counselling for the disorders of the mitochondrial respiratory chain remains one of the most challenging areas in the field of human genetics. Where there appears to be only a single member of a family affected, no family history of consanguinity, and no mutation is identified in either mitochondrial or nuclear DNA, it is impossible to predict with any confidence the likely recurrence risk and one needs to resort to empiric risk data. It has been suggested that the recurrence risk for the offspring of affected females where the mutation is unknown is in the order of 10-20%, whereas for the offspring of affected males the risk is -1-2% (Hammans and Morgan-Hughes, 1994). A woman in whom a specific mtDNA mutation has been identified would have a high recurrence risk in her offspring but, because the level of heteroplasmy is generally only loosely correlated with disease severity, it has been impossible to predict with confid- Mitochondrial respiratory chain disorders ence the likely severity of the disease in a fetus identified as having the mutation (Poulton and Marchington, 1996). However, as the body of experience builds, such risk data ought to be more reliably derived, as recently demonstrated for the T8993G and T8993C mutations (White et al, 1999). In addition, with refinements in tissue manipulation techniques and reproductive technologies, it is now possible to analyse the mitochondrial genome of single oocytes or preimplantation embryos to aid in the assessment of recurrence risks for individual females (Blok et al, 1997; De Boer, 1999). When there are difficulties in accurate genetic diagnosis such as those outlined above, prenatal testing based on molecular methods is generally not available. Respiratory chain activities in cultured skin fibroblasts probably best reflect those in cultured chorionic villus cells or cultured amniocytes (Munnich et al, 1996). Thus, if one is relying on functional enzyme assays of the respiratory chain in cultured cells, in the main only in those families where the parents are consanguineous (presumed autosomal recessive inheritance) and where the defect is clearly demonstrable in cultured skin fibroblasts, will prenatal diagnosis be most reliable. As more gene defects are identified, the usefulness of molecular prenatal testing will improve. Conclusions In summary, although much is known, four important questions remain to be answered in the field of human mitochondrial disease: (i) why is there so much phenotypic variability between patients seemingly independent of the degree of heteroplasmy?; (ii) what are the genetic bases of the broad group of metabolic defects for which no mitochondrial genomic mutation has been identified?; (iii) will it be possible to develop reliable methods of prenatal diagnosis?; and (iv) will it be possible to devise effective treatments? The complicated biology of this group of disorders makes their study particularly challenging. However, advances in molecular and reproductive technologies, coupled with the explosion of new biological data in humans and other species generated by the Human Genome Project, mean that the new millennium will see the evolution of methods that will allow us to define more of the mitochondrial respiratory chain disorders at the molecular level, and to evaluate their functional significance. It is in that climate that accurate prenatal and symptomatic testing, and possibly novel therapeutic strategies will be developed for this devastating group of disorders. Note added in proof Recently, mutations in the WFS1 gene have been reported in patients with DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness) syndrome (Hardy et al, 1999). References Adams, P.L., Lightowers, R.N. and Tumuli, D.M. (1997) Molecular analysis of cytochrome c oxidase deficiency in Leigh's syndrome. Ann. Neuroi, 41, 268-270. Agsteribbe, E., Huckriede, A., Veenhuis, M. et al. 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