8 Defects of the respiratory chain

8 Defects of the respiratory chain L. A. B I N D O F F D. M. T U R N B U L L

INTRODUCTION Defects of the mitochondrial respiratory chain give rise to a wide range of clinical disorders ranging from isolated myopathy to multisystem disease. These conditions are now being increasingly recognized, especially among the young, and are significant causes of morbidity and mortality. Our understanding of both respiratory-chain function and the disorders which result from its failure has grown enormously since Luft and colleagues described the first case in 1962 (Luft et al, 1962). However, owing to the complexity of the system we still have a great deal yet to learn. This chapter attempts to provide a general understanding of this unique and vital pathway in energy metabolism. We explore how its dysfunction may produce both local and systemic defects and what is known about the inheritance of these disorders. The last two sections deal exclusively with the methods used for investigation and describe some of the clinical presentations associated with defects of each individual complex.

Biochemistry The mitochondrial respiratory chain consists of a series of multi-subunit complexes, the function of which is to use energy derived from substrate oxidation to synthesize ATP (Figure la). Details of the subunit structure and enzyme function are given in Table 1 and readers are referred to Chapter 5 for a more detailed explanation of the bioenergetics. The respiratory-chain complexes are found within the inner mitochondrial membrane and form an integrated system of redox-couples along which reducing equivalents (electrons) pass: energy is generated at each step, but is sufficient at only three sites to allow the extrusion of protons and the formation of an electrochemical proton gradient across the inner mitochondrial membrane (Figure lb). This gradient is used by ATP synthase (complex V) to drive ATP synthesis (Figure lb). Linkage of substrate oxidation with phosphorylation of ADP, hence 'oxidative phosphorylation', is the unique feature of the mitochondrial respiratory chain; disruption of any aspect of this system may lead to a BailliOre' s Clinical Endocrinology and Metabolism--

Vol. 4, No. 3, September 1990 ISBN 0-7020-1464-8

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lowering of cellular ATP, slowed metabolism, and the accumulation of intermediates which may be toxic. The effects of an abnormality within the respiratory chain will depend on several factors: 1. 2.

The site and degree to which functional activity is lost. The capacity of the cell to derive energy from other metabolic processes and thus the dependence of that cell type on oxidative metabolism. The secondary effects that may result from a defect of respiratory chain function.

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Figure 1. Diagrammatic representation of the structure and function of the mitochondrial respiratory chain. (a) Oxidative phosphorylation. The oxidation of substrates yields energy which is used by the respiratory chain to drive ATP synthesis (phosphorylation). The process depends on cofactors (NAD + and FAD) to link substrate oxidation with the respiratory chain, and on the ultimate reduction of molecular oxygen, hence the term aerobic respiration. (b) Detail of electron transport through the respiratory chain. Three sites generate sufficient energy to drive proton translocation creating the electrochemical gradient (Apaa ÷) that is used by ATP synthetase to drive ATP synthesis. The complexes are large and probably relatively immobile but are linked by the smaller and freely mobile electron carriers ubiquinone (Q) and cytochrome c (c). Arrows show the route of electron flow. For diagrammatic purposes, cytochrome c is shown on the matrix side of the membrane.

Name

NADH-ubiquinone oxidoreductase

Succinate-ubiquinone oxidoreductase

Ubiquinol-cytochrome c oxidoreductase

Cytochrome c oxidase

ATP Synthetase (Mg 24 ATPase)

Complex

I

II

III

IV

V

Uses electrochemical gradient to drive reaction ADP + Pi---+ATP Reverses proton gradient

Re-oxidizes cytochrome c Passes electrons to molecular oxygen Proton pump

Re-oxidizes ubiquinol to ubiquinone Passes electrons to cytochrome c Proton pump

Oxidizes succinate to fumarate Transfers electrons to UQ: producing UQH2

Oxidizes NADH to NAD + Transfers electrons to ubiquinone (UQ) reducing it to ubiquinol (UQH2) Proton pump

Function

14

13

11

4

ca 25

None

Cytochrome a + a3 2 or 3 Cu atoms

Cytochromes b566/b562 Cytochrome cl FeS centre (Reiske)

FAD 3 FeS Cytochrome b558

8-9 FeS centres FMN

Subunits Prosthetic groups

Table 1. The respiratory chain complexes--structure and function.

1.

3.

1. 2.

3.

1. 2.

3.

2.

Two subunits coded by mtDNA. The remaining subunits are encoded by nuclear genes, often by more than one gene

Succinate dehydrogenase is part of complex Unique because involved in both respiratory chain and TCA cycle No subunits encoded by mtDNA Known as bcl complex Cytochrome b is one molecule with two active sites b566 and b562 Cytochrome b apoprotein is encoded by mtDNA Reduces molecular oxygen Debate over number of Cu, now thought to be 3 The three largest subunits are encoded by mtDNA and it is these which is involved in active sites

1.

3.

2.

Largest and most difficult of complexes to isolate Seven subunits encoded by mtDNA Other enzymes are capable of N A D H oxidation, therefore measurement of activity problematical

1.

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L. A. BINDOFF AND D. M. TURNBULL

Identification of the site of abnormality within the respiratory chain is now possible with a reasonable degree of accuracy. However, we are not yet at the stage where we can always identify the particular subunit involved; indeed, we are not even sure how many subunits are contained within complex I, which is perhaps the most commonly affected of the respiratory chain complexes. Little is known, moreover, about the degree of functional reserve contained within each complex and thus the degree to which its activity must decline before deleterious consequences ensue. The effects of an abnormality of the respiratory chain will reflect, to a large extent, the dependence of each cell on the supply of ATP from oxidative phosphorylation. ATP can also be generated anaerobically, by glycolysis, and this may be sufficient under certain circumstances to maintain cellular metabolism. Apart, perhaps, from skin fibroblasts, it seems that most tissues depend to a significant extent on oxidative phosphorylation to generate ATP. Tissues such as the central nervous system (CNS), skeletal muscle and heart appear very dependent on aerobic metabolism, which probably explains why these tissues are most commonly affected by disorders of respiratory-chain complexes. Aside from the direct effect on ATP concentration, a defect of the respiratory chain will lead to a variety of other primary and secondary effects. The cofactors nicotinamide adenine dinucleotide (NAD +) and flavin adenine dinucleotide (FAD) (Figure la) are required by numerous enzymes as acceptors of reducing equivalents. Failure to reoxidize these cofactors will have profound effects on cellular metabolism: for example, pyruvate dehydrogenase complex is inhibited by increasing N A D H concentration (Reed and Yeaman, 1987). Increasing concentrations of N A D H will also slow fatty-acid oxidation, an effect thought specifically to involve the NAD+-dependent hydroxyacyl-CoA dehydrogenases (Latip~i~ et al, 1986). Direct evidence that a defect involving complex I of the respiratory chain does affect [3-oxidation, almost certainly at this step, has recently been published (Watmough et al, 1990). In an attempt to overcome the elevated N A D H concentration, cells generate lactate from pyruvate by the reaction catalysed by lactate dehydrogenase (LDH): LDH Pyruvate

.....~Lactate NADH

NAD +

Lactate is exported from the cell giving rise to one of the commonest findings in these disorders, that of lactic acidaemia. From the blood it is transported to the liver. Under normal circumstances, lactate in the liver is used to form glucose or metabolized via the tricarboxylic acid (TCA) cycle. If the liver is itself affected, this will not be possible and lactate concentration in body fluids will rise, often to very high levels. Profound elevation of serum lactate may occur relatively quickly and may give rise to encephalopathy. Localized elevation of lactate may also be responsible for tissue dysfunction, producing symptoms such as muscle fatigue and cramps. It is also possible that,

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in regions of poor perfusion, such as the brain stem and basal ganglia, failure

to remove lactate may lead to tissue damage and focal signs (Robinson et al, 1987a). The role of free radical damage in disorders of the respiratory chain remains uncertain. Normal function of the respiratory chain involves the formation of highly reactive species, f o r example the semiquinone generated by complex III. Moreo~v~r, there is evidence that other free radicals are formed in vivo, especially if electron transport is compromised (Nohl and Hegner, 1978; Krishnamoorthy and Hinkle, 1988; Muscari et al, 1990). Once formed, free radicals are known to cause extensive damage not only to the respiratory chain itself, but also to other proteins, lipid membranes and DNA. It is possible, therefore, that once initiated, defects of the respiratory chain generate increasingly severe disorders by this process. We must restate that the extent to which this occurs is unknown; however, we feel that free-radical damage may be an important factor in the manifestation of respiratory-chain disorders and should not be overlooked. Genetics

Mitochondria are unique in that they contain their own DNA (mtDNA) (Schatz et al, 1964). This is a closed, circular molecular of 16.5 kilobases encoding 13 polypeptides, which are all components of respiratory-chain complexes, 2 ribosomal and 22 transfer RNAs (Anderson et al, 1981) (see Figure 1, Chapter 9). The genes encoded by mtDNA are not, as far as can be ascertained, duplicated in the nuclear genome as functional units, although some similar sequences have been found in non-coding regions (Corral et al, 1989). Several copies of mtDNA are found within each mitochondrion and, depending on cell type, this can vary between 3-4 in platelets and 10 or more in skeletal muscle. As cells such as skeletal muscle contain large numbers of mitochondria, it is clear that one cell can contain many (possibly over 1000) copies of mtDNA. Mitochondrial DNA is inherited exclusively from the mother (Giles et al, 1980). Consequently, if mutation of mtDNA is responsible for inherited disease, such disorders should demonstrate maternal transmission. The majority of respiratory-chain defects are sporadic, although in those cases with a positive family history, evidence of maternal transmission far exceeds that for paternal transmission. Interestingly, two disorders do exhibit clear maternal inheritance--Leber's Optic Atrophy and Myoclonus Epilepsy with Ragged Red Fibres (MERRF). In Leber's Optic Atrophy a mutation of mtDNA involving the ND4 gene has been found (Wallace et al, 1988a), although this occurs in only around 50% of families (Vikki et al, 1989) and is not apparently associated with any biochemical disturbance. In the case of MERRF, defects of several of the respiratory-chain complexes have been identified biochemically, but no abnormality of mtDNA has been shown. Deletions of mtDNA have been described in a number of conditions, but predominantly in the Kearns-Sayre syndrome (Holt et al, 1988; Zeviani et al, 1988). This has not been associated with any consistent biochemical

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lesion nor, in the majority of cases, shown maternal inheritance. These findings are discussed in more detail in Chapter 9. Although we have concentrated on the mitochondrial genome, it must be remembered that mitochondria contain a large number of proteins, relatively few of which are coded for by mtDNA. The majority are transcribed from nuclear genes and synthesized in the cytosol. Most will contain an N-terminal sequence (pre-protein) necessary for targeting (directing the protein to the mitochondrion) and for uptake and correct placement of the protein within the mitochondrion (Hartl et al, 1989) (see Chapter 5). Defects of the proteins encoded by nuclear genes will follow Mendelian patterns of inheritance. Clinical manifestations

This section is not intended to .be an exhaustive review of all reported associations; rather, we look at the major manifestations and review the types and categories of disease associated with respiratory-chain defects. Presentation will clearly depend on the type and number of tissues involved. As mentioned earlier, even though dysfunction of one tissue may dominate the clinical picture, other tissues may still harbour the defect. Two further difficulties associated with the clinical diagnosis of respiratory-chain defects must be highlighted. First, phenotypic expression may vary greatly, despite finding the same (or at least very similar) biochemical defects. Second, it is not yet possible to correlate the severity of the clinical disorder with the extent to which enzyme activity is lost. Both discrepancies may reflect the presence of other genetically determined differences leading to either better or worse handling of the consequences of mitochondrial respiratory-chain dysfunction, e.g. the disposal of lactate or free radicals. It is also fair to point out that these inconsistencies may also reflect our still relatively crude methods of investigation and may therefore be resolved as we improve the techniques and develop further the molecular evaluation. A further confusion has arisen from the descriptive terminology that has evolved for these disorders. The initial discovery that mitochondrial dysfunction could cause disease was made using skeletal muscle mitochondria taken from a woman with euthyroid hypermetabolism (Luft et al, 1962). The cases which followed also tended to have muscular disorders (but not hypermetabolism) plus characteristic histological findings in muscle (Olsen et al, 1972). This led to the commonly used term 'mitochondrial myopathy' (Morgan-Hughes et al, 1977; Petty et al, 1986). As more cases were discovered, involving tissues other than muscle, terms such as mitochondrial encephalomyopathy or cytopathy were coined (Schapira et al, 1977). Most recently, eponyms and acronyms have been introduced to describe subgroups of patients thought to have the same or similar clinical features. Examples of these are Kearns-Sayre Syndrome (KSS) (Berenberg et al, 1977), Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis and Stroke (MELAS) (Pavlakis et al, 1984) and Myoclonus Epilepsy with Ragged Red Fibres (MERRF) (Fukuhara et al, 1980). Unfortunately, despite the similarity of phenotypic expression, these disorders have displayed no consistent biochemically detectable lesion.

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Patients with defects of the respiratory chain can present at any age. Broadly speaking, children and adults present in one of three ways: weakness due to myopathy; multisystem disease which particularly involves CNS; or, less frequently, cardiomyopathy (Table 2). The very young tend to present in a different manner which, only partly, reflects their lack of Table 2. Clinical features found in patients with defects of the respiratory chain, a Infants and neonates

Children

Adults

Fatal lactic acidosis Benign lactic acidosis Encephalopathy Hypotonia Poor feeding/vomiting Failure to gain weight Convulsions Sudden death Psychomotor retardation Developmental delay Convulsions Focal Generalized Myoclonus Ataxia Progressive or transient motor/ sensory loss (stroke) External ophthalmoplegia/ptosis Movement disorder Dystonia Choreoathetosis Retinal degeneration Deafness Myopathy Proximal Fascioscapulohumeral Limb girdle Endocrine disturbance (hypothyroidism) Peripheral neuropathy Stroke Dementia Convulsions Focal Generalized Myoclonus Ataxia Movement disorder Chronic progressive external ophthalmoplegia Myopathy Deafness Optic atrophy (hereditary)

a Although the clinical features are grouped according to age this is an arbitrary distinction and there is considerable overlap. This is especially true between child and adult categories.

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physical and mental development and inability to verbalize. Two manifestations of respiratory-chain dysfunction appear to be exclusive to infants: these are fatal and benign lactic acidosis. Although lactic acidaemia is found in the older age groups, they tend to die from end-organ damage such as cardiorespiratory failure, rather than overwhelming acidosis. Fatal lactic acidosis of infancy has been described with defects of complex I (Moreadith et al, 1984), complex III (Birch-Machin et al, 1989) and complex IV (Minchom et al, 1983). The benign form of lactic acidosis has been described only with complex IV deficiency and is thought to be due to an abnormal fetal isoform; as this is replaced by the mature form the disease disappears (DiMauro et al, 1983; Zeviani et al, 1987). The myopathy associated with respiratory-chain defects has no particular diagnostic features. Fatigue is a common symptom; cramp is often present and this begins early and persists throughout exercise. The distribution of weakness can vary (Table 2) and may be associated with marked wasting. External ophthalmoplegia is a common occurrence and may occur in combination with myopathy. Some of these patients may also have retinal abnormalities such as pigmentation (Petty et al, 1986). Some patients who present with a purely myopathic disease can slowly develop symptoms of CNS involvement such as myoclonus, epilepsy and dementia. Those who manifest CNS disease early generally have a more severe disorder and many, if not most, will die before the age of 20. In those with mild systemic disease, ataxia is often seen and may be the sole symptom. Many of the children with predominantly CNS disease also have associated muscle involvement although, interestingly, this is frequently much less severe than in those with muscle disease alone. Patients with liver involvement tend to have high fasting lactate, but only rarely are found to have liver-cell damage detectable by measuring aminotransferase levels. General aminoaciduria, de Toni-Fanconi-Debr6 syndrome, has been described in several cases of complex IV deficiency (DiMauro et al, 1980; Ogier et al, 1988).

Tissue-specific defects Several of the disorders described in the preceding section are confined to one tissue. The suggestion that components of respiratory-chain complexes may differ from one tissue to another is an important observation and one that may provide insight into the tissue-specific regulation of respiratorychain activity. Explanation of this in molecular terms must, once again, take account of the presence of the two genomes--mitochondrial and nuclear-that encode mitochondrial proteins. As the entire complement of mtDNA is derived from the ovum, a genetic defect in this genome could either be present systemically in the mother, arise during oogenesis or occur during early embryogenesis. As the fetal cells divide and cellular organelles segregate, it is possible to see that an mtDNA mutation could become confined to one cell destined to produce one tissue or group of tissues. Persistence and, indeed, expression of the defect, should depend on the relative amount of abnormal versus normal

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mtDNA present; the presence of two populations of mtDNA is termed heteropIasrny. Wallace, who has extensively reviewed this topic (Wallace, 1987), has postulated that mtDNA mutations may manifest only when present in numbers above a certain 'threshold level'. This is a useful concept as it may be extended to explain why tissues are affected to different degrees, the degree of involvement being proportional to the amount of abnormal D N A over threshold level (i.e. the degree of heteroplasmy). Unfortunately, although deletions may be present systemically and at high frequency compared with normal mtDNA, no correlation has yet been found between the proportion of deleted mtDNA present and disease severity. More work is required to clarify this but at present it is difficult to explain tissue-specific disorders on the basis of mtDNA abnormalities (see Chapter 9). Evidence that abnormalities of nuclear genes produce tissue-specific disease is more compelling (see review by Lomax and Grossman, 1989). Isoforms of three of the smaller subunits of cytochrome oxidase (Via, VIIa and VIII) have been found in bovine and rat tissues (Kadenbach et al, 1982, 1990). The function of these subunits is not yet established, but Kadenbach and colleagues have suggested that one at least may be involved in the allosteric modification of cytochrome oxidase. Similar differences have been found in subunits of bovine complex I (Clay and Ragan, 1988). Although the differences in either complex have yet to be confirmed in human tissues, tissue-specific defects have been reported (Watmough et al, 1989). INVESTIGATION OF PATIENTS WITH RESPIRATORY-CHAIN DEFECTS

As with any other clinical problem, investigation of respiratory-chain defects will be directed by the age and clinical history. It should follow a logical sequence, beginning with simple procedures and advancing to the more specialized and often more invasive. Most of the so-called 'clinical' investigations define the presence only of abnormal mitochondrial function and give no indication of site or which enzyme(s) are involved. Therefore, the investigation of these disorders almost always involves taking tissue, although the tissue sampled will, of course, depend on the presentation and results of preliminary studies. Diagnosis of respiratory-chain defects is difficult and no consensus currently exists as to how this is best achieved. Nevertheless, we believe that by using the tests outlined below in a systematic way, most defects will be identified. Investigation is discussed under three main headings--clinical, biochemical and molecular. Clinical studies

The major diagnostic finding in blood is the presence of an abnormally high lactate concentration. This is often associated with high concentrations of pyruvate (the immediate precursor of lactate) and alanine (produced from pyruvate by transamination). In some instances, high lactate levels are not

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L. A. BINDOFF AND D. M. TURNBULL

found in Serum at rest, even when fasting, and in these patients it may be necessary to provoke a rise in lactate by exercise. This must be performed under strictly controlled conditions with the ready availability of resuscitation equipment; it is not recommended for young children. Even under these conditions, venous lactate may not rise and it appears that there is a group of patients with predominantly CNS disease in whom the elevated lactate may be confined to cerebrospinal fluid (CSF). Examination of CSF is also useful in children with suspected multisystem involvement, as those with KearnsSayre syndrome often have elevated protein levels. Radiological studies provide supporting evidence, but are rarely diagnostic. Both computerized X-ray tomography (CT) and nuclear magnetic resonance imaging (MRI) have been used to demonstrate CNS pathology. The commonest findings are abnormalities within the basal ganglia and cerebellum: on CT these appear as low-attenuation zones, whereas on MRI they produce a high signal, often indicating active disease. Basal ganglia calcification is also seen occasionally, usually in children with slowly progressive disease. In the majority of cases, CNS disease will be apparent on clinical examination; however, in a few, radiologically identified lesions may confirm the suspicion of a mitochondrial disorder and therefore help direct further investigation. The presence of radiological abnormalities within the basal ganglia or cerebellum has been claimed to be diagnostic of a condition called Leigh's disease or subacute necrotizing encephalomyopathy. As it is now known that Leigh's disease may be caused by several enzyme defects, this diagnostic category is unhelpful, apart from indicating the need for further investigation (see later). Electrophysiological studies--electromyography (EMG), nerve conduction studies (NCS) and evoked potential studies (EP)--all have a place in the investigation of mitochondrial disorders but, like radiological studies, they are confirmatory rather than diagnostic. EMG can demonstrate the presence of myopathy and may do so in patients with unsuspected muscle involvement. EP studies may allow preclinical detection of white-matter dysfunction; however, the correlation has not been fully evaluated and once again EP studies define only the presence of disease, not its nature. Much more detailed information, both clinical and biochemical, can be gained from two non-invasive, but time-consuming investigations--NMR spectroscopy and positron emission tomography (PET). 31P NMR spectroscopy permits the study of muscle energy metabolism by the measurement of ATP, phosphocreatinine and inorganic phosphate concentrations, and intracellular pH. By studying the changes in these parameters before and after exercise, it has been possible to identify abnormalities that are highly suggestive of mitochondrial disease (see Griffiths and Edwards, 1987, for review). PET uses isotopes to measure several aspects of cerebral energy metabolism. A recent study (Frackowiak et al, 1988) showed that, in patients with established respiratory-chain disorders, there was an uncoupling of the normal ratio of oxygen consumed per molecule of glucose metabolized. This suggests that some of the glucose was metabolized anaerobically, thus confirming, non-invasively within the CNS, the bio-

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chemical findings established in mitochondrial fractions isolated from skeletal muscle. Both NMR and PET provide much detailed information regarding the distribution of disturbed energy metabolism and may be of increasing value in preliminary diagnosis. However, as neither is yet able to define accurately which enzyme (or enzymes) is affected, tissue diagnosis remains an essential step. Morphological studies

Owing to the frequency with which it is involved, together with the ease of biopsy and low associated morbidity, skeletal muscle has been the preferred tissue to study. Historically, morphological abnormalities provided one of the first indications that disease may result from altered mitochondrial function. The presence of structurally abnormal mitochondria, in increased concentration around the periphery of the muscle fibre, is perhaps the classic morphological observation associated with the so-called mitochondrial disorders. Although many investigators use the Gomori trichrome stain, which prompted the term 'ragged red fibre' (Olsen et al, 1972), the changes are easily, and perhaps more specifically, demonstrated by mitochondriaspecific stains such as succinate dehydrogenase (Figure 2B). In association with the mitochondrial changes, various other structural abnormalities have been reported: atrophy is common, as is vacuolation; this latter finding is of particular importance because the vacuoles often contain lipid, suggesting, therefore, the presence of abnormallipid metabolism (see later) (Figure 2C). Ultrastructural studies have shown various mitochondrial changes, which include loss of internal architecture, abnormal cristae (which are often whorled) and the presence of very regular paracrystalline bodies which appear to be within the inner mitochondrial membrane (Figure 2E). Not all mitochondrial disorders are associated with structurally abnormal mitochondria, so that their absence should be interpreted with caution. In contrast to the purely morphological studies, histochemical and immunocytochemical techniques have identified specific enzyme deficits, namely lowered cytochrome oxidase activity or lowered succinate dehydrogenase (part of complex II) activity. Although it is often possible to diagnose complex IV deficiency histochemically (Figure 2D), cytochrome oxidasenegative fibres have been described in a number of conditions which are not thought to be associated with mitochondrial dysfunction. Immunocytochemistry allows an assessment of whether alteration of activity is associated with a steady-state decrease in immunodetectable protein (Johnson et al, 1988). Biochemical studies

These studies are best performed on fresh tissue; skeletal muscle is the commonest source, but mitochondria can also be isolated from tissues such as heart, liver, cultured skin fibroblasts and platelets. Frozen tissue can be used, but this is associated with a lower yield of mitochondria, which are often broken. A mitochondrial fraction is isolated from whole tissue by

Figure 2. Morphological and cytochemical changes seen in muscle from patients with disorders of the mitochondrial respiratory chain. (A) H/E: shows atrophic fibres in a patient with respiratory chain defect. (B) Succinate dehydrogenase: this shows the characteristically increased staining around the periphery of fibres due to the accumulation of mitochondria. (C) Oil Red O: this stain detects neutral lipid. This section shows abnormal accumulation in some fibres in a patient with complex I deficiency. (D) Cytochrome oxidase: D1, control--showing activity in all fibres; D2, patient--with cytochrome oxidase deficiency causing Leigh's disease. (E) Electron micrograph: shows abnormal mitochondria beneath the sarcolemma. The mitochondria are large and contain abnormal cristae and paracrystalline inclusions (arrow). We are grateful to Dr M. A. Johnson for Figures 2A-D and Dr M. J. Cullen for Figure 2E.

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Figure 3. Diagrammatic representation of respiratory chain showing substrates used to assay flux and sites of action of inhibitors used to assay complexes. Investigation of respiratory chain activity measures flux through the whole chain and the activity of the individual complexes. Flux can be measured in the oxygen electrode (polarographically) or spectrophotometrically. Different substrates feed electrons into different parts of the respiratory chain and this can provide information concerning the site of a defect in either complex I or II. Ferricyanide accepts electrons from cytochrome c and therefore assesses flux through at most three of the four complexes. The individual complex activities are measured after isolating the complex of interest by the use of specific inhibitors. In the example shown (inset), complex II activity alone is measured by following the flow of reducing equivalents from succinate to the dye DCPIP. Complexes I and III are inhibited by rotenone and antimycin, respectively, ensuring that only complex II activity is measured. Fe(III), ferricyanide; Fe(II), ferrocyanide; TFA, trifluoroacetone; DCPIP, dichlorophenolindophenol.

homogenization and differential centrifugation. The type of tissue will influence the method of homogenization: tissue such as skeletal muscle requires vigorous mechanical disruption, whereas liver, which is much softer, requires less. For example, we use an Ystral tissue homogenizer (setting 9, 4-5 s) for skeletal muscle and a power-driven Teflon-on-glass homogenizer (10 up-and-down strokes) for liver. Cell membranes and nuclear debris are removed by low-speed centrifugation and kept for analysis of DNA, while the mitochondrial fraction is isolated from the supernatant by high-speed centrifugation. Measurement of respiratory-chain activity is not straightforward. Historically, many investigators studied mitochondrial function using the oxygen electrode. By incubating mitochondrial fractions with different substrates (Figure 3) and limiting amounts of ADP, it is possible to monitor oxygen consumption during both active respiration/ATP formation (state 3 respiration) and the basal state (state 4). If there was significant slowing of oxygen consumption with NAD+-linked substrates (e.g. pyruvate), but not succinate, this would suggest a disorder of complex I. Alternatively, if the succinate rate alone was slowed, this would point to a defect of complex II. If both rates were abnormal, the defect lay further along the chain between coenzyme Q and cytochrome oxidase. Since such measurement is performed on intact mitochondria, oxygen consumption is reliant on more than respiratory chain function: substrate transport across the inner mitochondrial

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MEASUREMENT OF CYTOCHROME CONCENTRATIONSLow temperature reduced minus oxidised spectra

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FLUX MEASUREMENT

Figure 4. Schematic representation of our approach to biochemical diagnosis. Measurements of flux and cytochrome concentration require intact mitochondria and are therefore performed on day the mitochondrial fraction is isolated. In case of suspected involvement of other pathways, such as ~-oxidation, these also require fresh, unbroken mitochondria.

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membrane, the activity of various NAD+-linked and FAD-linked dehydrogenases, the adenine nucleotide translocator and complex V (ATP synthase) are all necessary for continued respiratory-chain activity under these conditions. Moreover, maximal activity of each respiratory chain complex differs; therefore, measurement of flux, such as that described above, depends on the slowest, that is to say, rate-limiting step. A genetic defect may still be important and result in defective respiratory-chain function in vivo, but may not be detected by flux measurement, which tends to average the activities of the complexes. This latter point is also important when considering any assay which measures activity in more than one complex, e.g. NADH-cytochrome c reductase and succinate cytochrome c reductase. Our approach to diagnosis is illustrated in Figure 4. We use flux measurement as a screening procedure, but do this spectrophotometrically using ferricyanide which accepts electrons from cytochrome c (Figure 3). Once again, the reasoning elaborated above holds true for these assays and abnormalities are detectable using this technique. We do not, however, rely on ferricyanide assays alone: whether or not we find slowing of N A D H ferricyanide or succinate-ferricyanide oxidation, we measure activity of the individual complexes in all cases of suspected respiratory-chain defects (Birch-Machin et al, 1989; Watmough et al, 1990). Ferricyanide assays do have an additional use: by using at least three different NAD÷-linked substrates, it is often possible to detect cases of pyruvate dehydrogenase complex deficiency that show abnormal rates of pyruvate but normal glutamate and oxoglutarate oxidation, unless the E3 component is affected. The activity of each complex can be measured separately by using inhibitors to isolate the complex of interest by blocking the remaining respiratory chain components (Figure 3). For example, complex II activity is measured in the presence of rotenone (a complex I inhibitor) and antimycin (a complex III inhibitor); electrons passed from succinate to an artificial electron acceptor, dichlorophenolindophenol (DCPIP), can then be monitored without loss via other parts of the respiratory chain (see inset, Figure 3). Measurement of complex I activity is complicated by the presence of at least one other enzyme able to oxidize NADH, the one thought to be most commonly involved being cytochrome b5 reductase found in the outer mitochondrial membrane (Kuwahara et al, 1978). For this reason, it is necessary to measure both total and rotenone-sensitive NADH oxidation and to calculate complex ! activity by subtraction. A further problem, both for complex I and the other complexes, is ensuring that the assay measures all the available enzyme activity. Respiratory-chain complexes are large structures, much of which is buried within the inner mitochondrial membrane. Generally, to release maximal enzyme activity, it is sufficient to disrupt the membrane by three cycles of freezing (in liquid nitrogen) and thawing. This is true for complexes II and IV and probably for complex I; however, maximal complex III activity requires additional treatment with detergent, perhaps because the analogue of ubiquinone used in the assay is not as lipid-soluble as that found in vivo (unpublished results). Whether similar treatment is required for complex I is still under investigation, but our initial studies have not substantiated this. Complexes I and II activities

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L. A. BINDOFF AND D. M. TURNBULL

CONTROL SUCCINATE + KCN

aa3

b

0.06

J i

I

I

I

i

SKELETAL MUSCLE (SUCCfNATE + KCN C

0.075

Z I

I

I

I

530

540

550

560

Figure 5. Reduced-minus-oxidizedspectra of the mitochondrial cytochromes. Reduced spectra are obtained in the presence of substrate, ADP and cyanide. Cyanide prevents terminal electron transport to oxygen so that the cytochromes are held in the reduced state by the continuous input of electrons from substrate (usually succinate), In the absence of substrate, the cytochromes remain oxidized and the difference (reduced-minus-oxidized) allows assessment of these respiratory-chain components separate from the many other compounds which absorb at similar wavelengths. The top scan shows the normal spectra with each of the five cytochromes marked. The lower scan is from a patient with cytochrome oxidase deficiency, which results in lowering of the aa3, peak.

599

DEFECTS OF THE RESPIRATORY CHAIN

are linear with time, whereas complexes III and IV are not and are best expressed in terms of first-order kinetics (k.s-L). Having found low activity in one or more complexes, we use lowtemperature reduced-minus-oxidized spectroscopy and/or Western blotting to evaluate steady-state levels of the components of each complex. These techniques measure the relative amounts of subunit at the moment of sampling; hence, they reflect the steady state, but give no indication of turnover (see later). A brief description of each is given, but for a more detailed account readers are referred to standard biochemical texts. Low-temperature reduced-minus-oxidized spectroscopy allows semiquantitative measurement of the concentrations of five haem-containing cytochromes--b and cl, in complex III, the mobile electron carrier cyto1

2

3

4

5

6

7 Mr 110

75 - -

51 49

- -

42 39

- -

30

- -

20

- -

18

- -

15

24

13 - -

10

Figure 6. Western blot of mitochondrial fractions--complex I. This immunoblot was probed with antibodies raised against complex I; molecular weight of the immunodetectable subunits shown on right. Purified complex I (different loadings) is in lanes i, 2 and 7. Patients with complex I deficiency are in lanes 4 and 5 (100~xg loaded), controls in lanes 3 and 6 (100txg loaded). There is a generalized decrease in all the immunodectable suhunits in both patients.

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L. A. BINDOFF A N D D. M. T U R N B U L L

chrome c and cytochromes a and a3 in complex IV (Figure 5). This technique is neither simple to perform nor easy to interpret. Much confusion still surrounds the choice of extinction coefficients used to calculate concentration, which in turn reflects the difficulties encountered in the original attempts to measure the relative amount of each cytochrome in the normal complex. We use the values given by Tervoort et al (1981) and calculate the concentrations of the cytochromes by solving the simultaneous equation given by these authors. This takes into account not only the absorption maxima but also the contribution of each cytochrome to the absorption of the others. The most commonly used values do not make this compensation and also use a coefficient for cytochrome aa3 that produces results 50% greater than expected (Bookelman et al, 1978). By combining this assessment of concentration and the results of activity measurement, it is possible to obtain an approximate turnover number for complexes III and IV (turnover no. = activity/unit enzyme). More detailed assessment of the subunit composition of each complex may be obtained by Western blotting. Antibodies to respiratory-chain complexes have generally been raised in rabbits against bovine proteins. This technique is entirely dependent on the quality of the antibody, i.e. the specificity with which it recognizes the proteins of interest (Figure 6). This in turn is dependent on the purity of material used for immunization. Due to the problems associated with extracting the large and often very hydrophobic, multicomponent complexes from mitochondria, good antibodies are difficult to raise. In addition to their use in Western blotting, which reflects only steady-state levels, antibodies are also useful in the study of protein turnover. This is performed with cells in culture and consists of labelling proteins by growing the cells in the presence of [35S-]methionine. The proteins of interest are then immunoprecipitated using complex-specific or even component-specific antisera, and then compared fluorographically with control after separation by SDS-PAGE.

Molecular biology Thus far, our discussion has concentrated on measuring activity and identifying the abnormal protein(s). Recent developments in molecular biology mean that it is now possible to extend the investigation to defining the genetic defect responsible for these changes. Mitochondrial D N A has been fully sequenced and coding regions have been assigned to 13 polypeptides and 24 RNAs (Anderson et al, 1981). The remaining components necessary for respiratory-chain synthesis and function are encoded by nuclear genes and an increasing number of these have been identified and sequenced. Having established abnormal respiratory-chain function and localized the defect to one or more components, it should, therefore, be possible to investigate the molecular nature of these defects. That this has not yet been possible is due to the complexity of the respiratory chain, which gives rise to two main difficulties: first, the respiratory chain is a functionally integrated system so that activity measurements often define more than one affected site; second, complex assembly appears to be an ordered process,

DEFECTS OF THE RESPIRATORYCHAIN

601

dependent on the presence of most, if not all, of the subunits. Loss of one m a y lead to misassembly and apparent lowering of all components when investigated by Western blotting. For complexes which contain large numbers of c o m p o n e n t s it is, therefore, difficult to know where to start looking for the genetic defect. Despite these problems, several interesting observations, primarily involving m t D N A , have been made. Southern blotting (Figure 7) and m o r e recently the polymerase chain reaction (PCR) have identified large deletions and insertions in a n u m b e r of patients with mitochondrial disorders (see C h a p t e r 9). The two main groups harbouring these changes are patients with K e a r n s - S a y r e Syndrome or Chronic Progressive External Ophthalmoplegia (Holt et al, 1988; Zeviani et al, 1988), although isolated

1234567

16.5-~

Figure 7. Southern blot analysis of mtDNA from patients with mitochondrial disorders and controls. Total DNA is digested with PvulI and electrophoresed through agarose. The DNA is then transferred to a nylon membrane and probed with radioactively labelled whole mtDNA. Only wild-type, normal mtDNA (16.5kb) is seen in controls (lanes 1 and 5) and two patients with mitochondrial myopathy (lanes 4 and 6), whereas smaller speciescan be seen in lanes 2 and 3 (patients with chronic progressive external ophthalmoplegia) and lane 7 (patient with mitochondrial myopathy).

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cases with other presentations have been reported (ROtig et al, 1988). No association between biochemical defect (which is often obscure in these groups) and missing mtDNA has yet been found. Moreover, although correlation between number of deleted copy and clinical effect has been demonstrated in animal-cell studies, this has not been established in human cases. Nevertheless, the relative specificity of deletions suggests some role in pathogenesis, even if the mechanism for this remains unclear (see Chapter 9; Holt et al, 1989; Schon et al, 1989 for discussion). In concluding this section we would like to reiterate our earlier statement that the investigation of respiratory chain disorders is far from straightforward. Diagnosis cannot rest on one abnormal investigation, but should always be substantiated by others, e.g. abnormal activity supported by low cytochrome concentration and/or low subunit concentration on Western blot. Following the protocol we have outlined above, diagnosis should follow a logical sequence, starting with clinical evaluation and proceeding through activity measurement (flux and individual complexes), assessment of steady-state subunit concentrations of the affected complex(es) and ending with investigation of the genetic abnormality.

Antenatal diagnosis Although many of the disorders resulting from respiratory-chain dysfunction are sporadic and therefore unexpected, antenatal diagnosis would be of great value in families who already have one affected child. All the activity measurements and Western blot analysis can be performed on very small amounts of tissue and could therefore be adapted to chorionic villus samples (Besley and Broadhead, 1989). This is also true for cultured amniocytes, which may provide even more material. Cultured amniocytes grown on cover-slips provide a quick method of diagnosis of some disorders in which good antibodies are available. Brown and colleagues used this technique to investigate a fetus with pyruvate dehydrogenase deficiency (Brown et al, 1989) and were able to show decreased immunoreactive material confirming, antenatally, the presence of a defect. REVIEW OF CLINICAL FEATURES ASSOCIATED WITH RESPIRATORY-CHAIN DEFECTS There are an increasing number of reports describing individuals with respiratory-chain defects. Some appear to be attributable to one affected complex, whereas others, and possibly the majority, have multiple abnormalities. In view of the integrated nature of the respiratory chain, the latter are, perhaps, more expected. Those with single defects may represent early cases in which only the primary defect is apparent; however, because of the difficulties surrounding the biochemical analysis of these disorders, it is also possible that they reflect incomplete investigation. Apart from the case of benign lactic acidosis, no relationship has been found between biochemical defect (either which abnormal complex or

D E F E C T S OF T H E R E S P I R A T O R Y C H A I N

603

which part thereof) and clinical phenotype. A detailed review of each case of respiratory-chain deficiency is therefore unnecessary. We briefly summarize the reported clinical features associated with both single and multiple defects. As outlined earlier, these broadly conform to three categories-those having myopathy as the major and possibly only feature, those with multisystem disease that especially affects the CNS, and those with apparent single-organ involvement such as cardiomyopathy. We review separately those disorders that have not been fully defined in biochemical terms (KSS) and those that can result from different defects (MELAS, M E R R F and Leigh's syndrome). Lastly, we discuss Luft's syndrome and other disorders which give rise to clinical syndromes indistinguishable from those caused by respiratory-chain defects.

Complex I Defects affecting complex I alone or in combination with other complexes are probably the most commonly identified disorders of the mitochondrial respiratory chain. This is, perhaps, hardly surprising as complex I is the largest of the complexes, with 25 or more subunits (see Table 1). However, as discussed earlier, owing to the significant problems associated with measurement of complex I activity, many case reports must be interpreted with caution. A list containing most of the reported cases is given in Table 3. It is not our intention to discuss the relevant investigation and findings reported, but readers are urged to interpret the data with reference to the preceding discussion. Recently, abnormal complex I activity has been reported in two conditions not previously considered to be caused by mitochondrial dysfunction. Studies have shown that tissue hypoxia leads to lowered complex I activity (DarleyUsmar and O'Leary, 1988). Interestingly, maximal damage is not sustained during oxygen starvation, but during re-perfusion. This strongly suggests that free radicals do, indeed, have a role in secondary damage of the respiratory chain as these species are abundant at this time. Moreover, this may also indicate that free radicals are part of the pathophysiology of respiratory-chain defects themselves. These studies also highlight the danger of investigation of the respiratory chain in tissue that has been poorly handled. The second condition in which abnormal complex I activity has been found is Parkinson's disease. The possibility that this disorder may be associated with abnormal respiratory-chain function arose from studies on drug-induced parkinsonism. A by-product of illicit meperidine synthesis, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), was found to induce symptoms of classic Parkinson's disease. Further study showed that this compound is metabolized to methylphenyl pyridinium (MPP+), which is actively taken up by the dopamine uptake system into striatal neurones. MPP + is also actively transported into mitochondria (from any tissue) where it is a competitive complex I inhibitor. With this model has come much recent speculation that idiopathic Parkinson's disease may be due to an environmental toxin which poisons the respiratory-chain complex I. Although Schapira and colleagues (1989) have shown lowered complex I

604

L. A. BINDOFF AND D. M. TURNBULL Table 3. Clinical presentation of disorders of complex I

Type of clinical presentation Myopathy

Encephalopathy

Infantile

Reference"

Comment

1-11

These cases have been grouped together even though some may show clinical features outside muscle, such as ophthalmoplegia and retinal pigmentation, Also, as investigation has improved, the presence of other abnormalities has become apparent: e.g. case in (11) showed a defect of complexes III and IV and, as would be predicted in complex I deficiency, a profound abnormality of fatty-acid oxidation

6, 10, 12-30 Several different clinical syndromes were reported within this category. Patients were reported as having MELAS, one with M E R R F and one with Leigh's. Multiple defects were common. The case described in (23) had a clear abnormality of fat metabolism 20, 26, 29 The cases in (26) were all investigated as cultured skin fibroblasts. In one of the members of the family described in (28) studies showed that the patient's mitochondria synthesized an abnormalsized mitochondrially encoded subunit of complex I

Tables 3-6 give a sample of the many cases reported with disorders of the mitochondrial respiratory chain. References are numbered and shown below in abbreviated form for all four tables; the complete reference is contained in the list at the end of this chapter. 1. Morgan-Hughes et al (1979) 2. Land et al (1981a) 3. Land et al (1981b) 4. Arts et al (1983) 5. Clark et al (1984) 6. Morgan-Hughes et al (1984) 7. Sherratt et al (1984) 8. Morgan-Hughes et al (1985) 9. Morgan-Hughes et al (1987) 10. Schapira et al (1988a) 11. Watmough et al (1990) 12. Tanaka (1987) 13. Senior and Jungas (1974) 14. Prick et al (1981) 15. Morgan-Hughes et al (1982) 16. Moreadith et al (1984) 17. Hayes et al (1985) 18. Kobayashiet al (1986) 19. Tanaka et al (1986) 20. Robinson et al (1986) 21. Van Erwen et al (1986) 22. Garcia Silva et al (1987) 23. Hoppel et al (1987) 24. Kobayashi et al (1987) 25. Ichiki et al (1988b) 26. Robinson et al (1987a) 27. Van Erwen et al (1987) 28. Wijburg et al (1989) 29. Byrne et al (1988) 30. Ichikiet al (1988a) 31. Rivner et al (1989) 32. Riggs et al (1984) 33. Fischer et al (1986) 34. Martin et al (1988) 35. Desnuelle et al (1989) 36. Sengers et al (1983) 37. Behbehani et al (1984) 38. Sperl et al (1988) 39. Morgan-Hughes et al (1977) 40. Hayes et al (1984) 41. Kennaway et al (1984) 42. Reichmann et al (1986) 43. Schapira et al (1988b) 44 Darley-Usmar et al (1983) 45. Darley-Usmar et al (1986) 46. Eleff et al (1984) 47. Argov et al (1986) 48. Spiro et al (1970) 49. Papadimitriou et al (1984) 50. Sengers et al (1984) 51. Birch-Machin et al (1989) 52. Przyrembel (1987) 53. Scholte et al (1987) 54. Rimoldi et al (1982) 55. Zeviani et al (1986) 56. Servidei et al (1987) 57. Koga et al (1988b) 58. Oldfors et al (1989) 59. Miyabayashi et al (1983) 60. Angelini et al (1986) 61. Maertens et al (1988) 62. Van Erwen et al (1988) 63. Willems et al (1977) 64. Miyabayashi et al (1983) 65. Arts et al (1987) 66. DiMauro et al (1987) 67. Miyabayashi et al (1987) 68. Ogier et al (1988) 69. DiRocco et al (1988) 70. Shepherd et al (1988) 71. Hayasaka et al (1989) 72. Miranda et al (1989) 73. Van Biervliet et al (1977) 74. DiMauro et al (1980) 75. Heiman-Patterson et al (1982) 76. Stansbie et al (1986) 77. Boustany et al (1983) 78. Minchom et al (1983) 79. Trijbels et al (1983) 80. Bresolin et al (1985) 81. Zeviani et al (1985) 82. DiMauro et al (1983) 83. Zeviani et al (1987) 84. Jerusalem et al (1973)

DEFECTS OF THE RESPIRATORYCHAIN

605

activity in h o m o g e n a t e s of striatum taken post m o r t e m from patients with Parkinson's disease, and Parker et al (1989) have shown abnormal N A D H oxidation in platelet mitochondria, other complexes may also be involved (Bindoff et al, 1989).

Complex II This is the simplest in structure of all the complexes (Table 1). Despite this simplicity, however, complex H occupies a unique position in cell metabolism, functioning in both the respiratory chain and T C A cycle. Abnormalities of complex II are rare, with only 10 cases having been reported (Table 4). Fewer problems surround the interpretation of abnormal complex II activity because it is less sensitive to poor handling and can be assessed both histochemically ( S D H - - s e e Figure 2B) and biochemically ( S D H ; succinate-ferricyanide and complex II assay). Table 4. Clinical presentation of disorders of complex II. Type of clinical presentation Myopathy

Reference" Comment 31

Encephalopathy

32-35

Infantile

36-38

Developed normally until 6 years then developed gradual weakness, ptosis, ophthalmoplegia and respiratory weakness The cases in (32) were siblings. Both presented when 5 years old with limb and gait incoordination and then developed seizures. The biochemical studies showed normal NADH cytochrome c reductase activity in both, low succinate cytochrome reductase activity in one and low cytochrome oxidase activity in the other. Symptoms in this group include psychomotor retardation, spastic paraparesis and seizures. The cases in (35) are non-identical twins with mental retardation and behavioural problems; one developed weakness around 18 years old Infants in this group presented with feeding and respiratory difficulties, failure to thrive and motor delay. All had marked lactic acidosis and all but one (36) died before 8 months old.

a See footnote to Table 3 for references.

Complex III Defects of complex III are, like those of complex II, relatively u n c o m m o n (Table 5). Analysis of complex III is facilitated by the presence of two c y t o c h r o m e s - - b and cl. Reduced-minus-oxidized spectra allow the concentration of these subunits to be estimated and hence permit direct assessment of the concentration of complex III. In some cases of complex I I I deficiency, cytochrome b (and occasionally c1) content has been found to be low, whereas in others it is normal. In those cases with low or absent cytochrome b, it is not possible to infer that it is this, mitochondrially encoded subunit, that is

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L. A. BINDOFF AND D. M. TURNBULL

Table 5. Clinical presentation of disorders of complex III Type of clinical presentation Myopathy

Reference a Comment 39-43

Encephalopathy

48, 15, 8

Cardiomyopathy

49

Infantile

50-53

The case in ref. 41 has been very thoroughly investigated with subunit analysis (44, 45), NMR (46, 47) and was thought to be specific to muscle (45). All but one of the cases had lowered concentration of cytochrome b. One patient (43) was thought specifically to have lost the Reiske iron-sulphur protein due to a block in uptake. This patient had more marked complex II deficiency In two studies (48 and 8) familial cases were reported: in (48), father and son and in (8) mother and daughter. Cytochrome b concentration was low in five (out of twelve) and cytochromes b and cl were low in one case The defect in this child was confined to the heart, liver and skeletal muscle being normal The case in (51) represents the most completely investigated patient to date. Low complex III activity was documented in several tissues and Western blotting showed disproportionate loss of the iron-sulphur protein and cytochrome b.

a See footnote to Table 3 for references. affected. Loss of the Reiske i r o n - s u l p h u r protein has b e e n shown to be associated with lower c y t o c h r o m e b c o n c e n t r a t i o n in the yeast Saccharomyces cerevisiae ( L j u n g d a h l et al, 1989). In addition, loss of a n o t h e r of the smaller, n u c l e a r - c o d e d subunits (VII) also results in the loss of c y t o c h r o m e b and F e - S protein ( S c h o p p i n k et al, 1989).

Complex IV A b n o r m a l i t i e s of c y t o c h r o m e oxidase are relatively c o m m o n a m o n g respiratory-chain defects. T h e same b r o a d categories that are seen in defects of c o m p l e x e s I - I I I m a y be defined for this complex, but in addition, a benign or reversible disorder has also b e e n d o c u m e n t e d (Table 6). This disorder presents in infancy with lactic acidosis, m y o p a t h y and low e n z y m e activity, but with n o r m a l a m o u n t s of i m m u n o r e a c t i v e protein. T h e course is o n e of progressive i m p r o v e m e n t and return of e n z y m e activity and it has b e e n postulated that the condition m a y be due to an a b n o r m a l fetal protein, i m p r o v e m e n t being due to the expression of a n o r m a l , adult f o r m of the affected subunit. Investigation of c o m p l e x I V is possible using a variety of m e t h o d s . Activity can be m e a s u r e d in tissue h o m o g e n a t e s and isolated mitochondrial fractions and the c o n c e n t r a t i o n estimated by redox spectroscopy (complex I V contains c y t o c h r o m e s a + a3) and W e s t e r n blotting; these m e t h o d s have b e e n discussed earlier. C y t o c h r o m e oxidase activity can also be m e a s u r e d cytochemically and this has p r o v i d e d s o m e interesting insights. L o w activity has b e e n f o u n d in individual fibres of skeletal muscle biopsies t a k e n f r o m

DEFECTS OF THE RESPIRATORY CHAIN

607

Table 6. Clinical presentation of disorders of complex IV. Type of clinical presentation

Reference"

Myopathy

54-58

Encephalopathy

59-62

+ Leigh syndrome

Cardiomyopathy Infantile Fatal

Benign

63-72

54, 55, 58

73-81

82-84

Comment These cases had symptoms confined to skeletal muscle except (55); of the cases described by (58), one had a sibling who was similarly affected while another had a normal dizygotic twin. Biochemical diagnosis was made in skeletal muscle These cases showed gradually progressive psychomotor retardation, muscle wasting and in two cases seizures and ataxia. The child described in (61) had intracranial calcification The majority of these cases had autopsy-proven subacute necrotizing encephalomyelopathy although occasionally authors relied only on CT scan findings. Cytochrome oxidase deficiency generally shown in skeletal muscle although in some instances several tissues were studied (66, 67). Generalized aminoaciduria (de Toni-FanconiDebrd syndrome) was found in (68) and (70) The cases had coexistent myopathy and cytochrome oxidase was measured in muscle Uniformly fatal course (case in 79) and all except the case reported in (80) had generalized aminoaciduria. Unless ventilated, all died before 6 months of age Although no biochemical studies were performed in (84), it seems to be same disorder.

See footnote to Table 3 for references.

patients with Kearns-Sayre syndrome and chronic progressive external ophthalmoplegia. However, when measured in homogenates or mitochondrial fractions, activity has been normal. Presumably, this reflects the mixing of normal with abnormal mitochondria that occurs during tissue disruption. Interestingly, cytochrome oxidase-negative fibres have been found in a variety of other conditions (e.g. diabetes mellitus, polymyositis) which are not clearly linked to mitochondrial dysfunction; it has been suggested, therefore, that these findings may reflect an external stimulus such as hypoxia (Stadhouders, 1981) or be part of the ageing process (Muller-H6cker, 1989).

Complex V Defects of complex V appear very uncommon. However, complex V activity has been measured in few cases and in these studies only ATP hydrolysis has been evaluated. Whether this assessment will detect abnormalities within the ATP synthase component (which contains the mitochondrially encoded subunits) is not clear. The patients described with defects are a 37-year-old woman with limb weakness and abnormal accumulation of mitochondria in muscle (Schotland

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et al, 1976) and a second patient with myopathy, dementia, ataxia, retinal degeneration and peripheral neuropathy (Hayes et al, 1982). In both, mitochondrial respiration was decreased with all substrates but returned to normal after addition of the uncoupler 2,4-dinitrophenol. Multiple deficits So far we have discussed defects of respiratory chain complexes separately. Although this is how many cases were described, it has become clear that, as our investigations become more sensitive, abnormalities of more than one complex are common (Ruitenbeek et al, 1989). This would fit with the integrated nature of the respiratory chain, but may also reflect the influence of secondary, toxic phenomena, e.g. free-radical formation. Often, one complex appears more severely affected than the others. Whether this can be interpreted as reflecting a primary site of abnormality is unknown. Certainly, the observation of one profound abnormality associated with one or two less-affected complexes suggests that the latter have occurred secondarily (see Watmough et al, 1990); however, it should also be borne in mind that all three may be secondary. For instance, iron deficiency has been shown to lower activity in complexes I, II and III in experimental animals (Ackrell et al, 1984). Other possibilities that must be considered include a defect involving ubiquinone (see later) or the phospholipid environment which is crucial for mobility (of UQ and cyt c) and electron transport. Various combinations of abnormal complexes have been described. Most commonly these involve complexes I and IV (Sherratt et al, 1984; Byrne et al, 1988) and complexes III and IV (Kennaway et al, 1987) although we are now seeing an increasing number of cases with defects of three complexes.

DESCRIPTIVE SYNDROMES

MERRF: Myoclonus Epilepsy with Ragged Red Fibres This acronym was coined by Fukuhara although the condition was first described by Tsairis et al in 1973. It is, as described, an encephalomyopathy with seizures, mental and physical retardation and limb weakness. Biochemical studies have shown that it may be caused by defects of a variety of complexes: complex I (Garcia Silva et al, 1987), complex IV (Berkovic et al, 1987; Mendell et al, 1987), complexes I and IV (Wallace et al, 1988b; Byrne et al, 1988) and complexes I, III and IV (personal observation). It is a multisystem disorder probably caused by several different biochemical defects. Only more detailed knowledge of the biochemical and molecular basis will allow accurate and useful subdivision of the clinical phenotypes.

MELAS: Myopathy, Encephalopathy, Lactic Acidosis and Stroke The presence of stroke-like episodes marks this syndrome as one of the most interesting of the multisystem disorders. Several points are worth reviewing

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609

in some detail: these include the occurrence of stroke-like events, the presence of migraine and the lack of clear biochemical definition. Strokes appear to be unusual in mitochondrial disease. CT evidence from several subclassifications of mitochondrial disease has, however, shown the presence of low-attenuation zones, predominantly involving basal ganglia and cerebellum. A recent study has shown that the vascular supply to the affected region remains intact, suggesting, therefore, that the deficit is due to abnormal metabolism (Seyama et al, 1989). The association of migraine, which often heralds an episode of infarction, is fascinating; such patients appear to have higher levels of lactate in CSF than those found in blood, reflecting possibly an abnormal capacity to deal with lactate (either generally or specifically within the CNS). Of equal interest is the suggestion of an overlap between isolated migraine and respiratory-chain disease (Montagna et al, 1989). Biochemical studies have shown that various defects can give rise to this phenotype (Pavlakis et al, 1984; Kobayashi et al, 1987; Montagna et al, 1988). The case reported by Byrne et al (1988) did have abnormal activity of both complexes ! and IV and also appears to have changed from one phenotype (MERRF) to another (MELAS). This latter point highlights the problems associated with too great a reliance on clinical syndromes!

Kearn-Sayre Syndrome~ChronicProgressiveExternal Ophthalmoplegia Considerable nosological debate surrounds these two clinical syndromes (see Berenberg et al, 1977; Bastiaensen et al, 1978). The primary features of KSS include progressive external ophthalmoplegia, atypical pigmentary degeneration of the retina and heart conduction block (Kearns and Sayre, 1958; Rosenberg et al, 1968); spongy degeneration of the brain underlies the progressive nature of the disorder (Daroff et al, 1966). In addition to the three cardinal features, there are numerous other, less constant, associations such as cerebellar ataxia, short stature, hearing loss, endocrine abnormalities, myopathy, elevated CSF protein, and cranial and peripheral neuropathies. Many of the same features are found in patients with CPEO, although not with the same frequency. Age has formed the major division between these two groups: Rowland and colleagues have suggested that KSS is a true disease entity characterized by the occurrence of the three primary clinical features before the age of 20. Whether these conditions are, indeed, separate or part of a spectrum is outside the scope of our discussion. Two interesting features of these conditions are, however, worth mentioning: most patients with KSS or CPEO have been found to have deletions or insertions of their mitochondrial DNA and biochemical investigation has shown no consistency of affected complex or complexes. A number of these patients do, however, have abnormal cytochrome oxidase activity demonstrated histochemically (Johnson et al, 1983).

Leigh syndrome Subacute necrotizing encephalomyelopathy or Leigh syndrome is a diagnosis based on the neuropathological findings in brain of spongy necrosis

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with glial reaction and vascular proliferation (Leigh, 1951). Ante mortem the syndrome may be suspected by the combination of lactic acidosis, encephalopathy and the presence of low-attenuation regions in the basal ganglia or cerebellum on CT scan. Unfortunately, like most of the other descriptive terms used in respiratory-chain disease, it carries no diagnostic implications concerning the underlying biochemical defect (Van Erwen et al, 1987a). Leigh syndrome has been definitely associated with defects of pyruvate dehydrogenase complex (Kretzschmar et al, 1987; Kerr et al, 1987), cytochrome oxidase (see Table 6), complex ! (Van Erwen et al, 1986) and biotinidase deficiency (Baumgartner et al, 1989). Other and allied conditions

Luft's disease Only two cases of this disorder have been described (Luft et al, 1962; DiMauro et al, 1976). The condition manifests as euthyroid hypermetabolism and is associated with structurally abnormal mitochondria which show poor respiratory control but normal phosphorylation capacity. This state of socalled 'loose coupling' is thought to be secondary to inability of mitochondria to retain calcium. Constant recycling of calcium results in sustained, maximal respiration and hypermetabolism.

Ubiquinone deficiency This condition, although hinted at for some time, was only recently described by Ogasahara and colleagues in two sisters with an encephalomyopathic disorder. They were able to show that, whereas state 3 oxidation of both NAD+-linked and FAD-linked substrate, was slow, activity in each complex measured separately was normal. Further, they measured the concentration of UQ10 in both patients' mitochondria and found this to be low (Ogasahara et al, 1989). Ubiquinone concentration has also been found to be low in one case of Kearns-Sayre syndrome, although in four other similarly affected cases and 12 cases of chronic progressive external ophthalmoplegia, UQ concentration was normal (Zierz et al, 1989).

Pyruvate dehydrogenase complex (PDC) Although not part of the respiratory chain, PDC is a key enzyme in intermediary metabolism. Defects of this enzyme give rise to clinical disorders very similar to those found in respiratory-chain disease. PDC deficiency is considered to be the commonest cause of primary lactic acidosis. Abnormalities of PDC have been reported in fatal congenital lactic acidosis. (Str6mme et al, 1976), subacute necrotizing encephalomyopathy--Leigh syndrome (see above)--and intermittent ataxia (Blass, 1980). (For a review of clinical findings in PDC deficiency see Stansbie et al, 1986). Of the three enzymes that comprise PDC, disorders of Ea are the most commonly reported. Robinson et al (1987b) investigated patients with E~ deficiency and were able to identify three broad categories:

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1.

2.

3.

611

Very low PDC activity associated with severe lactic acidaemia and death before 6 months of age. As with all groups, the central nervous system (CNS) bears the brunt of the damage, with agenesis of the corpus callosum and widespread cystic changes. A group with significant structural CNS damage and associated psychomotor retardation. These less severely affected children usually have significantly elevated lactate levels; however, in some cases this is not found in blood, only in CSF (Brown et al, 1988). CNS lesions found in this group include cystic changes, especially within basal ganglia, and changes compatible with Leigh syndrome. Mild lactic acidosis, which may not be manifested except during periods of stress, for example due to infection, when patients may also develop ataxia. Psychomotor retardation may be mild or unapparent. PDC activity in this group is often greater than 20%, whereas in groups 1 and 2 it is usually very low, but variable.

Fumarase deficiency is another rare cause of encephalomyopathy that is worthy of mention. Four cases have now been described and clinical presentation is again similar to that of respiratory-chain disorders (Zinn et al, 1986; Walker et al, 1989). Other TCA enzyme abnormalities may also present in this fashion and should be considered in undiagnosed cases. TREATMENT

As may be expected, treatment of disorders that affect so fundamental a pathway in energy metabolism is difficult. There have, however, been occasional reports of successful treatment, indicating that this area is not entirely without hope. Treatment strategy should follow these principles: 1. 2. 3.

Limitation of damage caused by toxic metabolites Stimulation of residual enzyme activity Use of alternative substrates and artificial electron acceptors (see Przyrembel, 1987)

Dietary manipulation, such as is used in disorders of fatty-acid metabolism, is probably inappropriate, although in those patients with lipid storage myopathy, carnitine therapy may be beneficial. Toxicity results from the accumulation of lactic acid and possibly from the presence of highly reactive 'free radicals'. The use of oral bicarbonate is not straightforward and some have used dialysis (peritoneal and haemodialysis) in acute, severe lactic acidosis. Damage resulting from free-radical formation is difficult to quantify and, therefore, to treat. Empirical use of antoxidants such as vitamins E and C may be helpful and should be tried. Vitamin C and menadione (vitamin K3) have also been used as electron acceptor/donor to overcome a defect in complex III (Argov et al, 1986). The naturally occurring form of ubiquinone (UQ 10) has also been used in several instances, including cases with complex III defects. Combination of UQ10 and succinate was reported to be successful in a patient with partial complex I, IV and V deficiency (Schoffner et al, 1989).

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Stimulation of endogenous enzyme activities has been tried with riboflavin and thiamine. Riboflavin is a precursor of flavin mononucleotide, a component of complex I, and has been successful in some cases (Arts et al, 1983). Although thiamine is a cofactor of pyruvate dehydrogenase complex, its use has been helpful in a few cases of respiratory-chain disease.

REFERENCES Ackrell BAC, Maguire J J, Dallman PR & Kearney EB (1984) Effect of iron deficiency on succinate- and NADH-ubiquinone oxidoreductase in skeletal muscle mitochondria. Journal of Biological Chemistry 159: 10053-10059. Anderson S, Bankier AT, Barrell BG et al (1981) Sequence organization of the human mitochondrial genome. Nature 290: 457-465. Angelini C, Bresolin N, Pegolo G e t al (1986) Childhood encephalomyopathy with cytochrome c oxidase deficiency, ataxia, muscle wasting and mental impairment. Neurology 36: 1048-1052. Argov Z, Bank W J, Maris Jet al (1986) Treatment of mitochondrial myopathy due to complex III deficiency with vitamins K3 and C: a 31p-NMR follow-up study. Annals of Neurology 19: 598-602. Arts WFM, Scholte HR, Bogaard JM, Kernebijn KJ & Luyt Houwen IEM (1983) NADH-CoQ reductase deficient myopathy: successful treatment with riboflavin. Lancet ii: 581-582. Arts WFM, Scholte HR, Loonen MCB et al (1987) Cytochrome oxidase deficiency in subacute necrotizing encephalomyelopathy. Journal of the Neurological Sciences 77: 103-115. Bastiaensen LAK, Joosten EMG, DeRooij JAM et al (1978) Ophthalmoplegia plus, a real nosological entity. Acta Neurologica Scandinavica 58: 9-34. Baumgartner ER, Suormala TM, Wick H et al (1989) Biotinidase deficiency: a cause of subacute necrotizing encephalomyelopathy (Leigh syndrome). Report of a case with lethal outcome. Pediatric Research 26: 260-266. Behbehani AW, Goebel H, Osse G et al (1984) Mitochondrial myopathy with lactic acidosis and deficient activity of muscle succinate cytochrome-c-oxidoreductase. European Journal of Pediatrics 143: 6%71. Berenberg RA, Pellock JM, DiMauro Set al (1977) Lumping or splitting? 'Ophthalmoplegia plus' or Kearns-Sayre syndrome? Annals of Neurology 1: 37-54. Berkovic SF, Carpenter S, Karpati G e t al (1987) Cytochrome c oxidase deficiency: a remarkable spectrum of clinical and neuropathologic findings in a single family. Neurology 37 (supplement 1): 223. Besley GTN & Broadhead DM (1989) Prenatal diagnosis of inherited metabolic disease by chorionic villus analysis: the Edinburgh experience. Journal of Inherited Metabolic Disease 12(supplement 2): 263-267. Bindoff LA, Birch-Machin M, Cartlidge NEF, Parker WD Jr & Turnbull DM (1989) Mitochondrial function in Parkinson's Disease. (Letter) Lancet i: 49. Birch-Machin MA, Shepherd IM, Watmough NJ et al (1989) Fatal lactic acidosis in infancy with a defect of complex III of the respiratory chain. Pediatric Research 25: 553-559. Blass JP (1980) Pyruvate dehydrogenase. In Burman D, Hotten JB & Pennock CA (eds) Disorders of Carbohydrate Metabolism, pp 239-267. Lancaster: MTP Press. Bookelman H, Trijbels JMF, Sengers RCA & Janssen AJM (1978) Measurement of cytochromes in human skeletal muscle mitochondria, isolated from fresh and frozen stored muscle specimens. Biochemical Medicine 19: 366--373. Boustany RN, Aprille JR, Halpern J, Levy H & DeLong GR (1983) Mitochondrial cytochrome deficiency presenting as a myopathy with hypotonia, external ophthalmoplegia and lactic acidosis in an infant and as fatal hepatopathy in a second cousin. Annals of Neurology 14: 462-470. Bresolin N, Zeviani M, Bonilla E et al (1985) Fatal infantile cytochrome-c-oxidase deficiency: decrease of immunologically detectable enzyme in muscle. Neurology 35: 802-812.

DEFECTS OF THE RESPIRATORY CHAIN

613

Brown RM, Dahl H-HM & Brown GK (1989) X chromosome localization of the functional gene for the Exc, subunit of the human pyruvate dehydrogenase complex. Genomics 4: 174-181. Brown SK, Haan EA, Kirby DM et al (1988) 'Cerebral' lactic acidosis: defects in pyruvate metabolism with profound brain damage and minimal systemic acidosis. European Journal of Pediatrics 147: 10-14. Byrne E, Trounce I, Dennett X et al (1988) Progression from MERRF to MELAS phenotype in a patient with combined complex I and IV deficiencies. Journal of the Neurological Sciences 88: 327-337. Clark JB, Hayes DJ, Morgan-Hughes JA & Byrne E (1984) Mitochondrial myopathies: disorders of the respiratory chain and oxidative phosphorylation. Journal of Inherited Metabolic Diseases 7(supplement 1): 62-68. Clay VJ & Ragan CI (1988) Evidence for the existence of tissue specific isoenzyme of mitochondrial NADH dehydrogenase. Biochemical and Biophysical Research Communications 157: 1423-1428. Corral M, Baffet G, Kitzis A et al (1989) DNA sequences homologous to mitochondrial genes in nuclei from normal rat tissues and from rat hepatoma cells. Biochemical and Biophysical Research Communications 162: 258-264. Darley-Usmar VM & O'Leary VJ (1988) The role of mitochondria in causing cell damage on reoxygenation of the hypoxic myocardium. Journal of Molecular Cell Cardiology 20(supplement IV): $53. Darley-Usmar VM, Kennaway NG, Buist NR & Capaldi RA (1983) Deficiency in ubiquinone cytochrome c reductase in a patient with mitochondrial myopathy and lactic acidosis. Proceedings of the National Academy of Sciences of the USA 80: 5103-5106. Darley-Usmar VM, Watanabe M, Uchiyama Y et al (1986) Mitochondrial myopathy: tissue specific expression of a defect in ubiquinol-cytochrome c reductase. Clinica Chimica Acta 158: 253-261. Daroff RB, Solitare GB, Pincus JH et al (1966) Spongiform encephalopathy with chronic external ophthalmoplegia: central ophthalmoplegia mimicking ocular myopathy. Neurology 16: 161-169. Desnuelle C, Birch-Machin M, Pellisier J-F et al (1989) Multiple defects of the respiratory chain including complex II in a family with myopathy and encephalopathy. Biochemical and Biophysical Research Communications 163: 695-700. DiMauro S, Bonilla E, Lee CP et al (1976) Luft's Disease. Further biochemical and ultrastructural studies of skeletal muscle in the second case. Journal of the Neurological Sciences 27: 217-232. DiMauro S, Mendell JR, Sahenk Z et al (1980) Fatal infantile mitochondrial myopathy and renal dysfunction due to cytochrome-c-oxidase deficiency. Neurology 30: 795-804. DiMauro S, Nicholson JF, Hays AP et al (1983) Benign infantile mitochondrial myopathy due to reversible cytochrome c oxidase deficiency. Annals of Neurology 14: 226-234. DiMauro S, Servidei S, Zeviani M e t al (1987) Cytochrome c oxidase deficiency in Leigh syndrome. Annals of Neurology 22: 498-506. DiRocco M, Veneselli E, Ciccone MO et al (1988) Cytochrome c oxidase deficiency in three patients with Leigh's disease. Journal of Inherited Metabolic Diseases ll(supplement 2): 189-192. Eleff S, Kennaway NG, Buist NRM et al (1984) 31p NMR study of improvement in oxidative phosphorylation by vitamins K3 and C in a patient with a defect in electron transport at complex III in skeletal muscle. Proceedings of the National Academy of Sciences" of the USA 81: 3529-3533. Fischer JC~ Ruitenbeek W, Gabre61s FJM et al (1986) A mitochondrial encephalomyopathy: the first case with an established defect at the level of coenzyme Q. European Journal of Pediatrics 144: 441-444. Frackowiak RSJ, Herold S, Petty RKH & Morgan-Hughes JA (1988) The cerebral metabolism of glucose and oxygen measured with positron tomography in patients with mitochondrial diseases. Brain 111: 1009-1024. Fukuhara N, Tokiguelin S, Shirakawa K & Tsubaki T (1980) Myoclonus epilepsy associated with ragged-red fibres (mitochondrial abnormalities): disease entity or a syndrome? Journal of the Neurological Sciences 47: 117-133. Garcia Silva MT, Aicardi J, Gouti6res F & Chevrie JJ (1987) The syndrome of myoclonic

614

L. A. BINDOFF AND D. M. TURNBULL epilepsy with ragged-red fibers: report of a case and review of the literature. Neuro-

pediatrics 18: 200-204. Giles RE, Blanc H, Cann NM & Wallace DC (1980) Maternal inheritance of human mitochondrial DNA. Proceedings of the National Academy of Sciences of the USA 77: 67156719. Griffiths RD & Edwards RHT (1987) Magnetic resonance spectroscopy in the recognition of metabolic disease. Journal of Inherited Metabolic Disease 10(supplement 1): 147-158. Hartl F-U, Pfanner N, Nicholson DW & Neupert W (1.989) Mitochondrial protein import. Biochimica et Biophysica Acta 988: 1-45. Hayasaka K, Brown GK, Danks DM, Droste M & Kadenbach B (1989) Cytochrome c oxidase deficiency in subacute necrotizing encephalopathy (Leigh syndrome). Journal of Inherited Metabolic Diseases 12: 247-256. Hayes D J, Summers BA, Morgan-Hughes JA & Clark JB (1982) A combined deficiency of muscle carnitine and mitochondrial ATPase in a patient with multisystem disease partially responsive to oral carnitine. Proceedings of the 5th International Congress of Neuromuscular Diseases, Marseille. Abstract 16.4. Hayes D J, Lecky BRF, Landon DN et al (1984) A new mitochondrial myopathy. Biochemical studies revealing a deficiency in the cytochrome b-Cl complex (complex III) of the respiratory chain. Brain 107: 1165-1177. Hayes DJ, Hilton-Jones D, Arnold DL et al (1985) A mitochondrial encephalomyopathy. A combined 31p magnetic resonance and biochemical investigation. Journal of the Neurological Sciences 71: 105-118. Heiman-Patterson TD, Bonilla E, DiMauro S, Foreman J & Schotland DL (1982) Cytochrome-c-oxidase deficiency in a floppy infant. Neurology 32: 898-900. Holt I J, Harding A E & Morgan-Hughes JA (1988) Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 331: 717-719. Holt I J, Harding A E & Morgan-Hughes JA (1989) Deletions of muscle mitochondrial DNA in mitochondrial myopathies: Sequence analysis and possible mechanisms. Nucleic Acid Research 17: 4465-4469. Hoppel CL, Kerr DS, Dahm SB & Roessmann U (1987) Deficiency of the reduced nicotinamide adenine dinucleotide dehydrogenase component of complex I of mitochondrial electron transport. Journal of Clinical Investigation 80' 71-77. Ichiki T, Kobayashi M, Wada Y, Tanaka M & Ozawa T (1988a) Clinical and molecular heterogeneity in patients with mitochondrial encephalomyopathies due to complex I deficiency. Journal of lnherited Metabolic Diseases 11: 333-336. Ichiki T, Tanaka M, Nishikimi M e t al (1988b) Deficiencies of subunits of complex I and mitochondrial encephalomyopathy. Annals of Neurology 23: 287-294. Jerusalem F, Angelini C, Engel A G & Groover RN (1973) Mitochondria-lipid-glycogen (MLG) disease of muscle. A morphologically regressive congenital myopathy. Archives of Neurology 29: 162-169. Johnson MA, Turnbull DM, Dick DJ & Sherratt HSA (1983) A partial deficiency of cytochrome c oxidase in chronic progressive external ophthalmoplegia. Journal of the Neurological Sciences 60: 31-53. Johnson MA, Kadenbach B, Droste M, Old SL & Turnbull DM (1988) Immunocytochemical studies of cytochrome oxidase subunits in skeletal muscle of patients with partial cytochrome oxidase deficiencies. Journal of the Neurological Sciences 87: 75-90. Kadenbach B, Hartmann R, Glanville R & Buse G (1982) Tissue-specific genes code for polypeptide Via of bovine liver and heart cytochrome c oxidase. FEBS Letters 138: 236-238. Kadenbach B, Stroh A, Becker A, Eckerskorn C & Lottspeich F (1990) Tissue- and speciesspecific expression of cytochrome c oxidase isoenzymes in vertebrates. Biochimica et Biophysica Acta 1015: 368-372. Kearns TP & Sayre GP (1958) Retinitis pigmentosa, external ophthalmoplegia and complete heart block. Archives of Ophthalmology 60: 280-289. Kennaway NG, Buist NRM, Darley-Usmar VM et al (1984) Lactic acidosis and mitochondrial myopathy associated with deficiency of several components of complex III of the respiratory chain. Pediatric Research 18: 991-999. Kennaway NG, Wagner ML, Capaldi RA et al (1987) Combined deficiencies of complexes III and IV of the respiratory chain, involving both nuclear and mitochondrial gene products,

DEFECTS OF THE RESPIRATORY CHAIN

615

in skeletal muscle of a patient with lactic acidosis. Journal of Inherited Metabolic Disease 10(supplement 2): 247-251. Kerr DS, Ho L, Berlin CM et al (1987) Systemic deficiency of the first component of the pyruvate dehydrogenase complex. Pediatric Research 22: 312-318. Kobayashi M, Morishita H, Sugiyama Net al (1986) Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes syndrome and NADH-CoQ reductase deficiency. Journal of Inherited Metabolic Disease 9: 301-304. Kobayashi M, Morishita H, Sugiyama N et al (1987) Two cases of NADH-coenzyme Q reductase deficiency: relationship to MELAS syndrome. Journal of Pediatrics 110: 223227. Koga Y, Nonaka I, Kobayashi M, Tojyo M & Nihei K (1988a) Findings in muscle in complex I (NADH coenzyme Q reductase) deficiency. Annals of Neurology 24: 749-756. Koga Y, Nonaka I, Sunohara N, Yamanaka R & Kumagai K (1988b) Variability in the activity of respiratory chain enzymes in mitochondrial niyopathies. Acta Neuropathologica 76: 135-141. Kretzchmar HA, DeArmond SJ, Koch TK et al (1987) Pyruvate dehydrogenase complex deficiency as a cause of subacute necrotizing encephalopathy (Leigh Disease), Pediatrics 79: 370-373. Krishnamoorthy G & Hinkle PC (1988) Studies on the electron transfer pathway, topography of iron-sulphur centers and site of coupling in NADH-Q oxidoreductase. Journal of Biological Chemistry 263: 17566-17575. Kuwahara S, Okada Y & Omura T (1978) Evidence for molecular identity of microsomal and mitochondrial NADH-cytochrome b5 reductase of rat liver. Journal of Biochemistry 83: !049-1059. Land JM, Hockaday JM, Hughes JT & Ross BD (1981a) Childhood mitochondrial myopathy with ophthalmoplegia. Journal of the Neurological Sciences 51: 371-382. Land JM, Morgan-Hughes JA & Clark JB (1981b) Mitochondrial myopathy. Biochemical studies revealing a deficiency of NADH-cytochrome b reductase activity. Journal of the Neurological Sciences 50: 1-13. Latipfi~ PM, Karki TT, Hiltunen JK & Hassinen IE (1986) Regulation of palmitoylcarnitine oxidation in rat liver mitochondria. Role of the redox state of NAD(H). Biochimica et Biophysica Acta 875: 293-300. Leigh D (1951) Subacute necrotizing encephalomyelopathy in an infant. Journal of Neurology, Neurosurgery and Psychiatry 14" 216-221. Ljungdahl PO, Beckman JD & Trumpower BL (1989) Mutational analysis of the mitochondrial Reiske iron-sulphur protein of Saccharomyces cerevisiae. II Biochemical characterization of temperature-sensitive RIP 1 mutations. Journal of Biological Chemistry 264:3723-3731. Lomax MI & Grossman LI (1989) Tissue-specific genes for respiratory proteins. Trends in Biochemistry 14: 501-503. Luft R, Ikkos D, Palmieri G, Ernster L & Afzelius B (1962) A case of severe hypermetabolism of nonthyroid origin with a defect in the maintenance of mitochondrial respiratory control: a correlated clinical, biochemical and morphological study. Journal of Clinical Investigation 41: 1776-1804. Maertens P, Richardson R, Bastian F, Williams JP & Hommes F (1988) A new type of mitochondrial encephalopathy with stroke-like episodes due to cytochrome oxidase deficiency. Journal of Inherited Metabolic Diseases ll(supplement 2): 186-188. Martin J J, Van de Vyver FL, Scholte HR et al (1988) Defect in succinate oxidation by isolated muscle mitochondria in a patient with symmetrical lesions in the basal ganglia. Journal of the Neurological Sciences 84: 189-200. Mendell JR, Barohn RJ, Yates AJ et al (1987) Autopsy case of myoclonic epilepsy and ragged-red fibers with cytochrome c oxidase deficiency: evidence for a maternally inherited biochemical defect. Annals of Neurology 22(supplement 1): 128. Minchom PE, Dormer RI, Hughes IA et al (1983) Fatal infantile mitochondrial myopathy due to cytochrome oxidase deficiency. Journal of the Neurological Sciences 60: 453-463. Miranda A, Ishii S, DiMauro S & Shay JW (1989) Cytochrome c oxidase deficiency in Leigh syndrome: genetic evidence for a nuclear DNA-encoded mutation. Neurology 39: 697702. Miyabayashi S, Narisawa K, Tada K et al (1983) Two siblings with cytochrome oxidase deficiency. Journal of Inherited Metabolic Disease 6: 121-122.

616

L. A. BINDOFF AND D. M. TURNBULL

Miyabayashi S, Ito T, Abukawa D et al (1987) Immunocytochemical study in three patients with cytochrome c oxidase deficiency presenting Leigh's encephalomyelopathy. Journal of Inherited Metabolic Diseases 10: 289-292. Montagrla R, Gallassi R, Medori R et al (1988) MELAS syndrome: characteristic migrainous and epileptic features and maternal transmission. Neurology 38: 751-754. Montagna P, Sacquengha T, Cortelli P & Lugaresi E (1989) Migraine as a defect of brain oxidative metabolism: a hypothesis. Journal of Neurology 236: 124-125. Moreadith RW, Batshaw ML, Ohnishi T et al (1984) Deficiency of the iron-sulfur clusters of mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase (complex I) in an infant with congenital lactic acidosis. Journal of Clinical Investigation 74: 685-697. Morgan-Hughes JA, Darveniza P, Kahn SN et al (1977) A mitochondrial myopathy characterized by a deficiency in reducible cytocbrome b. Brain 100: 617-640. Morgan-Hughes JA, Darveniza P, Landon DN, Land JM & Clark JB (1979) A mitochondrial myopathy with a deficiency of respiratory chain NADH-CoQ reductase activity. Journal of the Neurological Sciences 43: 27-46. Morgan-Hughes JA, Hayes D J, Clark JB et al (1982) Mitochondrial encephalomyopathies. Biochemical studies in two cases revealing defects in the respiratory chain. Brain 105: 553-582. Morgan-Hughes JA, Hayes DJ & Clark JB (1984) Mitochondrial myopathies. In Serratrice G, Cros D, Desnuelle C, Gastant J-L, Pellisier J-F, Pouget J & Schiano A (eds) Neuromuscular Diseases, pp. 79-85. New York: Raven Press. Mogan-Hughes JA, Hayes D J, Cooper M & Clark JB (1985) Mitochondrial myopathies: deficiencies localized to complex I and complex III of the respiratory chain. Biochemical Society Transactions 13: 648-650. Morgan-Hughes JA, Cooper JM, Schapira AHV, Hayes DJ & Clark JB (1987) In Ellingson R J, Murray NMF & Halliday AM (eds). The London Symposia, pp. 103-20t. Elsevier: Amsterdam. Miiller-H6cker J (1989) Cytochrome-c-oxidase deficient cardiomyocytes in the human heart-an age-related phenomenon. A histochemical ultracytochemical study. American Journal of Pathology 134: 1167-1173. Muscari C, Frascaro M, Guarnier C & Caldera CM (1990) Mitochondrial function and superoxide generation from submitochondrial particles of age d rat hearts. Biochimica et Biophysica Acta 1015: 200-204. Nohl H & Hegner D (1978) Do mitochondria produce oxygen radicals in vivo? European Journal of Biochemistry 82: 563-567. Ogasahara S, Engel AG, Freus D & Mack D (1989) Muscle coenzyme Q deficiency in familial mitochondrial encephalomyopathy. Proceedings of the National Academy of Sciences of the USA 86: 232%2382. Ogier H, Lombes A, Scholte HR et al (1988) de Toni-Fanconi-Debr6 syndrome with Leigh syndrome revealing severe muscle cytochrome c oxidase deficiency. Journal of Pediatrics 112: 734-739. Oldfors A, Sommerland H, Holme E, Tulinius M & Kritiansson B (1989) Cytochrome c oxidase deficiency in infancy. Acta Neuropathologica 77: 267-275. Olsen W, King-Engel W, Walsh GO & Einangler R (1972) Oculocraniosomatic neuromuscular disease with 'ragged red' fibers. Archives of Neurology 26:193-211. Papadimitriou A, Neustein HB, DiMauro S, Stanton R & Bresolin N (1984) Histiocytoid cardiomyopathy of infancy: deficiency of reducible cytochrome b in heart mitochondria. Pediatric Research 18: 1023-1028. Parker WD, Boyson SJ & Parks JK (1989) Electron transport chain abnormalities in idiopathic Parkinson's disease. Annuls of Neurology 26: 719-723. Pavlakis SG, Phillips PG, DiMauro Set al (1984) Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes: a distinctive clinical syndrome. Annals of Neurology 16: 481-488. Petty RKH, Harding AE & Morgan-Hughes JA (1986) The clinical features of mitochondrial myopathy. Brain 109: 915-938. Prick MJJ, Gabre~ls FJM, Renier WO et al (1981) Progressive infantile poliodystrophy. Association with disturbed pyruvate oxidation in muscle and liver. Archives of Neurology 38: 767-772.

DEFECTS OF THE RESPIRATORY CHAIN

617

Przyrembel H (1987) Therapy of mitochondrial disorders. Journal of Inherited Metabolic Disease 10(supplement 1): 129-146. Reed LJ & Yeaman SJ (1987) Pyruvate dehydrogenase. In Boyer PD & Krebbs EG (eds) The Enzymes, vol. 18, pp. 77-95. New York: Academic Press. Reichmann H, Rohkamm R, Zeviani Met al (1986) Mitochondrial myopathy due to complex III deficiency with normal reducible cytochrome b concentration. Archives of Neurology 43: 957-961. Riggs JE, Schochet SS, Fakadej AV et al (1984) Mitochondrial encephalomyopathy with decreased succinate-cytochrome c reductase activity. Neurology 34: 48-53. Rimoldi M, Bottacchi E, Rossi Let al (1982) Cytochrome-c-oxidase deficiency in muscles of a floppy infant without mitochondrial myopathy. Journal of Neurology 227" 201-207. Rivner MH, Shamsnia M, Swift TR et al (1989) Kearns-Sayre syndrome and complex II deficiency. Neurology 39: 693-696. Robinson BH, Ward J, Goodyer P & Baudet A (1986) Respiratory chain defects in the mitochondria of cultured skin fibroblasts from three patients with lactic acidaemia. Journal of Clinical Investigation 77: 1422-1427. Robinson BH, DeMeirleir L, Glerum M, Sherwood G & Becket L (1987a) Clinical presentation of mitochondrial respiratory chain defects in NADH-coenzyme Q oxidase: clues to pathogenesis of Leigh disease. Journal of Pediatrics 110: 216-222. Robinson BH, MacMillan H, Petrova-Benediet R & Sherwood G (1987b) Variable clinical presentation in patients with defect E1 component of pyruvate dehydrogenase complex. Journal of Pediatrics 111: 525-533. Rosenberg RN, Schotland DL, Lovelace RE & Rowland LP (1968) Progressive ophthalmoplegia. Archives of Neurology 19: 362-376. R6tig A, Colonna M, Bianche S e t al (1988) Deletions of blood mitochondrial DNA in pancytopaenia. Lancet ii: 567-568. Ruitenbeek W, Trijbels JMF, Fischer JC et al (1989) Mitochondrial myopatbies: multiple enzyme defects in the respiratory chain. Journal of Inherited Metabolic Disease 12 (supplement 12): 352-354. Schapira Y, Harel S and Russel A (1977) Mitochondrial encephalomyopathies. A group of neuromuscular disorders with defects in oxidative metabolism. Israeli Journal of Medical Science 13" 161-164. Schapira AHV, Cooper JM, Morgan-Hughes JA et al (1988a) Mitochondrial myopathy with defective translocation of the Reiske FeS protein into muscle mitochondria. Fifth European Bioenergetics Conference, Aberystwyth, pp 63. Schapira AHV, Cooper JM, Morgan-Hughes JA et al (1988b) Molecular basis of mitochondrial myopathies: peptide analysis in complex I deficiency. Lancet i: 500-503. Schapira AHV, Cooper JM, Dexter D et al (1989) Mitochondrial complex I deficiency in Parkinson's disease. Lancet i: 1269. Schatz G, Halsbrunner E & Tuppy H (1964) Deoxyribonucleic acid associated with yeast mitochondria. Biochemical and Biophysical Research Communications 15" 127-132. Schoffner JM, Lob MT, Voljavec AS et al (1989) Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slipreplication model and metabolic therapy. Proceedings of the National Academy of Sciences of the USA 86: 7952-7956. Scholte HR, Busch HFM, Luyt-Houwen IEM et al (1987) Defects in oxidative phosphorylation. Biochemical investigations in skeletal muscle and expression of the lesion in other cells. Journal of lnherited Metabolic Disease 10(supplement 1): 81-97. Schon EA, Rizzuto R, Moreas CT et al (1989) A direct repeat is a hotspot for large-scale deletions of human mitochondrial DNA. Science 244: 346-349. Schoppink PJ, Berden JA & Grivell LA (1989) Inactivation of the gene encoding the 14-kDa subunit VII of yeast ubiquinol cytochrome c oxidoreductase and analysis of the resulting mutant. European Journal of Biochemistry 181: 475-483. Schotland DL, DiMauro S, Bonilla E, Scarpa A & Lee CP (1976) Neuromuscular disorder associated with a defect in the mitochondrial energy supply. Archives of Neurology 33: 475-479. Sengers RCA, Fischer JC, Trijbels JMF et al (1983) A mitochondrial myopathy with a defective respiratory chain and carnitine deficiency. European Journal of Pediatrics 140: 332-337. Sengers RCA, Trijbels JMF, Bakkeren JAJM et al (1984) Deficiency of cytochromes b and aa3

618

L. A. BINDOFF AND D. M. TURNBULL

in muscle from a floppy infant with cytochrome oxidase deficiency. European Journal of Pediatrics 141: 178-180. Senior B & Jungas RL (1974) A disorder resulting from an enzymatic defect of the respiratory chain. Pediatric Research 8: 438a. Servidei S, Lazaro RP, Bonilla E e't al (1987) Mitochondrial encephalomyopathy and partial cytochrome c oxidase deficiency. Neurology 37: 58-63. Seyama K, Suzuki K, Mizuno Y e t al (1989) Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes with special reference to the mechanism of cerebral manifestations. Acta Neurologica Scandinavica 80: 561-568. Shepherd IM, Birch-Machin M, Johnson MA et al (1988) Cytochrome oxidase deficiency: immunological studies of skeletal muscle mitochondrial fractions. Journal of the Neurological Sciences 87: 265-274. Sherratt HSA, Cartlidge NEF, Johnson MA & Turnbull DM (1984) Mitochondrial myopathy with partial cytochrome oxidase deficiency and impaired oxidation of NADH-linked substrates. Journal of lnherited Metabolic Disease 7(supplement 2): 107-108. Sperl W, Ruitenbeek W, Trijbels JMF et al (1988) Mitochondrial myopathy with lactic acidaemia, Fanconi-De Toni-Debt6 syndrome and a disturbed succinate cytochrome c oxidoreductase activity. European Journal of Pediatrics 147: 418-421. Spiro A J, Moore CL, Prineas JW, Strasberg PM & Rapin I (1970) A cytochrome-related inherited disorder of the nervous system and muscle. Archives of Neurology 23: 103-112. Stadhouders AM (1981) Mitochondrial ultrastructural changes in mitochondrial diseases. In Busch HFM, Jennekens FGI & Scholte HR (eds) Mitochondria and Muscular Diseases, pp. 77-88. The Netherlands: Mefar by, Beetssterzwagg. Stansbie D, Dormer RL, Hughes IA et al (1986) Mitochondrial myopathy with skeletal muscle cytochrome oxidase deficiency. Journal oflnherited Metabolic Diseases 5(supplement 1): 27-28. Str6mme JH, Borud O & Moe PH (1976) Fatal lactic acidosis in a newborn attributable to a congenital defect of pyruvate dehydrogenase. Pediatric Research 10: 60-66. Tanaka M (1987) Variation in the levels complex I subunits among tissues in a patient with mitochondrial encephalomyopathy and renal dysfunction. Biochemistry International 14: 735-739. Tanaka M, Nishikimi M, Suzuki H et al (1986) Deficiency of subunits in heart mitochondrial NADH-ubiquinone oxidoreductase of a patient with mitochondrial encephalomyopathy and cardiomyopathy. Biochemical and Biophysical Research Communications 140: 88-93. Tervoort M J, Schilder LTM & van Gelder BF (1981) The absorbance coefficient of beef heart cytochrome c~. Biochimica et Biophysica Acta 637:245-251. Trijbels JMF, Sengers RCA, Monnens LAH et al (1983) A patient with lactic acidaemia and cytochrome oxidase deficiency. Journal of Inherited Metabolic Diseases 6(supplement 2): 127-128. Tsairis P, Engel WK & Kark P (1973) Familial myoclonic epilepsy syndrome associated with skeletal muscle mitochondrial abnormalities. Neurology 23: 408. Van Biervliet JPGM, Bruinvis L, Ketting D et al (1977) Hereditary mitochondrial myopathy with lactic acidemia, a deToni-Fanconi-Debr6 syndrome and a defective respiratory chain in voluntary striated muscles. Pediatric Research 11: 1088-1093. Van Erwen PMM, Ruitenbeek W, GabreEls FJM et al (1986) Disturbed oxidative metabolism in subacute necrotizing encephalomyelopathy (Leigh Syndrome). Neuropediatrics 17: 28-32. Van Erwen PMM, Cillessen JPM, Eekhoff EMW et al (1987a) Leigh Syndrome, a mitochondrial encephalo(myo)pathy. Clinical Neurology and Neurosurgery 89(4): 217-230. Van Erwen PMM, Gabre~ls FJM, Ruitenbeek W, Renier WO & Fischer JC (1987b) Mitochondrial encephalomyopathy. Association with an NADH dehydrogenase deficiency. Archives of Neurology 44: 775-778. Van Erwen PMM, Gabre61s FJM, Ruitenbeek W e t al (1988) A mitochondrial encephalomyopathy with a partial cytochrome c oxidase deficiency of muscle. Journal of Neurology, Neurosurgery and Psychiatry 51: 704-708. Vikki J, Savontaus M-L & Mikaskelainen EK (1989) Genetic heterogeneity in Leber Hereditary Optic Neuroretinopathy revealed by mitochondrial DNA polymorphism. American Journal of Human Genetics 45:206-211. Walker V, Mills GA, Hall MA et al (1989) A fourth case of fumarase deficiency. Journal of

DEFECTS OF THE RESPIRATORY CHAIN

619

Inherited Metabolic Disease 12: 331-332. Wallace DC (1987) Maternal genes: mitochondrial diseases. In McKusick VA, Roderick TH, Mori J & Paul NW (eds) Birth Defects: OriginalArticles Series, vol. 23, no 3, pp. 137-190. New York: Alan Liss. Wallace DC, Singh G, Lott MT et al (1988a) Mitochondrial DNA mutation associated with Leber's Hereditary Optic Neuropathy. Science 242: 1427-1430. Wallace DC, Zheng X, Lott MT et al (1988b) Familial mitochondrial encephalomyopathy (MERRF): genetic, pathophysiological and biochemical characterisation of a mitochondrial DNA disease. Cell 55: 601-610. Watmough N J, Birch-Machin MA, Bindoff LA et al (1989) Tissue specific defect of complex I of the mitochondrial respiratory chain. Biochemical and Biophysical Research Communications 160: 623-627. Watmough NJ, Bindoff LA, Birch-Machin MA et al (1990) Impaired mitochondria113-oxidation in deficiency of complex I of the respiratory chain: studies in skeletal muscle mitochondria. Journal of Clinical Investigation 85: 177-184. Wijburg FA, Barth PG, Ruitenbeek W e t al (1989) Familial NADH:Q1 oxidoreductase (complex I) deficiency: variable expression and possible treatment. Journal oflnherited Metabolic Disease 12(supplement 2): 349-351. Willems JL, Monnens LAH, Trijbels JMF et al (1977) Leigh's encephalomyelopathy in a patient with cytochrome c oxidase deficiency in muscle tissue. Pediatrics 60: 850-857. Zeviani M, Nonaka I, Bonilla E et al (1985) Fatal infantile mitochondrial myopathy and renal dysfunction caused by cytochrome c oxidase deficiency: immunological studies in a new patient. Annals of Neurology 17: 414-417. Zeviani M, van Dyke DH, Servidei S e t al (1986) Myopathy and fatal cardiopathy due to cytochrome c oxidase deficiency. Archives of Neurology 43: 1198-1202. Zeviani M, Peterson P, Servidei S, Bonilla E & DiMauro S (1987) Benign reversible muscle cytochrome c oxidase deficiency. Neurology 37: 64-67. Zeviani M, Moreas CT, DiMauro Set al (1988) Deletions of mitochondrial DNA in KearnsSayre syndrome. Neurology 38: 1339-1346. Zierz S, Jahns G & Jerusalem F (1989) Coenzyme Q in serum and muscle of 5 patients with ophthalmoplegia plus. Journal of Neurology 236: 97-101. Zinn AB, Kerr DS & Hoppel CL (1986) Fumarase deficiency: a new cause of mitochondrial encephalomyopathy. New England Journal of Medicine 315: 469-475. N o t e a d d e d in p r o o f S i n c e w r i t i n g this a r t i c l e , S h o f f n e r et al (Cell 1990; 61: 9 3 1 - 9 3 7 ) h a v e i d e n t i f i e d a m u t a t i o n in a m i t o c h o n d r i a l t R N A in p a t i e n t s w i t h M E R R F . T h i s is a n i m p o r t a n t o b s e r v a t i o n a n d h e l p s e x p l a i n w h y t h e d i s o r d e r is associated with multiple defects of the respiratory chain.