BASED ON A PAPER READ TO SEC77ON OF PAEDIATRICS, 25 JANUARY Table 1. Disease

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1 The investigation of mitochondrial respiratory chain disease A A M Morris MRCP M J Jackson MRCP L A Bindoff MD MRCP D M Turnbull MD FRCP J R Soc Med 1995;88:217P-222P Keywords: mitochondria; respiratory chain; biochemistry; histochemistry BASED ON A PAPER READ TO SEC77ON OF PAEDIATRICS, 25 JANUARY 1994 INTRODUCTION The mitochondrial respiratory chain couples the oxidation of fuels to the generation of cellular energy. It consists of five protein complexes embedded in the inner mitochondrial membrane. Each respiratory chain complex has multiple subunits; most are encoded by nuclear genes, induding all the subunits of complex II, but the other complexes also have subunits encoded by mitochondrial DNA (mtdna). The mitochondrial genome is inherited exclusively from the mother and many copies are present in each mitochondrion. Normal and mutant mtdna can be found in the same mitochondrion (heteroplasmy) and the proportions vary in different tissues1. Diseases of the mitochondrial respiratory chain are a major diagnostic challenge. They can present in an enormous variety of ways, making clinical recognition difficult. There are no reliable screening tests and the diagnostic tests are generally invasive, expensive and not widely available. In this paper we describe an approach to the investigation of these disorders. First, we outline the clinical and biochemical features helpful in selecting which patients to investigate. Next, we consider whether the initial investigation should be to look for a biochemical defect in the respiratory chain or a genetic defect in mtdna. Respiratory chain defects cannot be detected reliably in all tissues. In our third section we discuss which tissues should be examined and how they should be obtained. Finally, we compare the advantages of histochemistry and conventional biochemical tests. SELECTION OF APPROPRIATE PATIENTS TO INVESTIGATE Clinical clues The first step in investigating suspected disorders of the respiratory chain is patient selection. Despite the diversity of Division of Clinical Neuroscience, University of Newcastle upon Tyne, Newcastle upon Tyne, UK Correspondence to: Professor D M Tumbull, Division of Clinical Neuroscience, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK Table 1 Disease Presentations of respiratory chain disease Neurological MELAS syndrome MERRF syndrome NARP syndrome Leigh disease Alpers-Huttenlocher disease KSS CPEO Sensorineural deafness Muscle Benign infantile myopathy Fatal infantile myopathy Myopathy in children and adults Rhabdomyolysis Ophthalmological LHON Pigmentary retinopathy, optic atrophy (in KSS, Leigh disease, etc.) Heart Cardiomyopathy: hypertrophic, dilated or histiocytoid Barth syndrome Renal Fanconi syndrome Liver mtdna depletion syndrome Pearson syndrome Alpers-Huttenlocher disease Reference , Haematological Sideroblastic anaemia, pancytopenia 28 (Pearson syndrome) Neutropenia (Barth syndrome) 25 Gastro-intestinal Pancreatic exocrine dysfunction (Pearson syndrome) 28 Partial villous atrophy 29 Motility disorders 30 Endocrine Diabetes mellitus Parathyroid, thyroid dysfunction (KSS) Metabolic decompensation Lactic acidaemia (in many of the above, see text) MELAS=mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes; MERRF=myoclonic epilepsy and ragged-red fibres; NARP=neurogenic weakness, ataxia and retinitis pigmentosa; KSS=Kearns-Sayre Syndrome; CPEO=chronic progressive external ophthalmoplegia; LHON=Leber's hereditary optic neuropathy 217P

2 218P respiratory chain disease, patient selection is still based on recognizing the common clinical presentations. Table 1 summarizes these with references that give details of the various conditions. Four main dues in the clinical presentation may suggest respiratory chain disease. (1) Diagnosis is easiest when the presentation conforms to one of the characteristic syndromes that have been reported. It is important, however, to be aware that these syndromes show considerable variability: they may be incomplete, present in atypical ways or overlap with other syndromes. For example, the cardinal features of Kearns-Sayre syndrome (KSS) are progressive external ophthalmo-plegia and pigmentary retinopathy, but it can present with hypocalcaemia or short stature; other patients progress from Pearson syndrome in infancy to KSS in childhood. There is also overlap with adult onset chronic progressive external ophthalmoplegia (CPEO)2. (2) The described syndromes often include features in several apparently unrelated systems. This should suggest respiratory chain disease even if the particular combination does not form part of a previously described syndrome. Myopathy combined with an unrelated symptom is particularly characteristic. In infancy, respiratory chain myopathy is usually associated with lactic acidosis and often with de Toni-Fanconi-Debre syndrome or liver failure; later, it is often found with cardiomyopathy or CNS disease such as dementia, MERRF syndrome (myoclonic epilepsy with ragged-red fibres) or MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). (3) Within each system respiratory chain disorders cause certain patterns of disease and not others. Thus, de Toni- Fanconi-Debre syndrome is the only renal disease commonly associated with respiratory chain defects. Certain clinical features, such as progressive external ophthalmoplegia, are so strongly suggestive of respiratory chain pathology that investigation is warranted even in the absence of other features. Some investigation findings are equally suggestive (e.g. the MRI findings in Leigh disease3). The value of raised lactate concentrations in blood or CSF will be discussed later. Other features, such as cardiomyopathy, ataxia, myoclonus or stroke-like episodes, should lead to a high index of suspicion but it is not feasible to investigate the respiratory chain in all these patients unless there is an additional pointer to this aetiology. (4) A final clinical clue to respiratory chain dysfunction is a family history of mitochondrial disease. This may take the same form as in the index case but often is markedly different, particularly in cases caused by mtdna mutations. For example, relatives of patients with MELAS syndrome have been identified with myoclonus, pigmentary retinopathy or deafness4. Obviously, a maternal pattern of inheritance is particularly suggestive but any pattern may be found. Table 2 Non-respiratory chain causes of hyperlactateemia Metabolic diseases Pyruvate dehydrogenase deficiency Gluconeogenic defects: Fructose 1,6-bisphosphatase deficiency Pyruvate carboxylase, multiple carboxylase or biotinidase deficiency Phosphoenolpyruvate carboxykinase deficiency Glycogen storage disease type 1 Hereditary fructose intolerance Long-chain hydroxyacyl-coa dehydrogenase deficiency Organic acidaemias: propionic, methyl malonic and isovaleric acidaemias, maple syrup urine disease Secondary causes Tissue hypoxia: hypoxia (including crying) lschaemia Venous stasis Shock Exercise, seizures Hepatic failure The hardest cases of respiratory chain disease to identify are those in whom a single system is affected, without a characteristic finding such as ophthalmoplegia. Isolated skeletal myopathy is one such presentation: the aetiology is usually apparent if histochemistry is performed on the muscle biopsy. Deafness can also be an isolated finding in respiratory chain disease5, but the aetiology would seldom be suspected unless there are affected relatives. Biochemical clues A raised lactate concentration in blood or CSF is an important pointer to respiratory chain disease though its sensitivity and specificity are low. Hyperlactataemia is uncommon in adult onset respiratory chain disease apart from MELAS syndrome and mitochondrial myopathies. Hyperlactataemia seems to be more common in childhood and especially in infancy: raised lactate concentrations may reflect widespread disease, which is likely to present early in life. Thus, Pearson syndrome, KSS and CPEO are all associated with similar mtdna rearrangements. Pearson syndrome, a multisystem disorder that usually presents in infancy, is almost always associated with raised blood lactate concentrations. Raised levels are also sometimes found in KSS, which presents later in childhood or in young adults, but have not been described in CPEO. Again, cases of mtdna depletion syndrome presenting in infancy tend to have hyperlactataemia, whereas those presenting later do not6. Normal lactate levels should not discourage investigation of the respiratory chain if the clinical picture is otherwise suggestive. A raised blood lactate concentration strengthens the case for respiratory chain disease but is far from specific. Other causes of hyperlactataemia are summarized in Table 2. Many

3 of these are easy to distinguish but others can cause diagnostic confusion. In general, the alternative diagnoses should be excluded first, as establishing the presence of a respiratory chain disorder is likely to be harder and the therapeutic implications more limited. For example, hereditary fructose intolerance is a treatable cause of infantile hyperlactataemia, Fanconi syndrome and liver disease: the diagnosis is apparent as soon as fructose is withdrawn and confirmed by an intravenous fructose tolerance test. In children it can be difficult to obtain reliable blood lactate measurements. Taking blood from a small vein in a struggling child can easily turn into an inadvertent ischaemic lactate test! Even arterial lactate levels can be artifactually raised by screaming. A better solution is to obtain the sample through a cannula inserted at least 45 min previously into an artery or large vein: no occlusion should be applied. Reference ranges are normally established on fasting individuals so samples should be obtained from patients under the same conditions, but this may not be practical when they are unwell. Ideally, age-specific reference ranges should be used, normal values being slightly higher in neonates. In patients with suspected respiratory chain disease but normal blood lactate levels, the effect of oral glucose or intravenous pyruvate loading is sometimes measured7. This may induce an abnormal rise but the lactate concentration remains normal in other patients with respiratory chain defects. Similarly, in adults with respiratory chain disease exercise may induce an excessive rise in lactate concentration8 but many patients are too disabled to perform exercise protocols. CSF lactate concentrations are often raised in patients with neurological manifestations of respiratory chain disease (e.g. MELAS, Leigh disease), even when the blood level is normal. This is a particularly difficult group of patients and the measurement of CSF lactate is therefore of great value. However, the same reservations apply as for blood levels. The CSF lactate concentration can be normal in respiratory chain diseases (e.g. CPEO), it can be raised artifactually (e.g. up to 48 h following seizures) and it can be raised in other metabolic diseases (e.g. pyruvate dehydrogenase deficiency). Respiratory chain disorders are associated with impaired oxidation of NADH, which would be expected to increase the ratio of lactate to pyruvate concentrations. This is therefore sometimes used to distinguish between hyperlactataemia due to respiratory chain disease and other causes9. Unfortunately, yet again this is unreliable: ratios can be normal in respiratory chain disease and raised in hyperlactataemia due to other causes. Not surprisingly, the lactate to pyruvate ratio is oflittle normal, one reason being that relevant family history. Measurement of the blood or CSF lactate, or the lactate to pyruvate ratio, can increase one's suspicion of respiratory chain disease but can neither prove it nor exclude it. SHOULD THE INITIAL INVESTIGATIONS BE BIOCHEMICAL OR GENETIC? The next question is whether to attempt diagnosis at the biochemical or the molecular level. The latter has obvious attractions. DNA can easily be sent to centres performing the relevant tests and suitable specimens can sometimes be obtained from blood, though this is not always the case as there may be different proportions of mutant mtdna in different tissues. Biochemical abnormalities are occasionally the result rather than the cause of the disease process: this is less likely for molecular defects. Moreover, if a molecular defect is found it immediately gives a precise diagnosis and the opportunity for genetic counselling; biochemical studies may need to be followed by molecular ones. Unfortunately, for the majority of respiratory chain diseases the molecular defect is not known. Indeed no defects in nuclear genes have yet been identified, though these must be responsible for a number of respiratory chain disorders. Even if there is evidence for a mtdna defect, such as a maternal pattern of inheritance, identifying the mutation can pose formidable problems. The mitochondrial genome is 16.5kb long: sequencing this is a major undertaking. Multiple clones may need to be sequenced to detect heteroplasmic mutations that are only present in a small proportion of the mtdna. Furthermore, abnormalities found may be polymorphisms and not pathogenic. A pragmatic approach is to pursue molecular tests when the clinical picture suggests a syndrome known to be associated with one or a small number of mutations. Pearson syndrome, KSS and CPEO are associated with mtdna rearrangements: these change the size of restriction fragments and can be detected on Southern blots10. Leber's hereditary optic neuropathy (LHON), MERRF and MELAS syndromes are associated with particular mtdna point mutations; these can be detected by sequencing or PCR and restriction digestion11. Another point mutation, originally described in association with neurogenic weakness, ataxia and retinitis pigmentosa (NARP syndrome) is a common cause of Leigh disease. It is worth screening all cases ofleigh disease for this mutation, particularly as biochemical investigations give normal results in these cases12. In diseases not known to be associated with particular mtdna mutations, the primary investigation should be histochemistry or biochemistry. We think that a biochemical or histochemical abnormality still needs to be documented in all cases ofmtdna depletion syndrome, preferably in muscle: this condition is poorly understood and the normal levels of help when the lactate level is low levels of pyruvate are hard to measure accurately. In summary, patient selection is a clinical procedure based on recognizing reported syndromes or suggestive features, are involved or there is a 219P particularly if several systems

4 220P mtdna have yet to be documented in children of various ages and in patients with other diseases. WHAT IS THE MOST APPROPRIATE TISSUE TO INVESTIGATE? Most mtdna defects, apart from those in LHON, are heteroplasmic and the proportion of mtdna affected can vary in different tissues. The mtdna defects in Pearson syndrome, MERRF, NARP and most cases of MELAS syndrome can be detected in DNA from leukocytes. However, in KSS, CPEO and some cases of MELAS the proportion of mutant mtdna in blood is too small to detect and DNA from musde must be analysed. Tissue choice is even more important for biochemical assays. Defects in nuclear genes may affect tissue-specific isoforms and so, like mtdna defects, may not be expressed in all tissues. Furthermore, even if a defect is expressed, the difficulty of the assays may make it hard to detect. Attempts to demonstrate respiratory chain defects in readily accessible cells such as platelets or fibroblasts have proved time consuming and generally disappointing. However, there have been several reports of complex IV defects successfully demonstrated in fibroblasts, notably in Leigh disease9. This avoids the need for more invasive tests but introduces a delay of 4-6 weeks while fibroblasts are cultured. Anxiety to establish the diagnosis may justify more invasive investigations, but fibroblast assays have another merit: if a defect is detectable in fibroblasts it is also likely to be expressed in amniocytes, raising the possibility of antenatal diagnosis. Biochemical investigation ofthe respiratory chain is usually performed on muscle. There are several reasons why this is appropriate. Though more invasive than taking blood or a skin biopsy, muscle is relatively easy to obtain (compared with liver, kidney or brain for example). Moreover, muscle gives abnormal results in most cases of respiratory chain disease even when it is not clinically affected. This may reflect the reliance of muscle on oxidative metabolism. Alternatively it may be because the cellular population is relatively stable: mutant mtdna seems to accumulate in non-dividing tissues. Normal biochemical results in muscle do not exclude a respiratory chain defect restricted to a single tissue such as brain or heart, but such cases appear to be rare. If strongly suspected, further biochemical assays on the affected tissue may be appropriate though there are several problems, particularly with regard to control data. Plenty of control data is available for muscle but there is much less control data for other tissues, particularly brain. Another reason for choosing muscle is that it is relatively homogeneous. Samples from patients and controls are therefore comparable. This is less true of other tissues such as liver, kidney or brain, which contain many cell types that may be present in different proportions in different samples, especially once the tissue has been distorted by disease. Even in muscle, mtdna defects only affect a proportion of fibres: if fewer than 10% are affected the biochemical defect cannot be detected but these cases can still be identified by histochemistry. Muscle biopsies are generally obtained from adults using local anaesthesia but this is too distressing for children. It has been claimed that drugs used in general anaesthesia may interfere with the respiratory chain13. However, this has not been our experience. Ideally respiratory chain assays should be performed on fresh tissue, but some patients are too sick to be moved to referral centres or die elsewhere. Preliminary data from our laboratory and elsewhere show that reliable results can be obtained on musde that is frozen immediately in liquid nitrogen. It should then be stored at - 70 C and transported to the laboratory on dry ice. Some patients with respiratory chain disease die early in the neonatal period. Inevitably many of these patients are not fully investigated during life. Postmortem specimens for biochemical assays should be obtained within 1 h of death, and even then some artifactual lowering of respiratory chain activity remains possible. Despite these reservations it is important to pursue a diagnosis in these patients both for genetic counselling and to increase our understanding of these diseases. THE ADVANTAGES OF BIOCHEMICAL AND HISTOCHEMICAL INVESTIGATIONS In our laboratory 250 mg of muscle (after removal of fat or fascia) are required for biochemical evaluation. This quantity allows isolation of mitochondria, measurement of the protein concentration and assays of the respiratory chain complexes and citrate synthase, a mitochondrial matrix enzyme14. Assays of the individual complexes are preferred as these will detect partial defects, which may be missed by other tests15. If more tissue is available it allows polarographic measurement of the flux through the respiratory chain using various substrates. This will confirm the results of the complex assays but seldom alters the conclusions; it is usually omitted in children in whom large biopsies are difficult. Moreover, flux measurements can only be performed on fresh rather than frozen tissue. A number of laboratories assess biochemistry on smaller amounts of muscle. However, this involves using muscle homogenate rather than isolated mitochondria, reducing the reproducibility of the results. Histochemistry, the study of enzyme activity in tissue sections, is of great value in respiratory chain disease and sometimes makes full biochemical evaluation unnecessary. This reduces the cost of investigation and the size of the biopsy required. Only 25 mg of muscle are required for histochemistry and a further 25 mg will allow DNA preparation. Biochemical tests cannot be justified in patients

5 with KSS or CPEO in whom musde biopsy is primarily to demonstrate mtdna rearrangements; histochemistry is a worthwhile confirmatory test as it requires little extra tissue. In other patients there is a greater chance of normal histochemistry. The options in these patients are either to take an initial biopsy adequate for biochemistry and histochemistry or to take a small biopsy first, accepting this will need to be repeated if the histochemistry is normal. Reliable histochemical methods are available for the determination of succinate dehydrogenase and cytochrome c oxidase activity (complexes II and IV of the respiratory chain)16. Three abnormal patterns are found. First, succinate dehydrogenase preparations reveal sub-sarcolemmal accumulation of mitochondria in many patients with respiratory chain disease (a phenomenon that gives rise to 'ragged-red' fibres on Gomori trichrome staining). Subsarcolemmal accumulation of mitochondria is good evidence for respiratory chain defects but is absent in many such diseases (e.g. Leigh disease). It seems to be retricted to diseases involving defects of mtdna, and specifically those with impaired mitochondrial protein synthesis, i.e. mtdna depletion or mutations involving trna genes17. Even some cases of MELAS syndrome have normal histology: these patients may have fewer mutant mitochondrial genomes and sometimes develop sub-sarcolemmal accumulation of mitochondria later in the course of their disease. They can usually be detected by a second abnormality on histochemical preparations, namely a mosaic of cytochrome c oxidase positive and negative fibres. This finding is also restricted to patients with mtdna defects. The third histochemical abnormality found in respiratory chain disease is a generalized lowering of succinate dehydrogenase or cytochrome c oxidase activity. There are no reliable histochemical methods to detect defects of complex I or III. Biochemical assays are necessary to detect isolated defects of these complexes, combined defects or partial defects. CONCLUSIONS Respiratory chain disease remains underdiagnosed due to the diversity of presentations and the difficulty of investigation. Patient selection continues to be based on the clinical features, i.e. recognition of patients with specific syndromes or involvement of several unrelated tissues, and a high level of suspicion in patients with certain symptoms or a suggestive family history. Raised lactate concentrations in blood and CSF are a helpful pointer, but normal levels do not exclude the diagnosis. When the clinical picture suggests a syndrome known to be associated with particular mtdna mutations, the primary test is to look for these in blood or muscle. Histochemistry detects most respiratory chain defects, only requires small amounts of tissue and can provide evidence for a genetic defect in mtdna. However, full biochemical evaluation is necessary if histochemistry is normal and if defects of multiple respiratory chain complexes are to be detected. Direct assays ofeach respiratory chain complex should be performed, usually on muscle mitochondria. If the defect is demonstrable in fibroblasts, antenatal diagnosis may be possible in future pregnancies. Acknowledgments AAMM is an Action Research Training Fellow. We thank Dr Margaret Johnson for helpful discussion on the histochemical analysis of muscle. We are grateful to the Muscular Dystrophy Group of Great Britain and NIH for financial support in our investigation of respiratory chain disease. REFERENCES 1 Wallace DC. Mitochondrial diseases: genotype versus phenotype. TIG 1993;9(4): Harding AE, Hammans SR. Deletions of-the mitochondrial genome. 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