JOURNAL OF THE ROYAL SOCIETY OF MEDICINE Volume 88 April 1995

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 BASED ON A PAPER READ TO SEC77ON OF PAEDIATRICS, 25 JANUARY 1994 Keywords: mitochondria; respiratory chain; biochemistry; histochemistry

INTRODUCTION Table 1 Presentations of respiratory chain disease The mitochondrial respiratory chain couples the oxidation of Reference fuels to the generation of cellular energy. It consists of five Disease protein complexes embedded in the inner mitochondrial Neurological 4 membrane. Each respiratory chain complex has multiple MELAS syndrome subunits; most are encoded by nuclear genes, induding all the MERRF syndrome 18 subunits of complex II, but the other complexes also have NARP syndrome 19 mitochondrial DNA The Leigh disease 3,12 subunits encoded by (mtDNA). Alpers-Huttenlocher disease 20 mitochondrial genome is inherited exclusively from the KSS 2 mother and many copies are present in each . CPEO 2 Normal and mutant mtDNA can be found in the same Sensorineural deafness 5 mitochondrion (heteroplasmy) and the proportions vary in Muscle 21 different tissues1. Benign infantile myopathy Fatal infantile myopathy 21 Diseases ofthe mitochondrial respiratory chain are a major Myopathy in children and adults 22 diagnostic challenge. They can present in an enormous variety Rhabdomyolysis of ways, making clinical recognition difficult. There are no Ophthalmological reliable screening tests and the diagnostic tests are generally LHON 23 Pigmentary retinopathy, optic atrophy (in KSS, invasive, expensive and not widely available. In this paper we Leigh disease, etc.) describe an approach to the investigation of these disorders. Heart First, we outline the clinical and biochemical features helpful Cardiomyopathy: hypertrophic, dilated or 24 in selecting which patients to investigate. Next, we consider histiocytoid whether the initial investigation should be to look for a Barth syndrome 25 biochemical defect in the respiratory chain or a genetic defect in Renal mtDNA. Respiratory chain defects cannot be detected reliably Fanconi syndrome 26 Liver in all tissues. In our third section we discuss which tissues mtDNA depletion syndrome 27 should be examined and how they should be obtained. Finally, Pearson syndrome 28 we compare the advantages of histochemistry and Alpers-Huttenlocher disease 20 conventional biochemical tests. Haematological Sideroblastic anaemia, pancytopenia 28 (Pearson syndrome) SELECTION OF APPROPRIATE PATIENTS (Barth syndrome) 25 TO INVESTIGATE Gastro-intestinal Pancreatic exocrine dysfunction (Pearson syndrome) 28 Clinical clues Partial villous atrophy 29 The first step in investigating suspected disorders of the Motility disorders 30 respiratory chain is patient selection. Despite the diversity of Endocrine Diabetes mellitus 31 Parathyroid, thyroid dysfunction (KSS) 32 Metabolic decompensation Lactic acidaemia (in many of the above, see text)

Division of Clinical Neuroscience, University of Newcastle upon Tyne, Newcastle MELAS=, encephalopathy, lactic acidosis and stroke-like upon Tyne, UK episodes; MERRF=myoclonic epilepsy and ragged-red fibres; NARP=neurogenic to: Professor D M Tumbull, Division of Clinical Neuroscience, weakness, ataxia and retinitis pigmentosa; KSS=Kearns-Sayre Syndrome; Correspondence CPEO=chronic progressive external ophthalmoplegia; LHON=Leber's hereditary The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK optic neuropathy 217P JOURNAL OF THE ROYAL SOCIETY OF MEDICINE Volume 88 April 1995

respiratory chain disease, patient selection is still based on Table 2 Non-respiratory chain causes of hyperlactateemia recognizing the common clinical presentations. Table 1 summarizes these with references that give details of the Metabolic diseases Pyruvate dehydrogenase deficiency various conditions. Four main dues in the clinical Gluconeogenic defects: Fructose 1,6-bisphosphatase deficiency presentation may suggest respiratory chain disease. Pyruvate carboxylase, multiple carboxylase or biotinidase (1) Diagnosis is easiest when the presentation conforms to deficiency one of the characteristic syndromes that have been reported. Phosphoenolpyruvate carboxykinase deficiency Glycogen storage disease type 1 It is important, however, to be aware that these syndromes Hereditary fructose intolerance show considerable variability: they may be incomplete, Long-chain hydroxyacyl-CoA dehydrogenase deficiency present in atypical ways or overlap with other syndromes. For Organic acidaemias: propionic, methyl malonic and isovaleric example, the cardinal features of Kearns-Sayre syndrome acidaemias, maple syrup urine disease (KSS) are progressive external ophthalmo-plegia and Secondary causes present with Tissue hypoxia: hypoxia (including crying) pigmentary retinopathy, but it can lschaemia hypocalcaemia or short stature; other patients progress Venous stasis from Pearson syndrome in infancy to KSS in childhood. There Shock is also overlap with adult onset chronic progressive external Exercise, seizures ophthalmoplegia (CPEO)2. Hepatic failure (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. The hardest cases of respiratory chain disease to identify Myopathy combined with an unrelated symptom is are those in whom a single system is affected, without a particularly characteristic. In infancy, respiratory chain characteristic finding such as ophthalmoplegia. Isolated myopathy is usually associated with lactic acidosis and often skeletal myopathy is one such presentation: the aetiology is with de Toni-Fanconi-Debre syndrome or liver failure; later, usually apparent ifhistochemistry is performed on the muscle it is often found with cardiomyopathy or CNS disease such as biopsy. Deafness can also be an isolated finding in respiratory dementia, MERRF syndrome (myoclonic epilepsy with chain disease5, but the aetiology would seldom be suspected ragged-red fibres) or MELAS syndrome (mitochondrial unless there are affected relatives. myopathy, encephalopathy, lactic acidosis and stroke-like episodes). (3) Within each system respiratory chain disorders cause Biochemical clues certain patterns of disease and not others. Thus, de Toni- A raised lactate concentration in blood or CSF is an important Fanconi-Debre syndrome is the only renal disease commonly pointer to respiratory chain disease though its sensitivity and associated with respiratory chain defects. Certain clinical specificity are low. Hyperlactataemia is uncommon in adult features, such as progressive external ophthalmoplegia, are so onset respiratory chain disease apart from MELAS syndrome strongly suggestive of respiratory chain pathology that and mitochondrial myopathies. Hyperlactataemia seems to be investigation is warranted even in the absence of other more common in childhood and especially in infancy: raised features. Some investigation findings are equally suggestive lactate concentrations may reflect widespread disease, which (e.g. the MRI findings in Leigh disease3). The value of raised is likely to present early in life. Thus, Pearson syndrome, KSS lactate concentrations in blood or CSF will be discussed later. and CPEO are all associated with similar mtDNA Other features, such as cardiomyopathy, ataxia, myoclonus or rearrangements. Pearson syndrome, a multisystem disorder stroke-like episodes, should lead to a high index of suspicion that usually presents in infancy, is almost always associated but it is not feasible to investigate the respiratory chain in all with raised blood lactate concentrations. Raised levels are these patients unless there is an additional pointer to this also sometimes found in KSS, which presents later in aetiology. childhood or in young adults, but have not been described in (4) A final clinical clue to respiratory chain dysfunction is a CPEO. Again, cases of mtDNA depletion syndrome family history of . This may take the presenting in infancy tend to have hyperlactataemia, same form as in the index case but often is markedly different, whereas those presenting later do not6. Normal lactate particularly in cases caused by mtDNA mutations. For levels should not discourage investigation of the respiratory example, relatives of patients with MELAS syndrome have chain if the clinical picture is otherwise suggestive. been identified with myoclonus, pigmentary retinopathy or A raised blood lactate concentration strengthens the case deafness4. Obviously, a maternal pattern of inheritance is for respiratory chain disease but is far from specific. Other 218P particularly suggestive but any pattern may be found. causes of hyperlactataemia are summarized in Table 2. Many JOURNAL OF THE ROYAL SOCIETY OF MEDICINE Volume 88 April 1995 ofthese are easy to distinguish but others can cause diagnostic relevant family history. Measurement of the blood or CSF confusion. In general, the alternative diagnoses should be lactate, or the lactate to pyruvate ratio, can increase one's excluded first, as establishing the presence of a respiratory suspicion of respiratory chain disease but can neither prove it chain disorder is likely to be harder and the therapeutic nor exclude it. implications more limited. For example, hereditary fructose intolerance is a treatable cause of infantile hyperlactataemia, Fanconi syndrome and liver disease: the diagnosis is apparent SHOULD THE INITIAL INVESTIGATIONS as soon as fructose is withdrawn and confirmed by an BE BIOCHEMICAL OR GENETIC? intravenous fructose tolerance test. The next question is whether to attempt diagnosis at the In children it can be difficult to obtain reliable blood lactate biochemical or the molecular level. The latter has obvious measurements. Taking blood from a small vein in a struggling attractions. DNA can easily be sent to centres performing the child can easily turn into an inadvertent ischaemic lactate test! relevant tests and suitable specimens can sometimes be Even arterial lactate levels can be artifactually raised by obtained from blood, though this is not always the case as screaming. A better solution is to obtain the sample through a there may be different proportions of mutant mtDNA in cannula inserted at least 45 min previously into an artery or different tissues. Biochemical abnormalities are occasionally large vein: no occlusion should be applied. Reference ranges the result rather than the cause of the disease process: this is are normally established on fasting individuals so samples less likely for molecular defects. Moreover, if a molecular should be obtained from patients under the same conditions, defect is found it immediately gives a precise diagnosis and but this may not be practical when they are unwell. Ideally, the opportunity for genetic counselling; biochemical studies age-specific reference ranges should be used, normal values may need to be followed by molecular ones. being slightly higher in neonates. Unfortunately, for the majority of respiratory chain In patients with suspected respiratory chain disease but diseases the molecular defect is not known. Indeed no normal blood lactate levels, the effect of oral glucose or defects in nuclear genes have yet been identified, though these intravenous pyruvate loading is sometimes measured7. This must be responsible for a number of respiratory chain may induce an abnormal rise but the lactate concentration disorders. Even ifthere is evidence for a mtDNA defect, such remains normal in other patients with respiratory chain as a maternal pattern of inheritance, identifying the mutation defects. Similarly, in adults with respiratory chain disease can pose formidable problems. The mitochondrial genome is exercise may induce an excessive rise in lactate concentration8 16.5kb long: sequencing this is a major undertaking. Multiple but many patients are too disabled to perform exercise clones may need to be sequenced to detect heteroplasmic protocols. mutations that are only present in a small proportion of the CSF lactate concentrations are often raised in patients with mtDNA. Furthermore, abnormalities found may be neurological manifestations of respiratory chain disease (e.g. polymorphisms and not pathogenic. MELAS, Leigh disease), even when the blood level is normal. A pragmatic approach is to pursue molecular tests when This is a particularly difficult group of patients and the the clinical picture suggests a syndrome known to be measurement of CSF lactate is therefore of great value. associated with one or a small number of mutations. However, the same reservations apply as for blood levels. The Pearson syndrome, KSS and CPEO are associated with CSF lactate concentration can be normal in respiratory chain mtDNA rearrangements: these change the size of restriction diseases (e.g. CPEO), it can be raised artifactually (e.g. up to fragments and can be detected on Southern blots10. Leber's 48 h following seizures) and it can be raised in other metabolic hereditary optic neuropathy (LHON), MERRF and MELAS diseases (e.g. pyruvate dehydrogenase deficiency). syndromes are associated with particular mtDNA point Respiratory chain disorders are associated with impaired mutations; these can be detected by sequencing or PCR and oxidation ofNADH, which would be expected to increase the restriction digestion11. Another point mutation, originally ratio of lactate to pyruvate concentrations. This is therefore described in association with neurogenic weakness, ataxia and sometimes used to distinguish between hyperlactataemia due retinitis pigmentosa (NARP syndrome) is a common cause of to respiratory chain disease and other causes9. Unfortunately, Leigh disease. It is worth screening all cases ofLeigh disease for yet again this is unreliable: ratios can be normal in respiratory this mutation, particularly as biochemical investigations give chain disease and raised in hyperlactataemia due to other normal results in these cases12. causes. Not surprisingly, the lactate to pyruvate ratio is oflittle In diseases not known to be associated with particular help when the lactate level is normal, one reason being that mtDNA mutations, the primary investigation should be low levels of pyruvate are hard to measure accurately. histochemistry or biochemistry. We think that a biochemical In summary, patient selection is a clinical procedure based or histochemical abnormality still needs to be documented in on recognizing reported syndromes or suggestive features, all cases ofmtDNA depletion syndrome, preferably in muscle: particularly if several systems are involved or there is a this condition is poorly understood and the normal levels of 219P JOURNAL OF THE ROYAL SOCIETY OF MEDICINE Volume 88 April 1995

mtDNA have yet to be documented in children ofvarious ages especially once the tissue has been distortedby disease. Even in and in patients with other diseases. 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 WHAT IS THE MOST APPROPRIATE histochemistry. TISSUE TO INVESTIGATE? Muscle biopsies are generally obtained from adults using Most mtDNA defects, apart from those in LHON, are local anaesthesia but this is too distressing for children. It has heteroplasmic and the proportion of mtDNA affected can been claimed that drugs used in general anaesthesia may vary in different tissues. The mtDNA defects in Pearson interfere with the respiratory chain13. However, this has not syndrome, MERRF, NARP and most cases of MELAS been our experience. Ideally respiratory chain assays should be syndrome can be detected in DNA from leukocytes. performed on fresh tissue, but some patients are too sick to be However, in KSS, CPEO and some cases of MELAS the moved to referral centres or die elsewhere. Preliminary data proportion of mutant mtDNA in blood is too small to detect from our laboratory and elsewhere show that reliable results and DNA from musde must be analysed. can be obtained on musde that is frozen immediately in liquid Tissue choice is even more important for biochemical nitrogen. It should then be stored at - 70°C and transported assays. Defects in nuclear genes may affect tissue-specific to the laboratory on dry ice. isoforms and so, like mtDNA defects, may not be expressed in Some patients with respiratory chain disease die early in all tissues. Furthermore, even if a defect is expressed, the the neonatal period. Inevitably many ofthese patients are not difficulty ofthe assays may make it hard to detect. Attempts to fully investigated during life. Postmortem specimens for demonstrate respiratory chain defects in readily accessible biochemical assays should be obtained within 1 h ofdeath, and cells such as platelets or fibroblasts have proved time even then some artifactual lowering of respiratory chain consuming and generally disappointing. However, there activity remains possible. Despite these reservations it is have been several reports of complex IV defects successfully important to pursue a diagnosis in these patients both for demonstrated in fibroblasts, notably in Leigh disease9. This genetic counselling and to increase our understanding ofthese avoids the need for more invasive tests but introduces a delay diseases. 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 THE ADVANTAGES OF BIOCHEMICAL defect is detectable in fibroblasts it is also likely to be expressed AND HISTOCHEMICAL INVESTIGATIONS in amniocytes, raising the possibility of antenatal diagnosis. In our laboratory 250 mg of muscle (after removal of fat or Biochemical investigation ofthe respiratory chain is usually fascia) are required for biochemical evaluation. This quantity performed on muscle. There are several reasons why this is allows isolation ofmitochondria, measurement ofthe protein appropriate. Though more invasive than taking blood or a skin concentration and assays of the respiratory chain complexes biopsy, muscle is relatively easy to obtain (compared with and citrate synthase, a mitochondrial matrix enzyme14. liver, kidney or brain for example). Moreover, muscle gives Assays ofthe individual complexes are preferred as these will abnormal results in most cases of respiratory chain disease detect partial defects, which may be missed by other tests15. even when it is not clinically affected. This may reflect the If more tissue is available it allows polarographic reliance of muscle on oxidative metabolism. Alternatively it measurement of the flux through the respiratory chain may be because the cellular population is relatively stable: using various substrates. This will confirm the results of the mutant mtDNA seems to accumulate in non-dividing tissues. complex assays but seldom alters the conclusions; it is usually Normal biochemical results in muscle do not exclude a omitted in children in whom large biopsies are difficult. respiratory chain defect restricted to a single tissue such as Moreover, flux measurements can only be performed on brain or heart, but such cases appear to be rare. If strongly fresh rather than frozen tissue. A number of laboratories suspected, further biochemical assays on the affected tissue assess biochemistry on smaller amounts ofmuscle. However, may be appropriate though there are several problems, this involves using muscle homogenate rather than isolated particularly with regard to control data. Plenty ofcontrol data mitochondria, reducing the reproducibility of the results. is available for muscle but there is much less control data for Histochemistry, the study of enzyme activity in tissue other tissues, particularly brain. sections, is of great value in respiratory chain disease and Another reason for choosing muscle is that it is relatively sometimes makes full biochemical evaluation unnecessary. homogeneous. Samples from patients and controls are This reduces the cost ofinvestigation and the size ofthe biopsy therefore comparable. This is less true of other tissues such required. Only 25 mg of muscle are required for as liver, kidney or brain, which contain many cell types that histochemistry and a further 25 mg will allow DNA 220P may be present in different proportions in different samples, preparation. Biochemical tests cannot be justified in patients JOURNAL OF THE ROYAL SOCIETY OF MEDICINE Volume 88 April 1995 with KSS or CPEO in whom musde biopsy is primarily to However, full biochemical evaluation is necessary if demonstrate mtDNA rearrangements; histochemistry is a histochemistry is normal and if defects of multiple worthwhile confirmatory test as it requires little extra tissue. respiratory chain complexes are to be detected. Direct In other patients there is a greater chance of normal assays ofeach respiratory chain complex should be performed, histochemistry. The options in these patients are either to usually on muscle mitochondria. Ifthe defect is demonstrable take an initial biopsy adequate for biochemistry and in fibroblasts, antenatal diagnosis may be possible in future histochemistry or to take a small biopsy first, accepting this pregnancies. will need to be repeated if the histochemistry is normal. Reliable histochemical methods are available for the Acknowledgments AAMM is an Action Research Training determination of succinate dehydrogenase and cytochrome c Fellow. We thank Dr Margaret Johnson for helpful oxidase activity (complexes II and IV of the respiratory discussion on the histochemical analysis of muscle. We are chain)16. Three abnormal patterns are found. First, succinate grateful to the Muscular Dystrophy Group of Great Britain dehydrogenase preparations reveal sub-sarcolemmal and NIH for financial support in our investigation of accumulation of mitochondria in many patients with respiratory chain disease. respiratory chain disease (a phenomenon that gives rise to 'ragged-red' fibres on Gomori trichrome staining). Sub- REFERENCES sarcolemmal accumulation of mitochondria is good evidence 1 Wallace DC. Mitochondrial diseases: genotype versus phenotype. TIG for respiratory chain defects but is absent in many such diseases 1993;9(4): 128-33 (e.g. Leigh disease). It seems to be retricted to diseases 2 Harding AE, Hammans SR. Deletions of-the mitochondrial genome. J involving defects of mtDNA, and specifically those with Inher Metab Dis 1992;15:480-6 impaired mitochondrial protein synthesis, i.e. mtDNA 3 Van Coster R, Lonbes A, De Vivo D, et a). 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