Human Reproduction, Vol. 15, (Suppl. 2), pp.

57-67, 2000

Practical problems in detecting abnormal mitochondrial function and genomes

David R.Thorburn1
The Murdoch Institute, Royal Children's Hospital, Melbourne, Australia
lr

To whom correspondence should be addressed at: The Murdoch Institute, Royal Children's Hospital, Flemington Road, Parkville, Melbourne, Victoria 3052, Australia. E-mail: thorburd@cryptic.rch.unimelb.edu.au

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Mitochondrial respiratory chain dysfunction causes a wide range of primary diseases in adults and children, with highly variable organ involvement. Diagnosis involves weighing evidence from a number of sources, including the clinical presentation, metabolic measurements in vivo, imaging studies, analysis of respiratory chain function or enzyme activities in vitro, studies of mitochondrial morphology after biopsy, and mitochondrial (mt) DNA mutation analysis. Irrespective of the category of the information, it can be difficult to determine whether abnormal results are due to primary defects of the respiratory chain or to practical problems that complicate the diagnostic methodology. This review describes six sources of such problems: genetic complexity, tissue and temporal variation, methodological limitations, secondary effects, logistical issues, and questions of interpretation. When these issues are all addressed, a reliable categorization of the diagnosis as definite, probable, or possible respiratory chain defect becomes possible. Key words: diagnosis/mitochondrial disease/ mitochondrial DNA/mitochondrial function/ respiratory chain
European Society of Human Reproduction and Embryology

Introduction The mitochondrial respiratory chain is the central energy generating pathway in humans and all other eukaryotic species; oxidation of fuels such as sugar, fat, and protein feeds electrons into it. These electrons are transferred through respiratory chain complexes I-IV to generate an electrochemical proton gradient (the protons, or hydrogen ions, accumulate in the intermembranal space) and the gradient is then utilized by respiratory chain complex V to drive ATP synthesis. In virtually all tissues, this process of oxidative phosphorylation (OXPHOS) is responsible for the bulk of ATP synthesis. Thus it is not surprising that respiratory chain dysfunction can affect any organ in the body. Severe respiratory chain defects cause a wide range of primary diseases in children and adults; these are described elsewhere in this issue (Christodoulou, 2000). Their clinical heterogeneity means that selecting patients for mitochondrial evaluation is a significant conundrum (Chinnery and Turnbull, 1997). Mitochondrial respiratory chain defects together comprise one of the more common inborn errors of metabolism, with an estimated incidence of 1/10 000 births (Schuelke et al., 1999). Genetically based mitochondrial respiratory chain dysfunction also contributes to the
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pathogenesis of common degenerative diseases (Wallace, 1999), so clearly it would be desirable to have simple and robust methods for detecting abnormal mitochondrial respiratory chain function and the underlying defects in mitochondrial (mt) DNA. Presently it is likely that many symptomatic patients who have a respiratory chain defect are undiagnosed; furthermore, there are other patients who have been diagnosed with a respiratory chain defect but who in fact do not have one. The reason for this unsatisfactory state of affairs is the subject of this review. Clinical suspicion of a respiratory chain defect is typically based on the presence of suggestive clinical features, family history, and the results of imaging or metabolic studies, particularly the finding of elevated lactic acid in cerebrospinal fluid or in blood. Since my laboratory is located in a children's hospital and is presently referred tissues from most children in Australia and New Zealand suspected of a respiratory chain defect, our referral pattern makes our diagnostic experience different from that of laboratories focused on adult neurology patients. In most children we have investigated for a respiratory chain disorder, the family history is not suggestive of a particular mode of inheritance, except that consanguinity is present in -20% of our diagnosed cases, implying autosomal recessive inheritance in these families. Patients suspected of a respiratory chain defect are usually investigated by one or more of three main methods: (i) respiratory chain enzyme and functional studies on mitochondria or homogenates prepared from biopsied tissues and/or cultured cells; (ii) histology, enzyme histochemistry and ultrastructural studies of tissue sections; and (iii) mtDNA mutation analysis. In some cases one or more of these investigations will give a definitive diagnosis, but in others the results might just be supportive of a respiratory chain defect (Figure 1).
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Multiple (22) mtDNA (35) Morphology (14)

Figure 1. Diagnostic yield of the three major methods used to diagnose respiratory chain defects in children. Between 1992 and 1998, 843 patients were studied at the Murdoch Institute Mitochondrial Laboratory, of whom 199 had a definite diagnosis of respiratory chain disease made according to the criteria of Walker and colleagues (Walker et al., 1996), as modified by Thorburn and co-workers (Thorburn et al., 1998). The pie chart shows data for 165 patients who had a severe defect detected by one or more of the three main classes of investigation, namely respiratory chain enzymes, mitochondrial morphology, and mtDNA mutation analysis. Of our definite diagnoses, >80% had all three classes of testing, but in 86% of the definite diagnoses only one class of testing gave a definitively abnormal result, with the other methods giving normal or non-specific results. Not shown are data for 34 patients who were classified as having a definite defect because of moderate defects detected by two or more methods.

Enzymes (94)

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Confounding aspects of mitochondrial diagnosis

This review describes six classes of problems that complicate diagnosis of respiratory chain dysfunction. Many of these problems are relevant not only to making a diagnosis in a particular patient but to investigational studies generally of the respiratory chain in specific cell types or lineages, such as the female germ line. Genetic complexity The mitochondrial genome comprises 37 genes, including genes for 13 protein subunits of the respiratory chain. The majority of respiratory chain enzyme subunits are encoded by nuclear genes, of which there are at least 70. A large number of other nuclear gene products, probably in excess of 100, are required for normal respiratory chain function. Table I gives examples of respiratory chain disease genes, showing that these disorders can exhibit virtually any mode of inheritance, including

Detecting mitochondrial abnormalities

Table I. Mechanisms, genes, and inheritance of mitochondrial respiratory chain defects Inheritance Maternal Gene category 1.mtDNA tRNA 2.mtDNA rRNA 3.mtDNA RC subunit Single deletions encompassing multiple mtDNA genes RC complex I subunit RC complex II subunit Protein import/assembly mtDNA/nuclear DNA interaction Regulation of iron transport (?) Metalloprotease/chaperonin (?) mtDNA transcription/replication Free radical scavenging ATP/ADP exchange mtDNA/nuclear DNA interaction (multiple mtDNA deletions) Mitochondrial protein import Mitochondrial iron export (?) Unknown role mtDNA point mutation plus gender and environment Examples MELAS, MERRF/Leigh disease OMIM reference8 Gene or locus MTTL1, MTTK MTRNA1 MTATP6 Multiple mtDNA genes deleted NDUFS8b SDHA SURF1 ECGF1 FRDA SPG7 Tfam Sod2 Antl 10q23.3-q24.3 3pl4.1-p21.2 DDP1 ABC7 BTHS MTND1 MTND4 MTND6

Sporadic Autosomal recessive

Autosomal dominant X-linked Complex

590050 590060 Non-syndromic deafness 561000 NARP/Leigh disease 516060 CPEO/Kearns-Sayre syndrome/Pearson 530000 syndrome 557000 Leigh disease, mitochondrial 602141 cytopathy Leigh disease 600857 Leigh disease 185620 MNGIE 550900 Friedreich ataxia 229300 Hereditary spastic paraplegia 602783 (ATfam knockout mouse) 600438 (ASod2 knockout mouse) 147640 (AAntl knockout mouse) 103220 CPEO 157640 601226 Deafness dystonia syndrome 304700 Sideroblastic anaemia/ataxia 300135 Barth syndrome 302060 LHON 516000 516003 516006

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References to these disorders refer to the Online Mendelian Inheritance in Man (OMIM) catalogue, 1999. Other complex I subunit mutations have been reported in the NDUFV1 (OMIM 161015) and NDUFS4 (OMIM 602694) genes. CPEO = chronic progressive external ophthalmoplegia; LHON = Leber hereditary optic neuropathy; MELAS = mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes; MERRF = myoclonic epilepsy, ragged red fibres; MNGIE = myoneurogastrointestinal encephalopathy; NARP = neuropathy, ataxia, retinitis pigmentosa; RC = respiratory chain.
b

autosomal dominant, autosomal recessive, Xlinked recessive as well as maternal ('cytoplasmic' or mitochondrial), complex and sporadic patterns. The non-Mendelian, dosage (or 'population') effects of mtDNA genetics (including heteroplasmy, tissue variation, the threshold effect, and the mtDNA bottleneck) all confer complexity of a unique kind to mitochondrial inheritance. A large number of mtDNA point mutations and deletions have been reported (DiMauro et aU 1998; MITOMAP database, 1999), with a relatively small number of 'classical' mtDNA mutations identified now in multiple families. These families have well-characterized (if uncommon) mitochondrial disease syndromes that go by such acronyms and

abbreviations as MELAS, MERRF, LHON, CPEO, KSS and NARP (Wallace, 1999; Christodoulou, 2000). In adult patients with mainly neuromuscular symptoms suspected of these disorders, a routine molecular analysis for these classical mutations can result in up to 80% of patients having a mutation identified. The high diagnostic yield in these (relatively rare) . conditions can result in the misperception that mtDNA mutations are the most common cause of respiratory chain dysfunction. However, such molecular findings are relatively uncommon in children suspected of mitochondrial disease. Unlike adults, typically <5% of children investigated for a respiratory chain disorder will have a known mtDNA mutation identified, and most dia59

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gnoses are achieved by finding an enzyme defect in a tissue biopsy or cell line (Figure 1) (Shoffner, 1996; Lamont et al, 1998). Although some children undoubtedly have rare mtDNA mutations that have not been tested, it is likely that most children with respiratory chain defects have mutations in the nuclear rather than mitochondrial genome. To date, mutations of only a handful of nuclear genes (these include paraplegin, Surf-1, and thymidine phosphorylase; see Table I) have been shown to cause respiratory chain dysfunction in more than one or two families. As more genes are identified in the next few years, it ought to be possible to identify a genetic basis in more cases, but the large number of genes involved, and the blurred correlation between genotype and phenotype, will still make it difficult to decide which gene(s) to target for investigation in particular cases. Tissue and temporal variation When investigating a patient with a suspected respiratory chain defect, the first choices to be made are the methods to use and the tissue or tissues to study. Ideally, the tissue chosen would be able to be obtained in a minimally intrusive manner; the tissue of first choice for mtDNA testing could thus be blood, hair follicles, mouth squames, or skin fibroblasts. However, some mtDNA mutations, particularly deletions, can be absent from these tissues, despite being present in high amounts in post-mitotic tissues such as muscle or brain (Holt et al., 1989). Patients with the syndrome of mtDNA depletion often show marked tissue specificity, e.g. showing normal mtDNA:nuclear DNA ratios in muscle, but gross mtDNA depletion in liver or kidney (DiMauro et al., 1998). Inter-tissue variation can particularly affect enzyme studies and be vividly evident morphologically. Dramatic abnormality of mito60

chondrial morphology in one tissue but a completely normal appearance in another tissue is relatively common and has been described in detail elsewhere in this issue (Chow and Thorburn, 2000). In our experience, -50% of the children with a respiratory chain defect in skeletal muscle have normal enzyme activities in cultured skin fibroblasts or liver. Tissue specificity is even more marked for patients with a diagnosis based on a liver respiratory chain enzyme defect; of 36 such patients whom we have diagnosed where skeletal muscle was also available for enzyme analysis, only six had muscle respiratory chain enzymes that were diagnostic of respiratory chain dysfunction (a typical example is shown in Figure 2a,b). Respiratory chain defects can also show marked within-tissue variation; classic examples are mtDNA deletions and tRNA mitochondrial gene mutations in skeletal muscle. Enzyme histochemical staining of muscle sections for cytochrome c oxidase activity (COX, or respiratory chain complex IV) in such patients usually shows a mosaic of COX-negative and COX-positive fibres (in contrast to patients with nuclear-encoded COX defects, who have uniformly decreased staining). Single-fibre polymerase chain reaction (PCR) analysis typically reveals that the COXnegative fibres contain very high levels of mutant mtDNA, whereas adjacent COX-positive fibres show a higher proportion of wildtype mtDNA (Moraes et al, 1993). Withintissue variation is not restricted to skeletal muscle; it is observed in blood, skin fibroblasts, and other tissues. Within-tissue variation can be particularly pronounced in the ovaries; a striking difference in the mutant loads of individual oocytes has been reported from a carrier of the mtDNA T8993G mutation (Blok et al., 1997). This is presumably a germ cell segregation effect related to the mitochondrial bottleneck phenomenon. Some tissues can show dramatic changes in

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Detecting mitochondrial abnormalities

240 220 200 180 160 140 120 100 80 60 40 20 0

1 t ten

ni us cle

\
I

240 220 200 180 160 140 120 100 80 60 40200

(B) patient 1 liver

240 220 200 180 160 140 120 100 80 60 40 20 0

(C) patient 2 muscle

I I

III

IV C S

I I

III

IV C S

II

III

IV

CS

Figure 2. Respiratory chain enzyme profiles in two patients with mitochondrial disease. Data are shown for respiratory chain complexes I, II, III and IV, and for the mitochondrial marker enzyme citrate synthase (CS). Residual enzyme activities are expressed as the percentage of normal control mean value for (A, C) skeletal muscle or (B) liver. The vertical bars represent the observed range for normal control samples. Patient 1 (mitochondrial encephalopathy with chronic intestinal pseudoobstruction) had normal respiratory chain enzyme activities in muscle, but severe defects of complexes I, III and IV in liver. Patient 2 (Kearns-Sayre syndrome) had normal (average) enzyme activities in skeletal muscle despite having a mtDNA deletion present in 30% of mtDNA molecules (see Figure 3 in Blok et al., 1995) and substantial numbers of ragged red fibres on histology.

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the amount of mtDNA mutations over time. It has been reported (Poulton and Morten, 1993) that blood and muscle mutant loads of the MELAS A3243G mutation appear to be the same at birth, but diverge with increasing age. This appears to be mostly due to selection against the mutation in blood (a tissue undergoing active mitosis), in which the mutation load can decrease with age, whereas in muscle (a mostly post-mitotic tissue) the mutation load can increase with age (Weber et al., 1997). Temporal variation is not confined to measurements of mtDNA mutation load, but can apply to morphological changes and to respiratory chain enzyme activity estimation, as in the patient with Alpers syndrome described by Chow and Thorburn (2000). Tissue and temporal variation of respiratory chain defects should be borne in mind in choosing which tissue to study and in deciding whether to biopsy a new tissue or to re-biopsy a tissue studied at an earlier stage of the patient's disease. The absence of a mutation, enzyme defect or abnormal morphological finding in one tissue clearly will not exclude its presence in a tissue more closely implicated by the patient's symptoms. As a general rule, if there is a clinically-apparent myopathy, then skeletal muscle is the tissue most likely to

yield a diagnosis and a child with abnormal conventional liver function tests should have a liver biopsy studied. Methodological limitations A limitation of most respiratory chain enzyme and functional assays is that they represent the activity averaged over all the mitochondria or cells in the sample. Enzymology typically loses information because it relies on homogenized samples. Enzyme histochemical staining, on the other hand, retains spatial information about the distribution of enzyme activity among muscle fibres. This difference is exemplified by Figure 2c, which shows a normal muscle respiratory chain enzyme profile in a patient who had a mtDNA deletion present at -30% mutant load and had demonstrable numbers of COX-negative and ragged red fibres on histochemical study (Blok et al., 1995). The lack of spatial information in biochemical respiratory chain enzyme analysis is balanced by the fact that it is more quantitative than histochemical staining. Enzyme assays are usually performed in a dilute aqueous system that differs from circumstances in vivo in several ways (Table II). Any of these differences could result in 61

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Table II. Differences between the in-vitro assay system for respiratory chain complex I and the in-vivo situation as possible explanations for why some complex I defects do not affect the measured enzyme activity In-vitro assay Mitochondria Protein concentration Substrate concentration Quinone substrate Function Disrupted 1 mg/ml Saturating (i.e. > > K m ) 5-carbon saturated chain Electron transport In-vivo system Intact 500 mg/ml Low (i.e. <K m ) 50-carbon isoprenoid chain Electron transport and proton pumping

an abnormal kinetic variant appearing to have normal enzyme activity in the in-vitro assay system. An example of this is the mtDNA G11778A mutation in the NADH dehydrogenase (ND)4 subunit gene of complex I, which has little effect on assayed complex I activity, although it strongly inhibits oxidation of complex I substrates (Hofhaus et al, 1996). Studies of respiratory chain function require intact mitochondria, and so this problem can in principle be avoided; but respiratory chain function studies also suffer from limitations (e.g. the need for fresh tissue) and are less robust than enzyme assays because of artefacts (e.g. inhibition or uncoupling by anaesthetics, variation in the efficiency of isolating wellcoupled mitochondria, and other secondary effects discussed below). Mitochondrial morphology can be normal or might show only non-specific changes in patient biopsies (Chow and Thorburn, 2000). Although enzyme histochemical studies retain spatial information, they are unable to detect respiratory chain complex I defects, which are the most common respiratory chain enzyme defects in children and which typically show unremarkable histochemical or morphological changes (Kirby et al, 1999). Methods of detecting mtDNA point mutations are also subject to specific limitations related to sensitivity, specificity, expense, and ease of use. Sequencing of mtDNA is a highly specific approach to mutation detection, but is not a 'front line' method in most centres due to the work-load and expense of 62

sequencing the entire 16.5 kb mtDNA genome, together with the relatively low sensitivity, which means that mutations present at heteroplasmic mutant loads of <20% may not be distinguished from background signals. PCR with restriction fragment-length polymorphism analysis is one of the most common direct tests used to detect a specific mutation. This test detects mtDNA mutations that either introduce or remove a restriction site by digesting patient and control DNA with the appropriate restriction enzyme, followed by separation of the fragments using gel electrophoresis. If the mutation introduces a restriction site, then the mutant DNA fragment will be smaller than the control fragment and will migrate faster. If the mutation abolishes the site, the mutant DNA fragment will be larger and slower to migrate. These tests are relatively sensitive and can detect mtDNA mutations present in only a few percent of total mtDNA. However, they can give false positive or false negative results if a mtDNA polymorphism is present within the restriction enzyme site. It has been estimated for example that such polymorphisms gave a false-positive rate of 2-7% for mtDNA mutations associated with LHON (Johns and Neufeld, 1993). Polymorphisms further away from the mtDNA mutation can also potentially cause false positive results for mtDNA mutations associated with MELAS (Kirby et al, 1998) and Leigh syndrome (White et al, 1998). These false results are more likely with mtDNA than with nuclear DNA tests, because polymorphisms are more

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Detecting mitochondrial abnormalities

common in the mitochondrial genome than in the nuclear genome. Methodological limitations affecting respiratory chain enzyme, morphological and mtDNA analyses mean that none of these methods on their own can provide a benchmark for the detection of all patients with respiratory chain defects. In practice, most patients require an integrated approach to diagnosis, utilizing each of these methods either simultaneously or sequentially (Chinnery and Turnbull, 1997). Secondary effects Mitochondrial numbers and function are influenced by the intracellular milieu and by external environmental factors impinging on the cell and organ in which they are located. This can blur the distinction between changes due to a primary respiratory chain defect and those due to secondary effects (reviewed by Trijbels et al., 1993). Skeletal muscle respiratory chain enzyme levels, for example, can be influenced by a number of factors, e.g. inactivity of age, fitness, immobility, and variation in fibre type composition. In our experience, skeletal muscle homogenates from neonates tend to have only about one-third of the level of respiratory chain enzymes compared with older children (presumably reflecting the relatively anaerobic environment of the preceding fetus). There are numerous reports of declining skeletal muscle respiratory chain enzyme levels in old age, and this is at least partly due to the effects of detraining rather than of mitochondrial mutations or ageing per se (Brierley et al., 1997). To distinguish primary deficiencies in respiratory chain enzymes from secondary deficiencies, activity measurements are made relative to a marker enzyme such as citrate synthase (Kirby et al., 1999) or as internal ratios (Chretien et al., 1998), rather than relative to protein. Interestingly, our experience suggests that liver and cardiac muscle have less age-related sec-

ondary variation, perhaps because as organs they are used more constantly than skeletal muscle. Tissue pathology and other inborn errors of metabolism can also secondarily affect respiratory chain enzyme levels and mitochondrial morphology. Our experience is that healthy livers have quite narrow observed ranges for each of the respiratory chain enzymes (lowest activities are typically >70% of the mean value). Livers from patients who have a known inborn error of metabolism (e.g. pyruvate dehydrogenase deficiency, fatty acid oxidation defects, or organic acidaemias) or liver disease (e.g. chronic biliary cirrhosis or massive hepatic necrosis) can have much more variable respiratory chain enzymes. Different respiratory chain enzymes show different extents of secondary variation. In our experience, complexes II, III, and the combined 11+III and I+III assays show most variability (lowest activities are down to -30% of the mean value), followed by complex I and then complex IV, with citrate synthase being the most robust assay. This pattern of lability tends to hold for secondary effects generally, whether caused by other inborn errors of metabolism, liver pathology, post-mortem delay in sample collection, or exposure to free radical damage, as in Friedreich's ataxia (Rotig et al., 1997) or, experimentally, in the Sod-2 knockout mouse (Melov et al., 1999). Another potential cause of secondary effects on respiratory chain enzymes and function are drugs [particularly antiviral nucleoside analogues such as zidovudine (AZT) (Lewis and Dalakas 1995) and fialuridine (Lewis et al., 1996)], poisons (e.g. carbon monoxide or cyanide) and anaesthetic agents (particularly barbiturates, which inhibit complex I, and bupivacaine). A number of authors have suggested that purified mitochondria from frozen tissue samples should not be used for respiratory chain enzyme assays, because of potential 63

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artefacts (as examples: Zheng et al., 1990; of <20% of the mean normal control value. Scholte and Trijbels 1995). Our experience is This is probably due to muscle disuse rather that assays for respiratory chain complex V than primary mitochondrial involvement in and combined assays relying on endogenous the disease process. Similarly, we have studied ubiquinone (complex I+III and 11+III) are mtDNA:nuclear DNA ratios in post-mortem not reliable in frozen tissue homogenates. liver samples from patients with liver failure However, in our laboratory, the activities of (e.g. chronic biliary cirrhosis, massive hepatic complexes I, II, III, IV and citrate synthase in necrosis). Some samples had values less than homogenates prepared from frozen muscle or 20% of the mean normal control value, and liver appear to be stable for over a decade if could be mistaken for primary mtDNA the tissue samples have been frozen rapidly depletion. and stored at -70C. Since secondary effects on respiratory chain Mitochondrial morphology and mtDNA enzymes, mitochondrial morphology, and studies are also prone to secondary effects. mtDNA can potentially result in misdiagnosis, Markedly abnormal mitochondrial morpho- it is particularly important to consider whether logy has been reported in conditions as diverse a normal range for respiratory chain enzymes as fat oxidation defects (Tyni et al., 1996), that has been established on healthy control alcoholic liver disease (Inagaki et al., 1992), tissues is appropriate for comparison with cyclosporin therapy (Larner et al., 1994) and, tissues from patients in other circumstances of course, anoxic tissue damage of any cause. and with primary tissue pathology of other In an inexperienced laboratory, the increased cause. mitochondrial numbers in skeletal muscle of an elite athlete could be mistaken for abnormal Logistical issues mitochondrial proliferation. False positive and false negative results for mtDNA point muta- Logistical issues can influence what tests may tion tests due to polymorphisms have been be performed on a patient suspected of respirdescribed above. Another example of a sec- atory chain dysfunction. Common mtDNA ondary effect on mtDNA mutation analysis mutation tests and routine histology and morlies behind a recent claim that multiple hetero- phology tests are widely available. More plasmic mtDNA mutations were a major risk extensive mtDNA mutation analysis can be factor for late-onset Alzheimer's disease obtained at only a limited number of centres. (Davis et al., 1997). It was subsequently Similarly, the type of respiratory chain enzyme shown that this finding was an artefact of co- and functional assays available and the tissues amplifying nuclear-embedded mtDNA that can be studied vary widely between differpseudogenes rather than an identification of ent centres. authentic mtDNA mutations (Hirano et al., The patient's location and, in some rapidly 1997; Wallace et al., 1997). progressive cases, the time of his or her A substantial number of cases of mtDNA death can govern whether fresh tissue can be depletion have been reported in recent years. transported to the laboratory for functional While there is no doubt that this is a real studies or if only frozen tissue can be fursyndrome, a number of secondary factors can nished. The large muscle biopsies (> 0.5 g) affect the mtDNA:nuclear DNA ratio. It has needed for traditional enzyme and polarobeen reported (Berger et al., 1998) that 19 of graphic studies cannot be obtained from 20 patients with spinal muscular atrophy had patients presenting in the neonatal period. mtDNA.nuclear DNA ratios in skeletal muscle While it is preferable to study 'symptomatic' 64

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tissues, this is often not practical (for example a brain biopsy would be impractical for a patient whose only symptoms related to encephalopathy). Finally, economic issues can dictate what tests are available. Full testing of respiratory chain enzymes and function in multiple tissues, mitochondrial morphology and detailed mtDNA analysis may cost thousands of dollars for a patient, and the local centre might have only the resources to devote a certain amount of time and effort to each sample. In such situations, it may be necessary to ration the availability of testing to highsuspicion patients, or to focus on using only the methods that are likely to give the highest number of diagnoses per annum. Interpretation The last difficulty in the diagnosis of respiratory chain defects lies with the interpretation of the data obtained. The secondary effects discussed above mean that many patients will have respiratory chain enzyme levels or morphological changes that are moderately below the 'normal' range and which overlap with levels or changes caused by authentic, primary respiratory chain disorders. It is difficult or impossible to define absolute cut-offs for normal ranges that are at once realistic and yet can reliably distinguish primary from secondary changes. While some respiratory chain enzyme defects or morphological changes are sufficiently marked to be diagnostic, others should therefore be interpreted as only suggestive of respiratory chain dysfunction. It is frustrating to patients, their families, clinicians and the laboratory to label patients as having a probable rather than a definite defect. However, I believe it is better to err on the conservative side in making such diagnoses. Once a patient is regarded as having a primary defect of the mitochondria, the clinician's focus may shift to patient management and away from con-

sidering alternative, potentially treatable diagnoses. Problems in interpretation also extend to mtDNA studies. If a patient has a novel mtDNA nucleotide change identified, a substantial amount of work is required to confirm that the change is pathogenic (Moraes et al., 1993; Walker et ai, 1996). A classic example of interpreting the role of mtDNA mutations in pathology relates to the role of mtDNA mutations that accumulate in somatic tissues with age. Many of these studies have focused on the accumulation of the common 4977 bp deletion (AmtDNA4977), which is typically undetectable in muscle or other tissues from normal young individuals but becomes increasingly detectable in different postmitotic tissues with advancing age. Even in old individuals, the amount of this mutant mtDNA species is typically < 1 % of total mtDNA, and debate continues as to whether this mutation (and others like it) is a major contributor to the phenotypic expression of ageing and common degenerative diseases or simply a clinically unimportant epiphenomenon. Two recent reviews have argued both the case for (Nagley and Wei, 1998), and against (Lightowlers et ai, 1999), a causal relationship. Conclusions I do not intend to convey the impression that practical problems always provide an insurmountable barrier to accurate diagnosis of respiratory chain dysfunction. My own laboratory has been involved in diagnosing -200 children with definite respiratory chain defects, and other large centres would have the experience of having made a similar number of reliable diagnoses. It is clear that issues such as genetic complexity, tissue and temporal variation, methodological limitations, secondary effects, logistical issues and interpretation need to be addressed in the process of investi65

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gating suspected cases. The final process involves weighing evidence from sources which include clinical findings, metabolic and imaging investigations, respiratory chain enzymes and function, mitochondrial morphology, and DNA studies in order to achieve a diagnosis of definite, probable or possible respiratory chain dysfunction. A recent review of this process has been published (Walker et al., 1996). In many cases a definite diagnosis can be achieved, but it is sensible to retain a cautious view of any individual case as to whether such a diagnosis has been confirmed or excluded.

Acknowledgements
I wish to thank all the colleagues who have made major contributions to the diagnostic work of my laboratory, in particular Denise Kirby and Henrik Dahl, C.W.Chow, Xenia Dennett, Avihu Boneh, Maureen Cleary, Jim McGill, David Danks and Geoff Thompson, as well as the staff of the Murdoch Institute Diagnostic DNA and Tissue Culture Laboratories. I am also grateful to geneticists, neurologists, and paediatricians throughout Australia and New Zealand for referring patients for investigation. This work was supported in part by an Institute Grant from the National Health and Medical Research Council of Australia.

References
Berger, A., Kofler, B., Pilz, P. et al. (1998) Mitochondrial DNA depletion in patients with spinal muscular atrophy. J. Inker. Metab. Dis., 21 (Suppl 2), 82. Blok, R.B., Thorburn, D.R., Thompson, G.N. et al. (1995) A topoisomerase II cleavage site is associated with a novel mitochondrial DNA deletion. Hum. Genet., 95, 75-81. Blok, R.B., Gook, D.A., Thorburn, D.R. et al. (1997) Skewed segregation of the mtDNA nt8993 (T->G) mutation in human oocytes. Am. J. Hum. Genet., 60, 1495-1501. Brierley, E.J., Johnson, M.A., Bowman, A. et al. (1997) Mitochondrial function in muscle from elderly athletes. Ann. Neuroi, 41, 114-116. Chinnery, P.F. and Turnbull, D.M. (1997) Clinical features, investigation, and management of patients with defects of mitochondrial DNA. J.Neurol. Neurosurg. Psychiatry, 63, 559-563.

Chow, C.W. and Thorburn, D.R. (2000) Morphological correlates of mitochondrial dysfunction in children. Hum. Reprod., 15 (Suppl. 2), 68-78. Chretien, D., Gallego, J., Barrientos, A. et al. (1998) Biochemical parameters for the diagnosis of mitochondrial respiratory chain deficiency in humans, and their lack of age-related changes. Biochem. J., 329, 249-254. Christodoulou, J. (2000) Genetic defects causing mitochondrial respiratory chain disorders and disease. Hum. Reprod., 15 (Suppl. 2), 2 8 ^ 3 . Davis, R.E., Miller, S., Herrnstadt, C. et al. (1997) Mutations in mitochondrial cytochrome c oxidase genes segregate with late-onset Alzheimer disease. Proc. NatlAcad. Sci. USA, 94, 4526-4531. DiMauro, S., Bonilla, E., Davidson, M. et al. (1998) Mitochondria in neuromuscular disorders. Biochim. Biophys. Acta, 1366, 199-210. Hirano, M., Shtilbans, A., Mayeux, R. et al. (1997) Apparent mtDNA heteroplasmy in Alzheimer's disease patients and in normals due to PCR amplification of nucleus-embedded mtDNA pseudogenes. Proc. Natl Acad. Sci. USA, 94, 14894-14899. Hofhaus, G., Johns, D.R., Hurko, O. et al. (1996) Respiration and growth defects in transmitochondrial cell lines carrying the 11778 mutation associated with Leber's hereditary optic neuropathy. J. Biol. Chem., 271, 13155-13161. Holt, I.J., Harding, A.E., Cooper, J.M. et al. (1989) Mitochondrial myopathies: clinical and biochemical features of 30 patients with major deletions of muscle mitochondrial DNA. Ann. Neuroi, 26, 699-708. Inagaki, T., Kobayashi, S., Ozeki, N. et al. (1992) Ultrastructural identification of light microscopic giant mitochondria in alcoholic liver disease. Hepatology, 15, 46-53. Johns, D.R. and Neufeld, M.J. (1993) Pitfalls in the molecular genetic diagnosis of Leber hereditary optic neuropathy (LHON). Am. J. Hum. Genet, 53, 916920. Kirby, D.M., Milovac, T. and Thorburn, D.R. (1998) A false positive diagnosis for the common MELAS (A3243G) mutation caused by a novel variant in the ND1 gene of mtDNA. Mol. Diagn., 3, 211-215. Kirby, D.M., Crawford, M., Cleary, M.A. et al. (1999) Respiratory chain complex I deficiency. An underdiagnosed energy generation disorder. Neurology, 52, 1255-1264. Lamont, P.J., Surtees, R., Woodward, C.E. et al. (1998) Clinical and laboratory findings in referrals for mitochondrial DNA analysis. Arch. Dis. Child., 79, 22-27. Larner, A.J., Sturman, S.G., Hawkins, J.B. et al. (1994) Myopathy with ragged red fibres following renal transplantation: Possible role of cyclosporin-induced hypomagnesaemia. Acta Neuropathol, 88, 189-192.

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