Practical Problems in Detecting Abnormal Mitochondrial Function and Genomes (Thornburn, 2000). the Murdoch Institute, Australia 2000

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Human Reproduction, Vol. 15, (Suppl. 2), pp. 57-67, 2000

Practical problems in detecting abnormal mitochondrialfunction and genomesDavid R.Thorburn1

The Murdoch Ins t i tute, Royal Chi ldren 's Hospi tal , Melbourne, Austral ialrTo whom correspondence should be addressed at: The Murdoch Institute, RoyalChildren's Hospital, Flemington Road, Parkville, Melbourne, Victoria 3052,Australia. E-mail: [email protected]

Mitochondrial respiratory chain dysfunc-tion causes a wide range of primary diseasesin adults and children, with highly variableorgan involvement. Diagnosis involvesweighing evidence from a number ofsources, including the clinical presentation,metabolic measurements in vivo, imagingstudies, analysis of respiratory chain func-tion or enzyme activities in vitro, studiesof mitochondrial morphology after biopsy,and mitochondrial (mt) DNA mutation ana-lysis. Irrespective of the category of theinformation, it can be difficult to determinewhether abnormal results are due to prim-ary defects of the respiratory chain orto practical problems that complicate thediagnostic methodology. This reviewdescribes six sources of such problems:genetic complexity, tissue and temporalvariation, methodological limitations, sec-ondary effects, logistical issues, and ques-tions of interpretation. When these issuesare all addressed, a reliable categorizationof the diagnosis as definite, probable, orpossible respiratory chain defect becomespossible.Key words: diagnosis/mitochondrial disease/mitochondrial DNA/mitochondrial function/respiratory chain

IntroductionThe mitochondrial respiratory chain is thecentral energy generating pathway in humansand all other eukaryotic species; oxidation offuels such as sugar, fat, and protein feedselectrons into it. These electrons are trans-ferred through respiratory chain complexesI-IV to generate an electrochemical protongradient (the protons, or hydrogen ions, accu-mulate in the intermembranal space) and thegradient is then utilized by respiratory chaincomplex V to drive ATP synthesis. In virtuallyall tissues, this process of oxidative phospho-rylation (OXPHOS) is responsible for the bulkof ATP synthesis. Thus it is not surprisingthat respiratory chain dysfunction can affectany organ in the body. Severe respiratorychain defects cause a wide range of primarydiseases in children and adults; these aredescribed elsewhere in this issue(Christodoulou, 2000). Their clinical hetero-geneity means that selecting patients for mito-chondrial evaluation is a significantconundrum (Chinnery and Turnbull, 1997).

Mitochondrial respiratory chain defectstogether comprise one of the more commoninborn errors of metabolism, with an estimatedincidence of 1/10 000 births (Schuelke et al.,1999). Genetically based mitochondrial respir-atory chain dysfunction also contributes to the European S ociety of Human R eproduct ion and Embryology 57

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D.R.Thorburnpathogenesis of common degenerative dis-eases (Wallace, 1999), so clearly it would bedesirable to have simple and robust methodsfor detecting abnormal mitochondrial respirat-ory chain function and the underlying defectsin mitochondrial (mt) DNA. Presently it islikely that many symptomatic patients whohave a respiratory chain defect are undia-gnosed; furthermore, there are other patientswho have been diagnosed with a respiratorychain defect but who in fact do not have one.The reason for this unsatisfactory state ofaffairs is the subject of this review.

Clinical suspicion of a respiratory chaindefect is typically based on the presence ofsuggestive clinical features, family history,and the results of imaging or metabolic studies,particularly the finding of elevated lactic acidin cerebrospinal fluid or in blood. Since mylaboratory is located in a children's hospitaland is presently referred tissues from mostchildren in Australia and New Zealand sus-pected of a respiratory chain defect, our refer-ral pattern makes our diagnostic experiencedifferent from that of laboratories focused onadult neurology patients. In most childrenwe have investigated for a respiratory chaindisorder, the family history is not suggestiveof a particular mode of inheritance, exceptthat consanguinity is present in -20% of ourdiagnosed cases, implying autosomal recessiveinheritance in these families.

Patients suspected of a respiratory chaindefect are usually investigated by one or moreof three main methods: (i) respiratory chainenzyme and functional studies on mitochon-dria or homogenates prepared from biopsiedtissues and/or cultured cells; (ii) histology,enzyme histochemistry and ultrastructuralstudies of tissue sections; and (iii) mtDNAmutation analysis. In some cases one or moreof these investigations will give a definitivediagnosis, but in others the results might justbe supportive of a respiratory chain defect(Figure 1).

Multiple(22)mtDNA(35)Morphology(14)

Enzymes(94)

Figure 1. Diagnostic yield of the three major methods usedto diagnose respiratory chain defects in children. Between1992 and 1998, 843 patients were studied at the MurdochInstitute Mitochondrial Laboratory, of whom 199 had adefinite diagnosis of respiratory chain disease made accordingto 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 asevere defect detected by one or more of the three mainclasses of investigation, namely respiratory chain enzymes,mitochondrial morphology, and mtDNA mutation analysis. Ofour definite diagnoses, >80% had all three classes of testing,but in 86% of the definite diagnoses only one class of testinggave a definitively abnormal result, with the other methodsgiving normal or non-specific results. Not shown are data for34 patients who were classified as having a definite defectbecause of moderate defects detected by two or moremethods.

Confounding aspects of mitochondrialdiagnosisThis review describes six classes of problemsthat complicate diagnosis of respiratory chaindysfunction. Many of these problems are rele-vant not only to making a diagnosis in aparticular patient but to investigational studiesgenerally of the respiratory chain in specificcell types or lineages, such as the femalegerm line.

Genetic complexityThe mitochondrial genome comprises 37genes, including genes for 13 protein subunitsof the respiratory c hain. The majority of respir-atory chain enzyme subunits are encoded bynuclear genes, of which there are at least 70.A large number of other nuclear gene products,probably in excess of 100, are required fornormal respiratory chain function. Table Igives examples of respiratory chain diseasegenes, showing that these disorders can exhibitvirtually any mode of inheritance, including

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Detecting mitochondrial abnormalities

Table I. Mechanisms, genes, and inheritance of mitochondrial respiratory chain defectsInheritance

Maternal

SporadicAutosomalrecessive

AutosomaldominantX-linked

Complex

Gene category

1.mtDNA tRNA2.mtDNA rRNA3.mtDNA RC subunitSingle deletions encompassingmultiple mtDNA genesRC complex I subunitRC complex II subunitProtein import/assemblymtDNA/nuclear DNA interactionRegulation of iron transport (?)Metalloprotease/chaperonin (?)mtDNA transcription/replicationFree radical scavengingATP/ADP exchangemtDNA/nuclear DNA interaction(multiple mtDNA deletions)Mitochondrial protein importMitochondrial iron export (?)Unknown rolemtDNA point mutation plusgender and environment

Examples

MELAS, MERRF/Leigh diseaseNon-syndromic deafnessNARP/Leigh diseaseCPEO/Kearns-Sayre syndrome/PearsonsyndromeLeigh disease, mitochondrialcytopathyLeigh diseaseLeigh diseaseMNGIEFriedreich ataxiaHereditary spastic paraplegia(ATfam knockout mouse)(ASod2 knockout mouse)(AAntl knockout mouse)CPEODeafness dystonia syndromeSideroblastic anaemia/ataxiaBarth syndromeLHON

OMIMreference8

590050590060561000516060530000557000602141600857185620550900229300602783600438147640103220157640601226304700300135302060516000516003516006

Gene or locus

MTTL1, MTTKMTRNA1MTATP6Multiple mtDNAgenes deletedNDUFS8bSDHASURF1ECGF1FRDASPG7TfamSod2Antl10q23.3-q24.33pl4.1-p21.2DDP1ABC7BTHSMTND1MTND4MTND6

a References to these disorders refer to the Online Mendelian Inheritance in Man (OMIM) catalogue, 1999.b 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 redfibres; MNGIE = myoneurogastrointestinal encephalopathy; NARP = neuropathy, ataxia, retinitis pigmentosa; RC =respiratory chain.

autosomal dominant, autosomal recessive, X-linked recessive as well as maternal ('cyto-plasmic' or mitochond rial), complex and spor-adic patterns. The non-Mendelian, dosage (or'population') effects of mtDNA genetics(including heteroplasmy, tissue variation, thethreshold effect, and the mtDNA bottleneck)all confer complexity of a unique kind tomitochondrial inheritance.A large number of mtDNA point mutationsand deletions have been reported (DiMauroet aU 1998; MITOMAP database, 1999),with a relatively small number of 'classical'mtDNA mutations identified now in multiplefamilies. These families have well-character-ized (if uncommon) mitochondrial diseasesyndromes that go by such acronyms and

abbreviations as MELAS, MERRF, LHON,CPEO, KSS and NARP (Wallace, 1999;Christodoulou, 2000). In adult patients withmainly neuromuscular symptoms suspected ofthese disorders, a routine molecular analysisfor these classical mutations can result in upto 80% of patients having a mutation identi-fied. The high diagnostic yield in these (rela-tively rare) . conditions can result in themisperception that mtDNA mutations are themost common cause of respiratory chain dys-function. However, such molecular findingsare relatively uncommon in children suspectedof mitochondrial disease. Unlike adults, typic-ally


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D.R.Thorburngnoses are achieved by finding an enzymedefect in a tissue biopsy or cell line (Figure1) (Shoffner, 1996; Lamont et al, 1998).Although some children undoubtedly haverare mtDNA mutations that have not beentested, it is likely that most children withrespiratory chain defects have mutations inthe nuclear rather than mitochondrial genom e.To date, mutations of only a handful of nucleargenes (these include paraplegin, Surf-1, andthymidine phosphorylase; see Table I) havebeen shown to cause respiratory chain dys-function in more than one or two families. Asmore genes are identified in the next fewyears, it ought to be possible to identify agenetic basis in more cases, but the largenumber of genes involved, and the blurredcorrelation between genotype and phenotype,will still make it difficult to decide whichgene(s) to target for investigation in particu-lar cases.

Tissue and tem poral variationWhen investigating a patient with a suspectedrespiratory chain defect, the first choices tobe made are the methods to use and the tissueor tissues to study. Ideally, the tissue chosenwould be able to be obtained in a minimallyintrusive manner; the tissue of first choice formtDNA testing could thus be blood, hairfollicles, mouth squames, or skin fibroblasts.However, some mtDNA mutations, particu-larly deletions, can be absent from these tis-sues, despite being present in high amountsin post-mitotic tissues such as muscle or brain(Holt et al., 1989). Patients with the syndromeof mtDNA depletion often show marked tissuespecificity, e.g. showing normal mtDNA:nu-clear DNA ratios in muscle, but gross mtDN Adepletion in liver or kidney (DiMauro et al.,1998).

Inter-tissue variation can particularly affectenzyme studies and be vividly evident mor-phologically. Dramatic abnormality of mito-

chondrial morphology in one tissue but acompletely normal appearance in another tis-sue is relatively common and has beendescribed in detail elsewhere in this issue(Chow and Thorburn, 2000). In our experi-ence, -50% of the children with a respiratorychain defect in skeletal muscle have normalenzyme activities in cultured skin fibroblastsor liver. Tissue specificity is even more markedfor patients with a diagnosis based on a liverrespiratory chain enzyme defect; of 36 suchpatients whom we have diagnosed where skel-etal muscle was also available for enzymeanalysis, only six had muscle respiratory chainenzymes that were diagnostic of respiratorychain dysfunction (a typical example is shownin Figure 2a,b).

Respiratory chain defects can also showmarked within-tissue variation; classicexamples are mtDNA deletions and tRNAmitochondrial gene mutations in skeletalmuscle. Enzyme histochemical staining ofmuscle sections for cytochrome c oxidaseactivity (COX, or respiratory chain complexIV) in such patients usually shows a mosaicof COX-negative and COX-positive fibres (incontrast to patients with nuclear-encoded COXdefects, who have uniformly decreased stain-ing). Single-fibre polymerase chain reaction(PCR) analysis typically reveals that the COX-negative fibres contain very high levels ofmutant mtDN A, whereas adjacent COX-posit-ive fibres show a higher proportion of wild-type mtDNA (Moraes et al, 1993). Within-tissue variation is not restricted to skeletalmuscle; it is observed in blood, skinfibroblasts, and other tissues. Within-tissuevariation can be particularly pronounced inthe ovaries; a striking difference in the mutantloads of individual oocytes has been reportedfrom a carrier of the mtDNA T8993G mutation(Blok et al., 1997). This is presumably agerm cell segregation effect related to themitochondrial bottleneck phenomenon.Some tissues can show dramatic changes in

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Detecting mitochondrial abnormalities240220200180160140120100806040200

u 1 tten

\

ni us cle

I II III IV C S

240220200180160140120100806040-20-0

(B) patient 1 liver

I II III IV C S

240220200180160140120100806040200

(C) patient 2 muscle

I II III IV CS

Figure 2. Respiratory chain enzyme profiles in two patients with mitochondrial disease. Data are shown for respiratory chaincomplexes I, II, III and IV, and for the mitochondrial marker enzyme citrate synthase (CS). Residual enzyme activities areexpressed as the percentage of normal control mean value for (A, C) skeletal muscle or (B) liver. The vertical bars representthe observed rang e for normal control samp les. Patient 1 (mitochondrial enceph alopathy with chronic intestinal pseudo-obstruction) 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 mtDNAdeletion present in 30% of mtDNA molecules (see Figure 3 in Blok et al., 1995) and substantial numbers of ragged red fibreson histology.

the amount of mtDNA mutations over time.It has been reported (Poulton and Morten,1993) that blood and muscle mutant loads ofthe MELAS A3243G mutation appear to bethe same at birth, but diverge with increasingage. This appears to be mostly due to selectionagainst the mutation in blood (a tissue under-going active mitosis), in which the mutationload can decrease with age, whereas in muscle(a mostly post-mitotic tissue) the mutationload can increase with age (Weber et al.,1997). Temporal variation is not confined tomeasurements of mtDNA mutation load, butcan apply to morphological changes and torespiratory chain enzyme activity estimation,as in the patient with Alpers syndromedescribed by Chow and Thorburn (2000).Tissue and temporal variation of respiratorychain defects should be borne in mind inchoosing which tissue to study and in decidingwhether to biopsy a new tissue or to re-biopsya tissue studied at an earlier stage of thepatient's disease. The absence of a mutation,enzyme defect or abnormal morphologicalfinding in one tissue clearly will not exc ludeits presence in a tissue m ore closely implicatedby the patient's symptoms. As a general rule,if there is a clinically-apparent myopa thy, thenskeletal muscle is the tissue most likely to

yield a diagnosis and a child with abnormalconventional liver function tests should havea liver biopsy studied.Methodological limitations

A limitation of most respiratory chain enzymeand functional assays is that they representthe activity averaged over all the mitochondriaor cells in the sample. Enzymology typicallyloses information because it relies on homo-genized samples. Enzyme histochemical stain-ing, on the other hand, retains spatialinformation about the distribution of enzymeactivity among muscle fibres. This differenceis exemplified by Figure 2c, which shows anormal muscle respiratory chain enzyme pro-file in a patient who had a mtDNA deletionpresent at -30% mutant load and had demon-strable numbers of COX-negative and raggedred fibres on histochemical study (Blok et al.,1995). The lack of spatial information inbiochemical respiratory chain enzyme analysisis balanced by the fact that it is more quantitat-ive than histochemical staining.Enzyme assays are usually performed ina dilute aqueous system that differs fromcircumstances in vivo in several ways (TableII). Any of these differences could result in

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D.R.Thorburn

Table II. Differences between the in-vitro assay system for respiratory chain complex I and the in-vivo situation as possibleexplanations for why some complex I defects do not affect the measured enzyme activityIn-vitro assay In-vivo system

MitochondriaProtein concentrationSubstrate concentrationQuinone substrateFunction

Disrupted1 mg/mlSaturating (i.e. >>K m )5-carbon saturated chainElectron transport

Intact500 mg/mlLow (i.e.


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Detecting mitochondrial abnormalitiescommon in the mitochondrial genome than inthe nuclear genome.Methodological limitations affecting respir-atory chain enzyme, morphological andmtDNA analyses mean that none of thesemethods on their own can provide a benchm arkfor the detection of all patients with resp iratorychain defects. In practice, most patients requirean integrated approach to diagnosis, utilizingeach of these methods either simultaneouslyor sequentially (Chinnery and Turnbull, 1997).

Secondary effectsMitochondrial numbers and function areinfluenced by the intracellular milieu and byexternal environmental factors impinging onthe cell and organ in which they are located.This can blur the distinction between changesdue to a primary respiratory chain defect andthose due to secondary effects (reviewed byTrijbels et al., 1993). Skeletal muscle respira-tory chain enzyme levels, for example, canbe influenced by a number of factors, e.g.inactivity of age, fitness, immobility, and vari-ation in fibre type composition. In our experi-ence, skeletal muscle homogenates fromneonates tend to have only about one-thirdof the level of respiratory chain enzymescompared with older children (presumablyreflecting the relatively anaerobic environmentof the preceding fetus). There are numerousreports of declining ske letal muscle respiratorychain enzyme levels in old age, and this is atleast partly due to the effects of detrainingrather than of mitochondrial mutations orageing per se (Brierley et al., 1997). Todistinguish primary deficiencies in respiratorychain enzymes from secondary deficiencies,activity measurements are made relative to amarker enzyme such as citrate synthase (Kirbyet al., 1999) or as internal ratios (Chretienet al., 1998), rather than relative to protein.Interestingly, our experience suggests that liverand cardiac muscle have less age-related sec-

ondary variation, perhaps because as organsthey are used more constantly than skeletalmuscle.Tissue pathology and other inborn errorsof metabolism can also secondarily affectrespiratory chain enzyme levels and mitochon-drial morphology. Our experience is thathealthy livers have quite narrow observedranges for each of the respiratory chainenzymes (lowest activities are typically > 70 %of the mean value). Livers from patients whohave a known inborn error of metabolism (e.g.pyruvate dehydrogenase deficiency, fatty acidoxidation defects, or organic acidaemias) orliver disease (e.g. chronic biliary cirrhosis ormassive hepatic necrosis) can have much morevariable respiratory chain enzymes. Differentrespiratory chain enzymes show differentextents of secondary variation. In our experi-ence, complexes II, III, and the combined11+III and I+III assays show most variability(lowest activities are down to -30% of themean value), followed by complex I and thencomplex IV, with citrate synthase being themost robust assay. This pattern of labilitytends to hold for secondary effects generally,whether caused by other inborn errors ofmetabolism, liver pathology, post-mortemdelay in sample collection, or exposure to freeradical dam age, as in Friedreich 's ataxia (Rotiget al., 1997) or, experimentally, in the Sod-2knockout mouse (Melov et al., 1999).

Ano ther potential cause of secondary effectson respiratory chain enzymes and functionare drugs [particularly antiviral nucleosideanalogues such as zidovudine (AZT) (Lewisand Dalakas 1995) and fialuridine (Lewiset al., 1996)], poisons (e.g. carbon monoxideor cyanide) and anaesthetic agents (particu-larly barbiturates, which inhibit complex I,and bupivacaine).A number of authors have suggested thatpurified mitochondria from frozen tissuesamples should not be used for respiratorychain enzyme assays, because of potential

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D.R.Thorburnartefacts (as examples: Zheng et al., 1990;Scholte and Trijbels 1995). Our experience isthat assays for respiratory chain complex Vand combined assays relying on endogenousubiquinone (complex I+III and 11+III) arenot reliable in frozen tissue homogenates.However, in our laboratory, the activities ofcomplexes I, II, III, IV and citrate synthase inhomogenates prepared from frozen muscle orliver appear to be stable for over a decade ifthe tissue samples have been frozen rapidlyand stored at -70C.

Mitochondrial morphology and mtDNAstudies are also prone to secondary effects.Markedly abnormal mitochondrial morpho-logy has been reported in conditions as diverseas fat oxidation defects (Tyni et al., 1996),alcoholic liver disease (Inagaki et al., 1992),cyclosporin therapy (Larner et al., 1994) and,of course, anoxic tissue damage of any cause.In an inexperienced laboratory, the increasedmitochondrial numbers in skeletal muscle ofan elite athlete could be mistaken for abnormalmitochondrial proliferation. False positive andfalse negative results for mtDNA point muta-tion tests due to polymorphisms have beendescribed above. Another example of a sec-ondary effect on mtDNA mutation analysislies behind a recent claim that multiple hetero-plasmic mtDNA mutations were a major riskfactor for late-onset Alzheimer's disease(Davis et al., 1997). It was subsequentlyshown that this finding was an artefact of co-amplifying nuclear-embedded mtDNApseudogenes rather than an identification ofauthentic mtDNA mutations (Hirano et al.,1997; Wallace et al., 1997).

A substantial number of cases of mtDNAdepletion have been reported in recent years.While there is no doubt that this is a realsyndrome, a number of secondary factors canaffect the mtDNA:nuclear DNA ratio. It hasbeen reported (Berger et al., 1998) that 19 of20 patients with spinal muscular atrophy hadmtDN A.nuclear DNA ratios in skeletal muscle

of


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Detecting mitochondria! abnormalitiestissues, this is often not practical (for examplea brain biopsy would be impractical for apatient whose only symptoms related to ence-phalopathy). Finally, economic issues can dic-tate what tests are available. Full testing ofrespiratory chain enzymes and function inmultiple tissues, mitochondrial morphologyand detailed mtDNA analysis may cost thou-sands of dollars for a patient, and the localcentre might have only the resources to devotea certain amount of time and effort to eachsample. In such situations, it may be necessaryto ration the availability of testing to high-suspicion patients, or to focus on using onlythe methods that are likely to give the highestnumber of diagnoses per annum.

InterpretationThe last difficulty in the diagnosis of respirat-ory chain defects lies with the interpretationof the data obtained. The secondary effectsdiscussed above mean that many patients willhave respiratory chain enzyme levels or mor-phological changes that are moderately belowthe 'normal' range and which overlap withlevels or changes caused by authentic, primaryrespiratory chain disorders. It is difficult orimpossible to define absolute cut-offs for nor-mal ranges that are at once realistic and yet canreliably distinguish primary from secondarychanges. W hile some respiratory chain enzymedefects or morphological changes are suffi-ciently marked to be diagnostic, others shouldtherefore be interpreted as only suggestive ofrespiratory chain dysfunction. It is frustratingto patients, their families, clinicians and thelaboratory to label patients as having a prob-able rather than a definite defect. However, Ibelieve it is better to err on the conservativeside in making such diagnoses. Once a patientis regarded as having a primary defect of themitochondria, the clinician's focus may shiftto patient management and away from con-

sidering alternative, potentially treatable dia-gnoses.Problems in interpretation also extend tomtDNA studies. If a patient has a novelmtDNA nucleotide change identified, a sub-stantial amount of work is required to confirmthat the change is pathogenic (Moraes et al.,1993; Walker et ai, 1996). A classic exampleof interpreting the role of mtDNA mutationsin pathology relates to the role of mtDNAmutations that accumulate in somatic tissueswith age. Many of these studies have focusedon the accumulation of the common 4977 bpdeletion (AmtDNA4977), which is typicallyundetectable in muscle or other tissues fromnormal young individuals but becomesincreasingly detectable in different post-mitotic tissues with advancing age. Even inold individuals, the amount of this mutantmtDNA species is typically < 1 % of totalmtDNA, and debate continues as to whetherthis mutation (and others like it) is a majorcontributor to the phenotypic expression ofageing and common degenerative diseases orsimply a clinically unimportant epiphenom-enon. Two recent reviews have argued boththe case for (Nagley and Wei, 1998), andagainst (Lightowlers et ai, 1999), a causalrelationship.

ConclusionsI do not intend to convey the impressionthat practical problems always provide aninsurmountable barrier to accurate diagnosisof respiratory chain dysfunction. My ownlaboratory has been involved in diagnosing-200 children with definite respiratory chaindefects, and other large centres w ould have theexperience of having m ade a similar number ofreliable diagnoses. It is clear that issues suchas genetic complexity, tissue and temporalvariation, methodological limitations, second-ary effects, logistical issues and interpretationneed to be addressed in the process of investi-

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D.R.Thorburngating suspected cases. The final processinvolves weighing evidence from sourceswhich include clinical findings, metabolic andimaging investigations, respiratory chainenzymes and function, m itochondrial morpho -logy, and DNA studies in order to achieve adiagnosis of definite, probable or possiblerespiratory chain dysfunction. A recent reviewof this process has been published (Walkeret al., 1996). In many cases a definite d iagnosiscan be achieved, but it is sensible to retain acautious view of any individual case as towhether such a diagnosis has been confirmedor excluded.

AcknowledgementsI wish to thank all the colleagues who have made majorcontributions to the diagnostic work of my laboratory,in particular Denise Kirby and Henrik Dahl, C.W.Chow,Xenia Dennett, Avihu Boneh, Maureen Cleary, JimMcGill, David Danks and Geoff Thompson, as well asthe staff of the Murdoch Institute Diagnostic DNAand Tissue Culture Laboratories. I am also grateful togeneticists, neurologists, and paediatricians throughoutAustralia and New Zealand for referring patients forinvestigation. This work was supported in part by anInstitute Grant from the National Health and MedicalResearch Council of Australia.

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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 elderlyathletes. Ann. Neuroi, 41 , 114-116.Chinnery, P.F. and Turnbull, D.M. (1997) Clinicalfeatures, investigation, and management of patientswith defects of mitochondrial DNA. J.Neurol.Neurosurg. Psychiatry, 63 , 559-563.

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features of 30 patients with major deletions of musclemitochondrial DNA. Ann. Neuroi, 26 , 699-708.Inagaki, T., Kobayashi, S., Ozeki, N. et al. (1992)Ultrastructural identification of light microscopic giantmitochondria in alcoholic liver disease. Hepatology,15 , 46-53.Johns, D.R. and Neufeld, M.J. (1993) Pitfalls in themolecular genetic diagnosis of Leber hereditary opticneuropathy (LHON). Am. J. Hum. Genet, 53, 916-920.Kirby, D.M., Milovac, T. and Thorburn, D.R. (1998) Afalse positive diagnosis for the common MELAS(A3243G) mutation caused by a novel variant in the

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Practical Problems in Detecting Abnormal Mitochondrial Function and Genomes (Thornburn, 2000). the Murdoch Institute, Australia 2000