the investigation of mitochondrial respiratory...

JOURNAL OF THE ROYAL SOCIETY OF MEDICINE Volume 88 April 1995

The investigation of mitochondrial respiratory chaindiseaseA 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

INTRODUCTION

The mitochondrial respiratory chain couples the oxidation offuels to the generation of cellular energy. It consists of fiveprotein complexes embedded in the inner mitochondrialmembrane. Each respiratory chain complex has multiplesubunits; most are encoded by nuclear genes, induding all thesubunits of complex II, but the other complexes also havesubunits encoded by mitochondrial DNA (mtDNA). Themitochondrial genome is inherited exclusively from themother and many copies are present in each mitochondrion.Normal and mutant mtDNA can be found in the samemitochondrion (heteroplasmy) and the proportions vary indifferent tissues1.

Diseases ofthe mitochondrial respiratory chain are a majordiagnostic challenge. They can present in an enormous varietyof ways, making clinical recognition difficult. There are noreliable screening tests and the diagnostic tests are generallyinvasive, expensive and not widely available. In this paper wedescribe an approach to the investigation of these disorders.First, we outline the clinical and biochemical features helpfulin selecting which patients to investigate. Next, we considerwhether the initial investigation should be to look for abiochemical defect in the respiratory chain or a genetic defect inmtDNA. Respiratory chain defects cannot be detected reliablyin all tissues. In our third section we discuss which tissuesshould be examined and how they should be obtained. Finally,we compare the advantages of histochemistry andconventional biochemical tests.

SELECTION OF APPROPRIATE PATIENTSTO INVESTIGATE

Clinical clues

The first step in investigating suspected disorders of therespiratory chain is patient selection. Despite the diversity of

Table 1 Presentations of respiratory chain disease

Disease Reference

NeurologicalMELAS syndromeMERRF syndromeNARP syndromeLeigh diseaseAlpers-Huttenlocher diseaseKSSCPEOSensorineural deafness

MuscleBenign infantile myopathyFatal infantile myopathyMyopathy in children and adultsRhabdomyolysis

OphthalmologicalLHONPigmentary retinopathy, optic atrophy (in KSS,

Leigh disease, etc.)HeartCardiomyopathy: hypertrophic, dilated or

histiocytoidBarth syndromeRenalFanconi syndrome

LivermtDNA depletion syndromePearson syndromeAlpers-Huttenlocher disease

4

18193,12

20225

21

2122

23

24

25

26

272820

HaematologicalSideroblastic anaemia, pancytopenia 28

(Pearson syndrome)Neutropenia (Barth syndrome) 25

Gastro-intestinalPancreatic exocrine dysfunction (Pearson syndrome) 28Partial villous atrophy 29Motility disorders 30

EndocrineDiabetes mellitusParathyroid, thyroid dysfunction (KSS)

Metabolic decompensationLactic acidaemia (in many of the above, see text)

3132

MELAS=mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-likeepisodes; MERRF=myoclonic epilepsy and ragged-red fibres; NARP=neurogenicweakness, ataxia and retinitis pigmentosa; KSS=Kearns-Sayre Syndrome;CPEO=chronic progressive external ophthalmoplegia; LHON=Leber's hereditaryoptic neuropathy

BASED ON A PAPER READ TO SEC77ON OF PAEDIATRICS, 25 JANUARY 1994

Division of Clinical Neuroscience, University of Newcastle upon Tyne, Newcastleupon Tyne, UK

Correspondence to: Professor D M Tumbull, Division of Clinical Neuroscience,The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK 217P


respiratory chain disease, patient selection is still based onrecognizing the common clinical presentations. Table 1summarizes these with references that give details of thevarious conditions. Four main dues in the clinicalpresentation may suggest respiratory chain disease.

(1) Diagnosis is easiest when the presentation conforms toone of the characteristic syndromes that have been reported.It is important, however, to be aware that these syndromesshow considerable variability: they may be incomplete,present in atypical ways or overlap with other syndromes. Forexample, the cardinal features of Kearns-Sayre syndrome(KSS) are progressive external ophthalmo-plegia andpigmentary retinopathy, but it can present withhypocalcaemia or short stature; other patients progressfrom Pearson syndrome in infancy to KSS in childhood. Thereis also overlap with adult onset chronic progressive externalophthalmoplegia (CPEO)2.

(2) The described syndromes often include features inseveral apparently unrelated systems. This should suggestrespiratory chain disease even if the particular combinationdoes not form part of a previously described syndrome.Myopathy combined with an unrelated symptom isparticularly characteristic. In infancy, respiratory chainmyopathy is usually associated with lactic acidosis and oftenwith de Toni-Fanconi-Debre syndrome or liver failure; later,it is often found with cardiomyopathy or CNS disease such asdementia, MERRF syndrome (myoclonic epilepsy withragged-red fibres) or MELAS syndrome (mitochondrialmyopathy, encephalopathy, lactic acidosis and stroke-likeepisodes).

(3) Within each system respiratory chain disorders causecertain patterns of disease and not others. Thus, de Toni-Fanconi-Debre syndrome is the only renal disease commonlyassociated with respiratory chain defects. Certain clinicalfeatures, such as progressive external ophthalmoplegia, are sostrongly suggestive of respiratory chain pathology thatinvestigation is warranted even in the absence of otherfeatures. Some investigation findings are equally suggestive(e.g. the MRI findings in Leigh disease3). The value of raisedlactate concentrations in blood or CSF will be discussed later.Other features, such as cardiomyopathy, ataxia, myoclonus orstroke-like episodes, should lead to a high index of suspicionbut it is not feasible to investigate the respiratory chain in allthese patients unless there is an additional pointer to thisaetiology.

(4) A final clinical clue to respiratory chain dysfunction is afamily history of mitochondrial disease. This may take thesame form as in the index case but often is markedly different,particularly in cases caused by mtDNA mutations. Forexample, relatives of patients with MELAS syndrome havebeen identified with myoclonus, pigmentary retinopathy ordeafness4. Obviously, a maternal pattern of inheritance isparticularly suggestive but any pattern may be found.

Table 2 Non-respiratory chain causes of hyperlactateemia

Metabolic diseasesPyruvate dehydrogenase deficiencyGluconeogenic defects: Fructose 1,6-bisphosphatase deficiency

Pyruvate carboxylase, multiple carboxylase or biotinidasedeficiency

Phosphoenolpyruvate carboxykinase deficiencyGlycogen storage disease type 1Hereditary fructose intoleranceLong-chain hydroxyacyl-CoA dehydrogenase deficiencyOrganic acidaemias: propionic, methyl malonic and isovaleric

acidaemias, maple syrup urine disease

Secondary causesTissue hypoxia: hypoxia (including crying)

lschaemiaVenous stasisShockExercise, seizures

Hepatic failure

The hardest cases of respiratory chain disease to identifyare those in whom a single system is affected, without acharacteristic finding such as ophthalmoplegia. Isolatedskeletal myopathy is one such presentation: the aetiology isusually apparent if histochemistry is performed on the musclebiopsy. Deafness can also be an isolated finding in respiratorychain disease5, but the aetiology would seldom be suspectedunless there are affected relatives.

Biochemical clues

A raised lactate concentration in blood or CSF is an importantpointer to respiratory chain disease though its sensitivity andspecificity are low. Hyperlactataemia is uncommon in adultonset respiratory chain disease apart from MELAS syndromeand mitochondrial myopathies. Hyperlactataemia seems to bemore common in childhood and especially in infancy: raisedlactate concentrations may reflect widespread disease, whichis likely to present early in life. Thus, Pearson syndrome, KSSand CPEO are all associated with similar mtDNArearrangements. Pearson syndrome, a multisystem disorderthat usually presents in infancy, is almost always associatedwith raised blood lactate concentrations. Raised levels arealso sometimes found in KSS, which presents later inchildhood or in young adults, but have not been described inCPEO. Again, cases of mtDNA depletion syndromepresenting in infancy tend to have hyperlactataemia,whereas those presenting later do not6. Normal lactatelevels should not discourage investigation of the respiratorychain if the clinical picture is otherwise suggestive.

A raised blood lactate concentration strengthens the casefor respiratory chain disease but is far from specific. Othercauses of hyperlactataemia are summarized in Table 2. Many218P


relevant family history. Measurement of the blood or CSFlactate, or the lactate to pyruvate ratio, can increase one'ssuspicion of respiratory chain disease but can neither prove itnor exclude it.

SHOULD THE INITIAL INVESTIGATIONSBE BIOCHEMICAL OR GENETIC?

The next question is whether to attempt diagnosis at thebiochemical or the molecular level. The latter has obviousattractions. DNA can easily be sent to centres performing therelevant tests and suitable specimens can sometimes beobtained from blood, though this is not always the case asthere may be different proportions of mutant mtDNA indifferent tissues. Biochemical abnormalities are occasionallythe result rather than the cause of the disease process: this isless likely for molecular defects. Moreover, if a moleculardefect is found it immediately gives a precise diagnosis andthe opportunity for genetic counselling; biochemical studiesmay need to be followed by molecular ones.

Unfortunately, for the majority of respiratory chaindiseases the molecular defect is not known. Indeed nodefects in nuclear genes have yet been identified, though thesemust be responsible for a number of respiratory chaindisorders. Even if there is evidence for a mtDNA defect, suchas a maternal pattern of inheritance, identifying the mutationcan pose formidable problems. The mitochondrial genome is16.5kb long: sequencing this is a major undertaking. Multipleclones may need to be sequenced to detect heteroplasmicmutations that are only present in a small proportion of themtDNA. Furthermore, abnormalities found may bepolymorphisms and not pathogenic.

A pragmatic approach is to pursue molecular tests whenthe clinical picture suggests a syndrome known to beassociated with one or a small number of mutations.Pearson syndrome, KSS and CPEO are associated withmtDNA rearrangements: these change the size of restrictionfragments and can be detected on Southern blots10. Leber'shereditary optic neuropathy (LHON), MERRF and MELASsyndromes are associated with particular mtDNA pointmutations; these can be detected by sequencing or PCR andrestriction digestion11. Another point mutation, originallydescribed in association with neurogenic weakness, ataxia andretinitis pigmentosa (NARP syndrome) is a common cause ofLeigh disease. It is worth screening all cases ofLeigh disease forthis mutation, particularly as biochemical investigations givenormal results in these cases12.

In diseases not known to be associated with particularmtDNA mutations, the primary investigation should behistochemistry or biochemistry. We think that a biochemicalor histochemical abnormality still needs to be documented inall cases ofmtDNA depletion syndrome, preferably in muscle:this condition is poorly understood and the normal levels of

of these are easy to distinguish but others can cause diagnosticconfusion. In general, the alternative diagnoses should beexcluded first, as establishing the presence of a respiratorychain disorder is likely to be harder and the therapeuticimplications more limited. For example, hereditary fructoseintolerance is a treatable cause of infantile hyperlactataemia,Fanconi syndrome and liver disease: the diagnosis is apparentas soon as fructose is withdrawn and confirmed by anintravenous fructose tolerance test.

In children it can be difficult to obtain reliable blood lactatemeasurements. Taking blood from a small vein in a strugglingchild can easily turn into an inadvertent ischaemic lactate test!Even arterial lactate levels can be artifactually raised byscreaming. A better solution is to obtain the sample through acannula inserted at least 45 min previously into an artery orlarge vein: no occlusion should be applied. Reference rangesare normally established on fasting individuals so samplesshould 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 valuesbeing slightly higher in neonates.

In patients with suspected respiratory chain disease butnormal blood lactate levels, the effect of oral glucose orintravenous pyruvate loading is sometimes measured7. Thismay induce an abnormal rise but the lactate concentrationremains normal in other patients with respiratory chaindefects. Similarly, in adults with respiratory chain diseaseexercise may induce an excessive rise in lactate concentration8but many patients are too disabled to perform exerciseprotocols.

CSF lactate concentrations are often raised in patients withneurological 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 themeasurement of CSF lactate is therefore of great value.However, the same reservations apply as for blood levels. TheCSF lactate concentration can be normal in respiratory chaindiseases (e.g. CPEO), it can be raised artifactually (e.g. up to48 h following seizures) and it can be raised in other metabolicdiseases (e.g. pyruvate dehydrogenase deficiency).Respiratory chain disorders are associated with impairedoxidation ofNADH, which would be expected to increase theratio of lactate to pyruvate concentrations. This is thereforesometimes used to distinguish between hyperlactataemia dueto respiratory chain disease and other causes9. Unfortunately,yet again this is unreliable: ratios can be normal in respiratorychain disease and raised in hyperlactataemia due to othercauses. Not surprisingly, the lactate to pyruvate ratio is oflittlehelp when the lactate level is normal, one reason being thatlow levels of pyruvate are hard to measure accurately.

In summary, patient selection is a clinical procedure basedon recognizing reported syndromes or suggestive features,particularly if several systems are involved or there is a 219P


mtDNA have yet to be documented in children ofvarious agesand in patients with other diseases.

WHAT IS THE MOST APPROPRIATETISSUE TO INVESTIGATE?

Most mtDNA defects, apart from those in LHON, areheteroplasmic and the proportion of mtDNA affected canvary in different tissues. The mtDNA defects in Pearsonsyndrome, MERRF, NARP and most cases of MELASsyndrome can be detected in DNA from leukocytes.However, in KSS, CPEO and some cases of MELAS theproportion of mutant mtDNA in blood is too small to detectand DNA from musde must be analysed.

Tissue choice is even more important for biochemicalassays. Defects in nuclear genes may affect tissue-specificisoforms and so, like mtDNA defects, may not be expressed inall tissues. Furthermore, even if a defect is expressed, thedifficulty ofthe assays may make it hard to detect. Attempts todemonstrate respiratory chain defects in readily accessiblecells such as platelets or fibroblasts have proved timeconsuming and generally disappointing. However, therehave been several reports of complex IV defects successfullydemonstrated in fibroblasts, notably in Leigh disease9. Thisavoids the need for more invasive tests but introduces a delayof 4-6 weeks while fibroblasts are cultured. Anxiety toestablish the diagnosis may justify more invasiveinvestigations, but fibroblast assays have another merit: if adefect is detectable in fibroblasts it is also likely to be expressedin amniocytes, raising the possibility of antenatal diagnosis.

Biochemical investigation ofthe respiratory chain is usuallyperformed on muscle. There are several reasons why this isappropriate. Though more invasive than taking blood or a skinbiopsy, muscle is relatively easy to obtain (compared withliver, kidney or brain for example). Moreover, muscle givesabnormal results in most cases of respiratory chain diseaseeven when it is not clinically affected. This may reflect thereliance of muscle on oxidative metabolism. Alternatively itmay 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 arespiratory chain defect restricted to a single tissue such asbrain or heart, but such cases appear to be rare. If stronglysuspected, further biochemical assays on the affected tissuemay be appropriate though there are several problems,particularly with regard to control data. Plenty of control datais available for muscle but there is much less control data forother tissues, particularly brain.

Another reason for choosing muscle is that it is relativelyhomogeneous. Samples from patients and controls aretherefore comparable. This is less true of other tissues suchas liver, kidney or brain, which contain many cell types thatmay be present in different proportions in different samples,

especially once the tissue has been distorted by disease. Even inmuscle, mtDNA defects only affect a proportion of fibres: iffewer than 10% are affected the biochemical defect cannot bedetected but these cases can still be identified byhistochemistry.

Muscle biopsies are generally obtained from adults usinglocal anaesthesia but this is too distressing for children. It hasbeen claimed that drugs used in general anaesthesia mayinterfere with the respiratory chain13. However, this has notbeen our experience. Ideally respiratory chain assays should beperformed on fresh tissue, but some patients are too sick to bemoved to referral centres or die elsewhere. Preliminary datafrom our laboratory and elsewhere show that reliable resultscan be obtained on musde that is frozen immediately in liquidnitrogen. It should then be stored at - 70°C and transportedto the laboratory on dry ice.

Some patients with respiratory chain disease die early inthe neonatal period. Inevitably many of these patients are notfully investigated during life. Postmortem specimens forbiochemical assays should be obtained within 1 h of death, andeven then some artifactual lowering of respiratory chainactivity remains possible. Despite these reservations it isimportant to pursue a diagnosis in these patients both forgenetic counselling and to increase our understanding of thesediseases.

THE ADVANTAGES OF BIOCHEMICALAND HISTOCHEMICAL INVESTIGATIONS

In our laboratory 250 mg of muscle (after removal of fat orfascia) are required for biochemical evaluation. This quantityallows isolation of mitochondria, measurement of the proteinconcentration and assays of the respiratory chain complexesand citrate synthase, a mitochondrial matrix enzyme14.Assays of the individual complexes are preferred as these willdetect partial defects, which may be missed by other tests15.If more tissue is available it allows polarographicmeasurement of the flux through the respiratory chainusing various substrates. This will confirm the results of thecomplex assays but seldom alters the conclusions; it is usuallyomitted in children in whom large biopsies are difficult.Moreover, flux measurements can only be performed onfresh rather than frozen tissue. A number of laboratoriesassess biochemistry on smaller amounts of muscle. However,this involves using muscle homogenate rather than isolatedmitochondria, reducing the reproducibility of the results.

Histochemistry, the study of enzyme activity in tissuesections, is of great value in respiratory chain disease andsometimes makes full biochemical evaluation unnecessary.This reduces the cost ofinvestigation and the size of the biopsyrequired. Only 25 mg of muscle are required forhistochemistry and a further 25 mg will allow DNApreparation. Biochemical tests cannot be justified in patients220P


with KSS or CPEO in whom musde biopsy is primarily todemonstrate mtDNA rearrangements; histochemistry is aworthwhile confirmatory test as it requires little extra tissue.In other patients there is a greater chance of normalhistochemistry. The options in these patients are either totake an initial biopsy adequate for biochemistry andhistochemistry or to take a small biopsy first, accepting thiswill need to be repeated if the histochemistry is normal.

Reliable histochemical methods are available for thedetermination of succinate dehydrogenase and cytochrome coxidase activity (complexes II and IV of the respiratorychain)16. Three abnormal patterns are found. First, succinatedehydrogenase preparations reveal sub-sarcolemmalaccumulation of mitochondria in many patients withrespiratory chain disease (a phenomenon that gives rise to'ragged-red' fibres on Gomori trichrome staining). Sub-sarcolemmal accumulation of mitochondria is good evidencefor respiratory chain defects but is absent in many such diseases(e.g. Leigh disease). It seems to be retricted to diseasesinvolving defects of mtDNA, and specifically those withimpaired mitochondrial protein synthesis, i.e. mtDNAdepletion or mutations involving tRNA genes17. Even somecases of MELAS syndrome have normal histology: thesepatients may have fewer mutant mitochondrial genomes andsometimes develop sub-sarcolemmal accumulation ofmitochondria later in the course of their disease. They canusually be detected by a second abnormality on histochemicalpreparations, namely a mosaic of cytochrome c oxidasepositive and negative fibres. This finding is also restricted topatients with mtDNA defects. The third histochemicalabnormality found in respiratory chain disease is ageneralized lowering of succinate dehydrogenase orcytochrome c oxidase activity. There are no reliablehistochemical methods to detect defects of complex I or III.Biochemical assays are necessary to detect isolated defects ofthese complexes, combined defects or partial defects.

CONCLUSIONS

Respiratory chain disease remains underdiagnosed due to thediversity of presentations and the difficulty of investigation.Patient selection continues to be based on the clinicalfeatures, i.e. recognition of patients with specific syndromesor involvement of several unrelated tissues, and a high levelof suspicion in patients with certain symptoms or a suggestivefamily history. Raised lactate concentrations in blood andCSF are a helpful pointer, but normal levels do not excludethe diagnosis.

When the clinical picture suggests a syndrome known to beassociated with particular mtDNA mutations, the primary testis to look for these in blood or muscle. Histochemistry detectsmost respiratory chain defects, only requires small amounts oftissue and can provide evidence for a genetic defect in mtDNA.

However, full biochemical evaluation is necessary ifhistochemistry is normal and if defects of multiplerespiratory chain complexes are to be detected. Directassays ofeach respiratory chain complex should be performed,usually on muscle mitochondria. If the defect is demonstrablein fibroblasts, antenatal diagnosis may be possible in futurepregnancies.

Acknowledgments AAMM is an Action Research TrainingFellow. We thank Dr Margaret Johnson for helpfuldiscussion on the histochemical analysis of muscle. We aregrateful to the Muscular Dystrophy Group of Great Britainand NIH for financial support in our investigation ofrespiratory chain disease.

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(Accepted 20 June 1994)

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