Multiple defects of the mitochondrial respiratory chain in a mitochondrial encephalopathy (MERRF): A clinical, biochemical and molecular study

Journal oj the Neurological Sciences, 102 ( 1991 ) 17-24 Elsevier

17

JNS 03487

Multiple defects of the mitochondrial respiratory chain in a mitochondrial encephalopathy (MERRF): a clinical, biochemical and molecular study L a u r e n c e A. B i n d o f f ~, C l a u d e D e s n u e l l e 2, M a r k A. B i r c h - M a c h i n 1, J e a n - F r a n c o i s

Pellissier 3,

G e o r g e s S e r r a t r i c e 2, C h a r l o t t e D r a v e t 3, M i c h e l l e B u r e a u 3, N e i l H o w e l l 4 a n d Douglass M. Turnbull ~ ~Division ~?[(Tinical Neuroscience and Human Metabolism Research Centre, University of Newcastle upon Tyne, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH (U.K.), 2Clinique des Maladies du SystOme Nerveux, CHU La Timone, 13385 Marseille Cedex 5 (France), 3Centre Saint Paul, 300 Bd. de Sainte Marguerite, 13009 Marseille (France), 4Department of Radiation Therapy, Biology Division, UniversiO' of Texas Medical Branch at Galveston, TX 77550-2780 (U.S.A.)

(Received 15 June, 1990) (Revised, received 22 October, 1990) (Accepted 25 October, 1990)

Key words." Respiratory chain; Mitochondrial encephalopathy; Mitochondrial DNA

Summary We describe a young man with a progressive neurological disorder including myoclonus, mental retardation, muscle weakness and a mitochondrial myopathy (myoclonus epilepsy and ragged red fibres - MERRF). Multiple abnormalities of the mitochondrial respiratory chain in skeletal muscle are shown by direct measurement of the flux through the individual complexes, low-temperature redox spectroscopy and decreased immunodetectable subunits of complexes I and IV by immunoblotting. No abnormality of mitochondrial DNA was found. This is the first report of combined defects of complexes I, III and IV as a cause of this clinical syndrome. However, we propose that the occurrence of multiple respiratory chain defects may be more common than previously recognised and that this particular combination of defects, involving complexes I, III and IV, may be the predominant biochemical abnormality in MERRF.

Introduction There is increasing interest in the multisystem diseases caused by defects of mitochondrial function. Such disorders are associated with a variety of clinical syndromes with little apparent correlation between clinical expression and biochemical defect. Many authors believe that the overlap between the clinical syndromes is too great to permit clear distinction ; these have tended to employ generic terms such as mitochondrial myopathy (Petty et al. 1986) or mitochondrial encephalomyopathy (Schapira etal. 1977; Pavlakis et al. 1988). Others have recognised a constancy of clinical associations and accepted classifications such as Kearns-Sayre syndrome (Rowland et al. 1983), Leigh's disease (Leigh, 1951), mitochondrial encephalopathy lactic

Correspondence to." D.M. Turnbull, MD, Division of Clinical Neuroscience, University of Newcastle upon Tyne, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, U.K. Telephone: 091 222 6000 Ext. 7051: Fax: 091 222 7424.

acidosis and stroke (MELAS; Pavlakis et al. 1984), and myoclonus epilepsy with ragged red fibres (MERRF; Fukuhara et al. 1980). Myoclonic epilepsy associated with abnormalities of skeletal muscle was first described by Tsairis et al. (1973). The acronym myoclonus epilepsy with ragged red fibres (MERRF) was suggested by Fukuhara (1983) when reviewing 8 additional cases. There have now been reports of over 30 patients with clinical features compatible with MERRF. Relatively few have been characterised biochemically, and in those who have been studied, no consistent abnormality has been associated with this clinical syndrome. We describe a patient with myoclonic epilepsy, mental retardation, ataxia, hearing loss and skeletal muscle involvement, in whom we have demonstrated multiple defects (complexes I, III and IV) of the mitochondrial respiratory chain. We suggest that multiple defects of the respiratory chain may be more common than previously recognised and that the combined defects of complexes I, Ill and IV may be the predominant biochemical abnormality causing MERRF.

0022-510X/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

Case report The patient was male, 17 years old and the second son of non-consanguinous, healthy parents. There was no family history of neurological or metabolic disease. He developed normally until 3 years of age when speech delay was noted. Sensorineural deafness was discovered when aged 5 and by 8 he was mildly mentally retarded. At the age of 11 he started having repetitive jerking movements involving the face and all four limbs; these movements were severe enough to cause him to fall, but were never associated with loss of consciousness. An EEG at this stage showed spontaneous, generalised spikes and he was treated with carbamazepine and then sodium valproate, but with no improvement. His condition has subsequently deteriorated, the myoclonic jerks have increased in severity, he has developed intention tremor and noticeable muscular fatigue when walking. Physical examination showed there was generalised muscle wasting and dry icthyotic skin around his neck. He had scanning speech, but his eye movements and facial muscles were normal. Proximal weakness was present in both arms and legs; he had diminished vibration sense in the lower limbs and all tendon reflexes were depressed; plantar responses were flexor, his gait ataxic and there was bilateral dysdidochokinesia, but no dysmetria. No myoclonus was visible at rest, but could be observed during movement, especially at the outset of movement requiring axial balance. The myoclonus was worse in the morning and exacerbated by fatigue and emotion. Serum lactate concentration at rest was 2.1 mM (normal range < 1.5 mM), and pyruvate 92/~M (normal range < 60/~M). CSF protein concentration was elevated at 1 g/1 (normal range < 0.5 g/l). An EEG showed a slow (6-7 Hz) background activity, normally distributed, but with bursts of diffuse, posterior slow waves. Paroxysmal abnormalities consisted of diffuse sharp and spike waves and polyspike waves, either isolated or in short bursts. These abnormalities were increased in amplitude and frequency by hyperventilation, but photic stimulation produced no effect. Physiological sleep patterns were present with normal cyclic organisation; paroxysmal abnormalities activated by drowsiness disappeared during slow wave sleep. Fast spikes, isolated in 10-15 sec bursts, were noted in the central and vertex areas during rapid eye movement sleep. Polygraphic recording confirmed the absence ofmyoclonus at rest, the intention myoclonus was fragmentary, and associated with brief atonia which usually corresponded to spike wave activity on the EEG. Evoked potential studies showed normal visual responses, no auditory response and blunted sensory responses. Cranial magnetic resonance imaging revealed calcification of the basal ganglia, scattered white-matter lucencies, but no atrophy.

Muscle biopsy was performed for morphological and biochemical analysis.

Methods

Muscle biopsy and morphology Muscle samples (0.4-1.6 g) were obtained by open biopsy under local anaesthesia, from left biceps (patient), deltoid or quadriceps (controls). Controls were patients in whom no muscle disease was detected. A portion of each biopsy was immediately frozen in isopentane chilled in liquid nitrogen and processed for histochemistry and electronmicroscopy (Desnuelle etal. 1982). Cytochemical staining was performed for NADH-tetrazolium reductase, succinate dehydrogenase (Dubowitz and Brooke 1973) and cytochrome oxidase (Seligman et al. 1968). The remaining muscle was stored at - 8 0 °C until used for biochemical and immunological studies.

Preparation of mitochondria A mitochondrial fraction was prepared as previously described (Watmough et al. 1988) except that the penultimate wash was omitted. Protein concentration was determined using a modified Lowry method (Sherratt et al. 1988).

Assays of individual respiratory chain complexes Mitochondrial fractions were freeze-thawed 3 times to completely disrupt the mitochondrial membranes; 60-90/~g of mitochondrial protein was required for each assay. All optical measurements were made with a Hitachi 557 dual wavelength spectrophotometer, Complex I (NADH:ubiquinone oxidoreductase). N A D H oxidation by complex I was measured using ubiquinone 1 (UQ~) as electron acceptor, in the presence and absence of rotenone. The assay medium contained 25 mM :potassium phosphate, 5 m M MgC12, 2 m M KCN, 2 , 5 m g ' m l -~ defatted bovine serum albumin (BSA), 2 /~g. ml-1 antimycin, 6 5 # M UQ~ and 0.13raM NADH, pH 7.2 and 30°C. The reaction was started with protein and the decrease in absorption was followed at 340 mm (425 nm reference). Complex I activity was calculated using an extinction coefficient of 6.22 mM :k ~. cm - ~for N A D H and the difference in rate before and after the addition of 2 #g. ml- ~ rotenone. Complex H(succinate :ubiquinone oxidoreductase). Succinate oxidation by complex II was measured using UQ~ as electron acceptor. The assay medium was similar to that used for the complex I assay except that BSA and N A D H were omitted and rotenone 2 #g. ml- ~ included. The reaction was started with 10 mM succinate and the rate calculated from the decrease in absorption at 280 nm

19 (465 nm reference) using the reduced minus oxidised extinction coefficient 13 mM J • cm- ~ for UQ~ (Lenaz et al. 1981 ). Thenoyltrifluoroacetone inhibited complex 1I activity by 65-85!!,,.

('omple.v 11I mbiquinol:cytochrome c oxidoreductase). Ubiquinol (UQzH2) was prepared by adding excess solid sodium borohvdride to 7 mM UQj in ethanol. Addition of 0.1 M HCI stabilized the U Q I H : and the excess borohydride was removed by centrifugation at 12 000 x &,v for 4 rain. Ubiquinol was made freshly on each occassion and its UV spectrum recorded to confirm that no ubiquinone was present. Assay conditions were as described for the complex I assay except that antimycin, ubiquinone and NADH were omitted and 15/~M cytochrome c (Ill) and rotenone, 2/~g' ml 1 were added in a final volume of 1 ml. Before starting the reaction with protein, the non-enzymatic reduction of cytochrome c was recorded for 1 rain and this rate was subtracted from the enzymatic rate. The increase in absorption was followed at 550 nm (580 nm reference) and the rate was expressed as an apparent first order rate constant after all the cytochrome c was reduced by a few grains of ascorbic acid. Inhibition by antimycin ( > 94°o) was checked for every patient. Contplex lV(
Low temperature cytochrome spectra The low temperature reduced minus oxidised spectra of mitochondrial cytochromes were recorded after reduction with both succinate and dithionite. The extinction coefficients and simultaneous equations quoted by Tervoort et al. (1981) and the intensification factors quoted by Wilson (1967) were used to calculate cytochrome concentration s.

Immunobb)t analysis of peptide subunits Complex IV. Immunoblot analysis of the cytochrome oxidase in mitochondrial fractions was performed using antibodies to the holo-complex IV as recently described (Shepherd et al. 1988). Complex I. Mitochondrial fractions were incubated with 3 mM p-aminobenzamidine before separation on a 15°;, SDS-polyacrylamide gel with 5"o stacking gel. Proteins were transferred to nitrocellulose (0.45/~m pore size) according to the method described by Towbin et al. (1979) with the addition of 0.1 i~,, SDS to the transfer buffer. Antisera to holo-bovine complex I was raised in rabbits and used at a dilution of 1 : 500. Detection of immunoreactive peptides used the immunoperoxidase method with 4-chloro-l-naphthol as substrate (Domin et al. 1984).

Southern blotting Total cellular DNA was extracted from the pellet obtained following the disruption of tissue during mitochondrial preparation. This homogenate was diluted with TE buffer (10 mM Tris/1 mM EDTA, pH 8) and incubated with 1°; SDS (end concentration) and 3 mg proteinase K (Boehringer Mannheim) at 55 °C for 2 h. The DNA was extracted with phenol/chloroform/isoamyl alcohol (25: 24: 1) and precipitated with absolute ethanol (2 volumes) and 3 M sodium acetate (0.1 volume). Approximately 2.5/~g of total DNA was digested with PvulI (Northumbria Biologicals) and then electrophoresed through an 0.6 °~o agarose gel at 30 V overnight. Alkaline Southern transfer was performed; the gel was soaked in denaturing solution (0.4 M NaOH/0.6 M NaCl) for 30 min and the DNA transferred to the nylon membrane (Gene Screen Plus, Dupont) overnight, in the same solution. Following transfer, the membrane was neutralized (1.5 M NaCI/0.5 M Tris, pH 7.5) for 15 rain and then air dried.

Labelling probe and hybridization Whole mtDNA (kindly provided by Dr. J. Poulton, Oxford, U.K.) was digested with PvulI and labelled by the random primer method (Feinberg and Vogelstein 1983) using a Pharmacia kit. Prehybridization was performed in a solution containing 1 ° o SDS, 1 M NaCI and 10 ,°,o dextran sulphate at 65°C in a Hybaid oven. After 30min, 400#g/ml denatured salmon sperm DNA (Sigma) and labelled probe (I × 106dpm) were added and the filter incubated overnight at 65 °C with this solution. The filter was washed at 65°C in SSC (0.15 M NaCI/0.015 M sodium citrate) and 1 ,°~oSDS for 1 h with 3 changes of wash solution. Autoradiography (Fuji X-ray film) was performed for 4-24 h.

PCR analysis DNA from the patient was screened for the common deletion (Schon et al. 1988) using the polymerase chain reaction (PCR). Oligonucleotide primers were synthesized which were identical to those described by Schon et al. (1988). PCR was carried out under standard manufacturers conditions (Perkin Elmer Cetus) using the following profile: 94°C for 8min followed by 25cycles of 94°C for 1.5 rain/55 °C for 2.5 rain/72 °C for 3 rain. An aliquot of the amplification mixture was electrophoresed through 0.8 g0 agarose stained with ethidium bromide and inspected under UV light to look for products. Results

Histochemistry and electronmicroscopy Approximately 4 0 oo of fibres had subsarcolemmal accumulation of mitochondria. Both fibre types were

2O TABLE 2 C Y T O C H R O M E C O N C E N T R A T I O N S 1N SKELETAL M U S C L E MITOCHONDRIAL FRACTIONS Results are expressed as nmol. mg protein are mean _+ SD for 5 subjects.

Cytochrome

aa 3

Cytochrome b

'. The figures for the controls

Reductant

Patient

Controls

Succinate Dithionite

0.011 0.019

0.067 _+ 0.023 0.084 +_ 0.021

Succinate Dithionite

0.036 0.063

0~045 + 0.01 0.11 + 0.021

Cytochrome concentrations (Tab& 2) Cytochrome aa 3 concentration was much lower in the

Fig. t, Skeletal muscle morphology. Skeletal muscle sections were stained for cytochrome c oxidase and show a large number of cytochrome c oxidase negative fibres.

patient than in controls. The concentration ofcytochrome b was also lower than controls and this was most apparent following reduction with sodium dithionite, The concentration of cytochrome c could not be assessed in either the

1

2

3

affected, but the changes were more marked in type I fibres. Fibres with abnormal accumulations of mitochondria were negative when stained for cytochrome oxidase activity (Fig. 1). Electron-microscopy revealed numerous enlarged mitochondria with either abnormal cristae or granular matrix. There were no paracrystalline inclusions.

Activity of the individual respiratory chain complexes (Table 1) Complex IV activity was 18.2~0, complex l activity 25.5% and complex III activity 31.4~o of control values (Table I). Complex II activity was high-normal (155% of control values).

II/111 IV Vab

TABLE 1

VI a

ACTIVITIES OF T H E I N D I V I D U A L R E S P I R A T O R Y C H A I N C O M P L E X E S IN S K E L E T A L M U S C L E M I T O C H O N D R I A L FRACTIONS

VI bc

Results are expressed as nmol substrate transformed m i n - ' -mg protein - ' (complex I), nmols of ubiquinone reduced min - i. mg protein (complex II) or as apparent first order rate constants ( s - ~.mg protein-- 1) (complexes III and IV). The figures shown for controls are mean + SD and represent the values for 4 control subjects.

Complex Complex Complex Complex

I II III IV

Patient

Controls

35 124 0.030 0.08

149 _+ 44 80 + 32 0.096 + 19 0.42 + 0.19

VII abc

Fig. 2. Immunoblot analysis of complex IV in human skeletal muscle mitoehondria. Mitochondrial proteins were separated on a 16% SDSpolyacrylamide gel containing 6M urea, transferred to nitrocellulose and incubated with antibodies against holocomplex IV. These antibodies were raised in rabbit against bovine cytochrome oxidase and used diluted 1 : 500 in 5% BSA/Tris-buffered saline solution. Lane 1, mitochondria from control. 140 #g loaded; lane 2. mitochondria from patient, 140/~g loaded: lane 3, purified cytochromec oxidase. 5 # g loaded. All the immunodetectabte peptides are present, but in lower amounts than in the control fraction,

21 1

2

3

4

5

6

Subunit

12

3

Mr .... - 1 1 0 -75 57 ¸

l

40 ¸

-39

= il;iiiil

29

0

16.5

• -30 -24 -20 O - 1 8 15 13

12.5

10

Fig. 3. Immunoblot analysis of complex I in human skeletal muscle mitochondria. Lanes 1 and 6, purified bovine complex I (7.25 #g); Lane 2, mitochondria from patient (200#g); lane3, control mitochondria (150 #g); lane 4, mitochondria from patient (150 ~g); lane 5, control mitochondria (150 #g). Proteins were separated on a 15 % SDS-polyacrylamide gel and transferred to nitrocellulose. Subunits were identified following incubation with holocomplex I antibodies (raised in rabbits against bovine complex I). All immunodetectable subunits (apart from the 51 kDa band) are present but in lower amounts than in control fractions. The I I0 kDa band represents the pyridine nucleotide transhydrogenase which co-purifies with complex 1. This is present in similar amounts in both patient and controls.

patient or controls because the preparation of mitochondrial fractions from frozen samples is associated with the loss of this cytochrome (Bookelman et al. 1978).

hnmunoblot analysis (Figs'. 2 and 3) Analysis of both complexes I and IV by immunoblotting with antisera to the holoenzymes showed low amounts of all detectable subunits (Figs. 2 and 3) compared with controls. Southern blotting (Fig. 4) and PCR analysis No deletions of mtDNA were found in the DNA prepared from the patient. Amplification by PCR did not reveal any mtDNA containing the common deletion.

Discussion

When Fukuhara first used the term myoclonus epilepsy with ragged-red fibres (MERRF), he recognised that the

?ii

Fig. 4. Southern blot analysis of mitochondrial DNA. Lanes 1 and 2 (controls) and lane3 (patient show only the wild-type mtDNA (16.5 kb).

clinical features closely resembled dyssynergia cerebellaris myoclonica (Ramsay-Hunt syndrome) combined with Friedreich's ataxia (Fukuhara et al. 1980). However, he felt that the combination of these clinical features with skeletal muscle mitochondrial abnormalities was sufficiently distinct to warrant a new classification (Fukuhara 1983). Our case had many similar features to those reported by Fukuhara, including myoclonic epilepsy, action and posrural myoclonus, intention tremor, mental retardation, sensorineural deafness, impaired deep sensation, depressed tendon reflexes and muscle weakness. The myoclonus and the EEG findings in our patient closely resembled those reported in dyssynergia cerebellaris myoclonica (Roger et al. 1968; Tassinari et al. 1974), but in contrast to previous observations (Roger et al. 1982) there was no resting myoclonus or disorganisation of sleep pattern. Also in contrast to previous reports, both photic-induced myoclonic jerking and photo-paroxysmal responses on conventional EEG were absent. The observed morphological abnormalities were characteristic ofa mitochondrial myopathy. Cytochrome c oxidase deficiency was demonstrated cytochemically and confirmed by direct measurement of enzyme activity, low concentration of cytochrome aa3 and Western blot analysis. In addition to the defect in complex IV, we found low complex I and III activities with the complex I deficiency also confirmed by immunoblotting. Southern blotting (and

?9 s h o w e d the a b s e n c e o f large deletion in

c o m p l e x is affected b e c a u s e the a u t h o r s have: n o t m e a s u r e d

k e e p i n g with o t h e r studies ( L o m b e s et al. 1989; W a l l a c e

the activities o f the i n d i v i d u a l c o m p l e x e s , but h a v e relied

limited P C R )

u p o n t e c h n i q u e s w h i c h a s s a y s e g m e n t s o f the r e s p i r a t o r y

et al. 1988). C o m b i n e d defects o f the r e s p i r a t o r y c h a i n h a v e been reported

in a s s o c i a t i o n

with

C o m p l e x I a n d IV deficiency

c h a i n t h a t c o n t a i n t w o o r m o r e c o m p l e x e s . A s s a y s such as

a variety o f p h e n o t y p e s .

NADH : cytochrome c reductase, succinate cytochrome c

with

r e d u c t a s e a n d p o l a r o g r a p h i c m e a s u r e m e n t o f flux, especi-

chronic

progressive

e x t e r n a l o p h t h a l m o p l e g i a ( S h e r r a t t et al. 1984; Bleistein

ally w h e n u s e d in isolation, m a y n o t d e t e c t d e c r e a s e d activ-

a n d Z i e r z 1989), fatal infantile lactic a c i d o s i s ( Z h e n g et al.

ity in o n e c o m p l e x d u e to it h a v i n g a low c o n t r o l strength

1989) and M E R R F

o n the overall r e a c t i o n .

( W a l l a c e et al. 1988; B y r n e et al. 1988)

and c o m p l e x III a n d IV deficiency w i t h a fatal infantile

T h e r e are n o w o v e r thirty r e p o r t e d c a s e s with clinical

e n c e p h a l o m y o p a t h y ( K e n n a w a y et al. 1987). In m o s t o t h e r

features compatible with the MERRF

r e p o r t s it is i m p o s s i b l e to asses w h e t h e r m o r e t h a n o n e

In e l e v e n o f the sixteen c a s e s w h i c h h a v e b e e n investigated

p h e n o t y p e ( T a b l e 3),

TABLE 3 REVIEW OF REPORTED CASES OF MERRF Although the initial reports of this syndrome were entirely clinical, the later studies do contain biochemical analyses. In cases 8, 10 and 13 the investigation relied solely on techniques that measure segments of the respiratory chain containing two or more complexes. The studies in cases 14 and 15 are much more complete and show the same combination of defects involving complexes I and IV. However, as we have indicated, in many reports the information is incomplete and in others there are instances in which significant abnormalities appear to have been overlooked. Authors

No. of cases

Biochemical defect

Comments

1. Tsairis et al. 1973

1

-

(a) Sisters with other affected family members (see Mendell et al. 1987)

2. 3. 4. 5. 6. 7. 8.

2 1 1 9 1 1 1

-NADH:cytochrome bc~

(a) 2 cases reported previously, 1 case called incomplete

9. Holliday et al. 1983 10. Riggs et al. 1984

3 2

?Succinate-cytochrome c reductase

11. Berkovic et al. 1987

7

Cytochrome oxidase

12. Mendell et al. 1987

1

Cytochrome oxidase

13. Garcia Silva et al. 1987

1

Complex 1

14. Wallace et al. 1988

9

Complex 1 + IV

15. Byrne et al. 1988 16. Lombes et al. 1989

2 l

Complex I + IV Complex IV

17. Berkovic et al. 1989

6

Complex III Complex 11I + IV

Fukuhara et al. 1980 Fitzsimmons et al. 1981 Roger et al. 1983 Fukuhara et al. 1983 Felt et. al. 1983 Sasaka et al. 1983 Busch et al. 1983

(a) limited information on which assay performed, but presumably will have measured complexes I + 1II (a) Patients were siblings and the most affected child did not have a significant abnormality (b)Used succinate cytochrome c reductase assay which measures (complexes 11 + Ill) (c) Variable results in the same family (d) Results of succinate dehydrogenase assay were not significantly abnormal (a) Family of affected individuals (b) Cytochrome oxidase deficiencY shown by cytochemistry and polarography in homogenate (a) Diagnosis only on cytochemistry (b) Patient was brother of cases in Tsairis et al. (1973) (a) Measured NADH oxidase (b) Do not mention low cytochrome oxidase activity (a) Biochemical findings on patients (b) Cases vary in degree of clinical disease (a) Used NADH:cytochr0me c reductase (I + II1) (a) Comprehensively investigated complex IV (b) Used NADH- and succinate cytochrome c reductase assays to measure complexes I + Iit and II + III respectively (a) 6 family members over 2 generations first described in 1987 (above) (b) Used NADH and succinate: cytochrome c reductase assays on homogenates (c) No consistent biochemical findings even in one family

23 biochemically,

the

characterisation

o f the

defect

has

References

d e p e n d e d either on c y t o c h e m i s t r y or assays which m e a s u r e activity in t w o or m o r e c o m p l e x e s . In the c a s e r e p o r t e d by' Garcia

Silva e t a l .

(1987} the b i o c h e m i c a l defect was

d e s c r i b e d as c o m p l e x I deficiency. H o w e v e r , their results also s h o w low c o m p l e x IV activity. Six o f the cases r e p o r t e d by B e r k o v i c et al. (1989) were studied b i o c h e m i c a l l y by m c a s u r i n g activity o f r e s p i r a t o r y c h a i n e n z y m e s in m u s c l e h o m o g e n a t e s . T h e studies i n c l u d e d assays which m e a s u r e d two or m o r e c o m p l e x e s and they r e p o r t e d defects o f c o m plexes III and IV in o n e patient, defects o f c o m p l e x III in 2 patients, and no d e t e c t a b l e a b n o r m a l i t y in 3 patients. T h e m o s t c o m p l e t e studies are t h o s e r e p o r t e d by W a l l a c e et al. (1988), B y r n e et al. (1988) and L o m b e s et al. (1989). In the latter case,

only c o m p l e x IV a p p e a r e d

to be affected

w h e r e a s in the o t h e r t w o studies, defects w e r e found in both c o m p l e x e s I and IV. A p a r t f r o m the c a s e d e s c r i b e d by L o m b e s et al. (1989), our c a s e and the others which h a v e been investigated b i o c h e m i c a l l y w o u l d seem to lend s u p p o r t to the p r o p o s a l that the M E R R F

p h e n o t y p e is a s s o c i a t e d

with a d i s o r d e r o f several r e s p i r a t o r y chain c o m p l e x e s . C o m p l e x e s I, lII, IV and V ( A T P s y n t h a s e ) c o n t a i n subunits e n c o d e d

by b o t h

the m i t o c h o n d r i a l

and

nuclear

g e n o m e s . A l t h o u g h the genetic defect r e s p o n s i b l e for the d i s o r d e r seen in o u r patient c o u l d involve either g e n o m e , the c o m b i n a t i o n o f defects (I, III and IV) suggests that m t D N A m a y be the site o f a b n o r m a l i t y . This is entirely c o m p a t i b l e with the recent finding that there is a p o i n t m u t a t i o n in the m t D N A t R N A ~y~ in three i n d e p e n d e n t M E R R F

pedigrees

( S c h o f f n e r et al. 1990), F u r t h e r genetic studies are required to d e t e r m i n e the m o l e c u l a r defect in o u r cases and t h o s e d e s c r i b e d previously. H o w e v e r , we h a v e s h o w n the m o l e c u lar defect in our c a s e is a s s o c i a t e d with e x t e n s i v e a b n o r malities o f the m i t o c h o n d r i a l r e s p i r a t o r y chain.

N o t e added in proof ( R e c e i v e d 24 D e c e m b e r , 1990) F u r t h e r to the findings o f S c h o f f n e r et al. (1990) we h a v e s e q u e n c e d the region o f t R N A jy~ f o u n d a b n o r m a l in their patients. N o m u t a t i o n was f o u n d in our patient.

Acknowledgements We are grateful for the financial support from The Muscular Dystrophy Group of Great Britain, Newcastle Health Authority Research Committee, The Wolfson Foundation and Newcastle University Research Committee (M. B-M., LA.B., D.M.T.), Association Franqaise contre les Myopathies (A.F.M.) (C.D., J-F.P., G.S.), NIH (HD 08315) and John Sealy Memorial Endowment Fund (N.H.). We are grateful to Dr. C.I. Ragan for his generous gift of purified complex I and the holocomplex I antibodies, to Professor B. Kadenbach for the generous gift of antibodies to complex IV and to Dr. J. Poulton for the whole mtDNA used to make the probe. We would also like to thank Dr. H.S.A. Sherratt for his helpful comments and Mrs. S. Lowe for help with the manuscript.

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