Findings in muscle in complex I (NADH coenzyme Q reductase) deficiency

Findings in Muscle in Complex I (NADH Coenzyme Q Reduct&) Deficiency Yasutoshi Koga, MD," Ikuya Nonaka, MD,' Masanori Kobayashi, MD,? Megumu Tojyo, MD,S and Kenji Nihei, MD§ Thirteen of 15 patients with complex I deficiency had the multisystemic form, with strokelike episodes and other symptoms that fulfilled the diagnostic requirements for MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes), and 2 had only muscle fatigability and weakness, having the purely myopathic form. I n the multisystemic form, 12 patients had ragged-red fibers. All multisystemic patients had myopathic histochemical abnormalities that consisted of mild to moderate variation in fiber size (1 3), disorganized intermyofibrillar networks (121, type 2 fiber atrophy (lo), and an increased number of type 2C fibers (9). Five of 13 multisystemic patients had decreased cytochrome c oxidase (CCO) activity in extrafusal fibers, with sparing of intrafusal muscle fibers. I n the myopathic form, pathological findings were similar to those in the multisystemic form. In addition to complex I and NADH dehydrogenase activities being decreased, the CCO activity was significantly decreased (less than 50% of control value) in 8 patients, especially when the disease was in its advanced stages, suggesting that CCO enzyme might be secondarily affected as the disease progresses. Koga Y , Nonaka I, Kobayashi M, Tojyo M, Nihei K. Findings in muscle in complex 1 (NADH coenzyme Q reductase) deficiency. Ann Neurol 1988;24:747-756

Mitochondrial myopathy with a deficiency of respiratory chain activity was first reported by Spiro and colleagues fl} in 1770. In 1979, Morgan-Hughes and colleagues 121 reported the first case of deficiency in respiratory N A D H :ubiquinone reductase (complex I), characterized clinically by muscle weakness, marked exercise intolerance, fluctuating lactic acidemia, and strokelike episodes. More than 14 patients with complex I deficiency have since been reported [3-151. Based on clinical observations on 15 patients in the present study, we agree that complex I deficiency can be classified into two major subtypes, as proposed by Morgan-Hughes and colleagues [16} in 1785: one a myopathic form, and the other a multisystemic (encephalomyopathic) form. We examined muscle biopsy specimens from 13 patients with the multisystemic form and 2 with the myopathic form histochemically and biochemically.

Materials and Methods Clinical Observations The clinical features of the 15 patients with complex 1 deficiency are summarized in Table 1. In 13 patients, including 8 male and 5 female patients, with the multisystemic form the age at onset ranged from 6 months to 12 years, the mean age being 7 years 8 months. All 13 patients exhibited

From the 'Division of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo; the tDepmment of Pediatrics, CirY University School of Medicine, Nagoya; the $Department of Pediatrics, Niigata Urniversity School of Medicine, Niigata; and the §Department of Neurology, National Center of Child Health, Tokyo, Japan.

easy fatigability and limb muscle weakness; 12 had convulsions. Both intellectual deterioration and short stature were characteristic of 11 patients. All patients had strokelike episodes such as hemiparesis, hemianopsia, or hemiconvulsions. Ataxia, myoclonic jerks, and cortical blindness were each found in 3 patients, and sensorineural hearing loss was found in 6, whereas only 1 patient manifested retinal degeneration and heart block, and 2 had ophthalmoplegia. Family history was negative in all except Patient 2, who had a sibling, an older brother, with similar clinical symptoms but who did not have a muscle biopsy. In half of the patients deep tendon reflexes were hypoactive to absent. All patients had abnormal electroencephalograms consisting of asymmetrical background activity and occasional paroxysmal spike-and-wave discharges. Computed tomographic scans of the brain were abnormal in 12. Abnormalities consisted of focal low-density areas in cortex (6) and in brainstem (1) and cortical atrophy (7). All patients had high lactate levels in both serum and cerebrospinal fluid that were more than twice control values. N o patient had a protein level in cerebrospinal fluid over 100 mg/dl. Patients 14 and 15, who had the myopathic form of complex I deficiency, had slowly progressive muscle weakness and easy fatigability from the age of 3 and 2 years, respectively. They were cousins. Their mothers also had similar symptoms, but were not investigated biochemically. Their serum lactate was elevated, but CSF lactate was normal in both patients. Electroencephalograms and computed tomog-

Received Mar 2 1, 1988, and in revised form Jun 14. Accepted for publication Jun 16, 1988. Address correspondence to Dr Koga, Division of Uitrastmcturd Research, ~ ~ ~~~~~i~~~ i ~of Neuroscience, ~ a l NCNp, Kodaira, Tokyo 187, J ~ ~ ~ ~ ,

Copyright 0 1988 by the American Neurological Association 749

Table 1 , Clinical Summary

of PatientJ- with

Complex I Deficiency Patients with i:he Myopa thic Form (patient Patients with the Multisystemic Form (patient no.)

Clinical Feature

1

Sex Male Female 14 Age at biopsy (yr) Age at onset (yr) Neonate- 1 1-5 5-10 10-15 > 15 Family history Symptoms Ophthalmoplegia Limb weakness Convulsions Hearing loss Blindness Ataxia Myoclonic jerks Mental retardation Easy fatigability Short stature Vomiting Hemiplegia Strokelike episodes EKG heart block EEG abnormality Retinal degeneration High lactate levels Serum"

+

3

2

+ + 3

13

+

+

CSFh C T findings Cortical atrophy Low density in cortex in basal ganglia Calcification

+

4

5

6

+ +

+

9

8

14

+ +

7

+ 15

+ +

8

+ 14

+

9

10

+

+

10

+

11

+ 13

27

+

+

no.?

____-

--

12

13

Total

+

+

8

5 15

+

7

+

1 1 10 1

1.4

+

15 t

5

3

+

t

-

-

0

+

+ +

+

+

+ +

+

+

+

+

+

t-

+ +

+ + + +

+ +

+ +

+ + -

+ + +

+ +

+

+ + + + - +

+ +

-

-

-

-

+

+

+ + + + + +

13

13 li

+

+

-

-

-

-

"Normal: 4.0-16.0 mgidl. 'Normal: < 18 rnddl. EKG

=

electrocardiogram; EEG

=

electroencephalogram; CSF = cerebrospinal fluid; CT

raphy scans were unremarkable. The intelligence quotients for Patients 14 and 15 were 102 and 106, respectively.

Muscle Preparation Muscle biopsy specimens were frozen in isopentane cooled in liquid nitrogen. Serial frozen sections were stained with hematoxylin-eosin, modified Gomori's trichrome, and a battery of histochemical stains [17], including those for cytochrome c oxidase (CCO) and succinate dehydrogenase. O n sections stained for adenosine triphosphatase each fiber was

750 Annals of Neurology

=

computed tomography

classified as type 1, 2A, 2B, or 2C 118). In sections stained with modified Gomori's trichrome scain, the numbers and fiber types of ragged-red fibers (RRFs) were evaluated. For electron microscopy, small specimens were immersed in 2% glutaraldehyde in 0.1 M cacodylate buffer with 0.05 mM calcium chloride (pH 7.3) for 2 hours. The tissues were then rinsed in cold cacodylate buffer and postfixed in a medium containing 1.3% osmium tetroxide in 0.2 M s-collidine buffer (pH 7.4) and 1.3% lanthanum nitrate at room temperature with vibratory agitation according t o the method of Revel and Karnovsky 1191.

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Mitochondria Prepuration

Muscle mitochondria were isolated from muscle homogenates by the procedure of Bookelman and colleagues [201. The crude mitochondria pellet was resuspended in the incubation medium containing 0.2 M sucrose, 20 mM KC1, 3 mM MgC12, 0.2 mM ethylene diaminetetraacetate, and 10 mM potassium phosphate buffer, pH 7.4. Mitochondria were immediately stored at - 70°C until biochemical analysis.

CHANGE I N FIBER SIZE Variation in fiber size

I

Small angular f i b e r s Fiber a t r o p h y

~ y p eI

2A

1

Type

28

1

Fiber hypertrophy

Spectrophotometric assays were used to measure rotenonesensitive NADH-cytochrome c reductase (complex I + 111) 1211, succinate-cytochrome c reductase (complex I1 + 111) 1211, CCO (complex IV) 1221, and NADH dehydrogenase (231. Oxygen Consumption Oxygen consumption of some freshly prepared mitochondria specimens was measured using a Clark-type oxygen electrode (Yellow Spring Instruments Co, Yellow Spring, OH) and the method of Chance and Williams 1241. The protein content was measured by the method of Lowry and colleagues 1251.

Results Muscle Pathological Findings

The histochemical findings in muscle biopsies from patients with the multisystemic form of complex I deficiency are summarized in Figure 1. All specimens showed mild to moderate variation in the size of both type 1 and 2 fibers. In Patients 1, 5 , and 10 there were scattered small angulated fibers (Fig 2). Five patients showed type 2 A fiber atrophy, and 8 showed type 2 B fiber atrophy. A selective type 1 fiber atrophy was seen in Patient 2. There were no hypertrophic fibers. Nine patients showed increased numbers of type 2C fibers, the increase ranging from 1.0 to 5.0%. Patients 1 and 4 showed type 1 fiber predominance (more than 55%), and Patients 1 and 6 type 2 A or 2B fiber deficiency (Fig 3). Patient 6 showed mild fiber-type grouping of type 1 fibers. There was no group atrophy. None of the biopsy specimens had necrotic fibers. O n modified Gomori's trichrome stain, all patients except Patient 2 had RRFs that stained darkly with the oxidative stains, particularly that for succinate dehydrogenase (Fig 4 A ) . With oil red 0, an increase in number and size of lipid droplets was found, especially in RRFs (Fig 4B), which usually also contained an increased amount of periodic acid-Schiff-positive granules. Patients 3 , 4,11, and 12 showed a disorganized intermyofibrillar network with a moth-eaten appearance in both RRFs and non-RRFs. O n C C O staining Patients 3, 5 , and 10 had scattered fibers with no enzyme activity (focal CCO deficiency) (Fig 4C). The enzyme deficiency was seen in both RRFs and nonRRFs. In Patients 2 and 8, the enzyme MULTISYSTEMIC FORM.

0

CHANGE IN DISTRIBUTION Fiber t y p e d e f i c i e n c y

3 7

Fiber t y p e grouping

1

T y p e 1 fiber p r e d o m i n a n c e

Determination of Respiratory Chain Enzyme Activities

1

Type

Inc. number of t y p e 2C fibers Group atrophy

0

DEGENERATION N e c r o t i c fibers

REGENERATION CELLULAR RESPONSE Fibrosis

0 0

0

ARCHITECTURAL CHANGES Targetoid I core

0

Moth-eaten

I 1

Ragged-red fibers Inc. amount of lipid droplets

1

CCO STAIN Almost totally absent

7

Focally positive

1

Unstained intrafusal fibers

0 100

50 Incidence

(96)

Fig 1 . Findings in analyses of muscle biopsiej from patients with the mtrltisystemic (encephalomyopathic)fom of complex I deficiency. CCO = cytochrome c oxidase.

activity was diffusely decreased. The intrafusal fibers had normal enzyme activities. Intramuscular blood vessels and nerve fibers appeared normal. MYOPATHIC FORM. Pathological findings in muscle biopsies from patients with the myopathic form of complex I deficiency were similar to those seen in the multisystemic form. There was a moderate variation in size of both type 1 and type 2 fibers. Type 2 A fiber atrophy and scattered RRFs were recognized in both patients. O n oxidative enzyme staining, there were many fibers, usually RRFs, with a disorganized intermyofibrillar network. O n CCO staining, the enzyme activity in Patient 14 was diffusely decreased, except in the intrafusal muscle fibers. Twelve biopsy specimens that contained RRFs were examined by electron microscopy. There were increased numbers of enlarged (giant) mitochondria, which were largely aggregated in the subsarcolemmal areas. The cristae were markedly proliferated, forming complicated structures. Paracrystalline inclusions in mitochondria were present in one specimen. In the severely affected fibers, myofibrils were disorganized, lipid droplets and glycogen particles were increased,

Koga et al: Muscle in Complex I Deficiency 751

Fig 2. Biceps brachii muscle from Patient 5 at age 14 years. Scattered small angulated fibers (arrow) stained Ark for reduced nicotinamide adenine dinucleotide-tetrazolium and nonspecific esterase, suggesting denervation. (Stainfor nonspecific esterase; x 500 before 24% reduction.)

Fig 3. Biceps brachii muscle from Patient 6 .rhoiu.c mild mriation in size of both type 1 and 2fibers. Type 1 .fibers are nnifirmly larger in diameter than type 2 fibers. Type 1 (1), 2 A (A), and 28 (B)fibers are distributed in a mosaic pattern. compriJing 68, 27, and J%, respectiaely. oj. the muscle sample. (Stain for adenosine triphosphatasc with preincubation at p H 4.6; x 400 before 24% reduction.)

and cytoplasmic bodies and autophagic vacuoles were seen (Fig 5). Smooth muscle cells in the small arteries were occasionally filled with numerous large mitochondria (Fig 6), though the endothelial cells were well preserved.

tients with the myopathic form, the biochemical features were similar to those seen in the multisystemic form. Patient 14 showed a marked decrease in both complex I + 111 and complex IV activities. In Patients 9, 10, 12, and 13, with the multisystemic form, and 14 and 15, with the myopathic form, state 3 respiration was not detected and state 4 respiration was significantly decreased when N ADH-dependent respiratory substrates ( 5 mM pyruvate and 5 mM malate) were added to the incubation medium (see Table 3). The respiratory control ratio and adenosine diphosphate-oxygen ratio could not be evaluated. When flavin adenine dinucleotide-dependent respiratory substrates ( 5 mM succinate) were added, the oxygen consumption rates of both states 3 and 4 and the adenosine diphosphate-oxygen ratio were norm;alized. Respiratory control ratio in all patients examined tended to be lower than in controls.

Biochemical Analysis The biochemical characteristics of all patients are shown in Tables 2 and 3. In all patients with the multisystemic form of complex I deficiency, the enzyme activities of complex I + I11 component and NADH dehydrogenase were markedly decreased (see Table 2), particularly in patients with a longer duration of disease. Mean enzyme activity was 16.7% of the normal value. In addition, the mean enzyme activity of complex IV was significantly decreased. The I + IIVII I11 and IV/II I11 ratios of enzyme activities were significantly decreased. In the pa-

+

752 Annals of Neurology

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Fig 5 . Biceps brachii muscle of Patient 5. One of two muscle fibers is filled by giant mitochondria with markedly proliferated cristae (arrow). The myojibrils (Mf) are decreased in number and disorganized. A n autopbagic vacuole containing cytoplasmic debris (arrowhead) is seen. (Lanthanum nitrate stain; x 11,000 before 36% reduction.)

~

Fig 4. Serial sections from the biceps brachii muscle of Patient 10 with accumulations of mitochondria in the subsarcolemmai areas (ragged-redfibers) clearly shown with succinate debydrogenase stain (A). Most ragged-red fibers have an increased number and size of oil red 0-positive lipid droplets (B). With a stain for qtochrome c oxidase, there are scattered fibers with no enzyme activities vocal deficiency) which are usuaib advanced ragged-red fibers (asterisk) (C). (A-C x 300 before 34% reduction.)

Discussion Although the clinical features in mitochondrial myopathies are heterogenous, Kearns-Sayre syndrome [261, MELAS (a syndrome characterized by mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) [ 2 7 ] , and myoclonus epilepsy associated with ragged-red fibers (MERRF) 128) have

been thought to be distinct clinical entities [291. In Kearns-Sayre syndrome a particular enzyme defect in the electron transport system may, at least in part, play a pathogenetic role; the focal CCO deficiency on histochemical analysis is the most common and consistent finding [30]. The focal deficiency may progress and involve more muscle fibers and organs, as seen at autopsy of a patient with Kearns-Sayre syndrome in whom a mild decrease in CCO activity in a biopsy sample progressed to a marked decrease in the autopsy material [3 11. A patient with MELAS reported by Pavlakis and colleagues [27) had no proven enzyme defects, although recent studies [12, 141 suggest a close relationship between MELAS and complex I deficiency. In the present study, all patients with the multisystemic form of complex I deficiency had clinical characteristics of MELAS. Although MELAS might be closely associated with complex I deficiency, the syndrome could be caused by different enzyme defects, including CCO deficiency. Although Morgan-Hughes and colleagues [l6] reported that pigmentary retinal degeneration was

Koga et al: Muscle in Complex I Deficiency

753

Fig 6. Biceps brachii muscle from Patient 7. Enlarged mitoi-hondria with occasional complicated cristae (arrow) are aggregated in the smooth muscle cells (Sm) of a small artery. The endothelial cells are wellpreservrd and the lumen (Lu) is not occhded. (Lanthanum nitrate stain: x 8.000 before 36C/o reduction.)

the most common feature of the multisystemic form of complex I deficiency, retinal degeneration was found in only 1 of our patients. This difference may reflect the duration of disease, since all but 1 of our patients were less than 15 years old, while Morgan-Hughes's patients were all much older. The present study has confirmed that the multisystemic form of complex I deficiency is closely associated with MELAS, but not with Kearns-Sayre syndrome, in which ophthalmoplegia, retinal degeneration, and heart block are the diagnostic triad. Although myoclonic jerks and ataxia were present in 4 patients with the multisystemic form, the overall clinical symptom complexes in our patients were different from those in patients with MERRF 1281. We found typical RRFs in all patients but 1. Although Patient 2 had no RRFs, they may appear later in development, since the RRF formation is a secondary reaction to the mitochondrial dysfunction. After reviewing previous reports and our own study, we believe the incidence of RRF in complex I deficiency to be the highest among mitochondrial myopathies with respiratory chain enzyme deficiencies. In addition to RRF, all patients had myopathic histochemical abnor754 Annals of Neurology

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malities such as mild to moderate variation in size of both type 1 and 2 fibers, disorganized intermyofibrillar networks, type 2 fiber atrophy, and increased numbers of undifferentiated type 2C fibers. Two patients had type 1 fiber predominance and 1 had fiber-type grouping suggesting the coexistence of a neuropathic process. Both myelinated and unmyelinated fibers are known to be involved in mitochondrial myopathy, especially in Kearns-Sayre syndrome {32]. An increased number of lipid droplets, predominantly in RRFs, was also found by Morgan-Hughes and Landon IS}. Clark and Hayes [l 11 described a mother and daughter with muscle weakness and fatigability who showed the typical features of lipid storage myopathy histochemically and complex I deficiency biochemically. Patients 14 and 15, with the myopathic form, also had an increased amount and size of lipid droplets. O n CCO staining, 6 patients showed decreased enzyme activities. Although intrafusal fibers in the encephalomyopathic form of CCO deficiency studied by Nonaka and colleagues 133) occasionally showed decreased activity (approximately 50% ), all intrafusal 6bers in the present study had normal enzyme activity. Decreased CCO and normal complex I1 plus 111 activities were also confirmed by chemical analysis. As shown in Table 2, complexes I1 plus 111 appeared to be preserved, even in the severely affected muscles, since the enzyme activities of complex I1 + 111 in patients were not significantly different from those in controls. Therefore, if one calculates the ratio of complexes I plus I11 and IV to complex I1 + I11 as a reference, the enzyme abnormality is more pronounced. From the present data, it can be said that complexes I and IV are more readily affected and variable than complex 111, even though all of the complexes are coded in part by mitochondrial DNA. In advanced stages, the CCO activity might be secondarily decreased in complex I deficiency [341.The explanation for the variability in respiratory chain enzymes has not been clarified. Why complex I deficiency causes the syndrome of MELAS is also not known. Kobayashi and colleagues [ 3 5 ] postulated that long-term ischemia resulting from occlusive changes in the capillary wall is responsible for the mitochondrial alterations. The twofold to threefold thickening of the basal lamina of the endomysial capillaries 171 and the increase in the number and size of mitochondria in the small- to medium-size arteries [36} may interfere with the normal blood supply to the brain. Since the smooth muscle cells of small arteries in our biopsies showed accumulation of mitochondria of varied size and shape, with occasional complicated cristae, a systemic angiopathy involving multiple organs that causes strokelike episodes in the brain may be present in complex I deficiency. Morphometric analyses of the mitochondria in blood vessels are under way in our laboratory.

No 6 December 1988

Table 2. Biochemical Features of Patients with Complex I Deficiency

Enzyme Activities" Patient No. Muitisystemic form 1 2 3

4 5 6 7

8 7 10 11 12 13 Mean t SD Myopathic form 14 15 Control(N = 14) Mean t SD

+ 111

+ 111

I1

17.3 19.8 12.7 18.2 5.5 30.7 11.6 21.5 58.1 52.8 58.7 57.6 67.6 33.4' 22.4

207.8 267.3 260.0 484.1 100.3 207.5 187.9 82.2 310.1 131.3 293.0 578.1 466.8 276.8 155.8

112.6 358.4 123.2 110.3 97.8 140.2 156.2 11.6 107.7 72.5 376.0 260.0 206.4 164.1' 108.3

6.8 59.6

245.0 515.8

202.1 83.8

272.5 113.0

I

IV

Ratio of Enzyme Activities

NADH dehydrogenase

I

+ IIYII + I11

IVlII

+ I11

I

+ IIYIV

I1

+ IIYIV

50.6 38.6 26.9b 14.2

0.08 0.07 0.05 0.04 0.05 0.15 0.06 0.26 0.17 0.40 0.20 0.10 0.14 0. 14b 0.10

0.54 1.34 0.47 0.23 0.95 0.68 0.83 0.14 0.35 0.55 1.28 0.94 0.44 0.67' 0.38

0.17 0.06 0.22 0.07 1.85 0.54 0.73 0.16 0.11 0.33 0.35b 0.49

1.86 0.75 2.11 4.39 1.05 1.48 1.20 7.09 2.88 1.81 0.78 1.07 2.26 2.21b 1.77

72.5 174.3

7.0 49.7

0.03 0.12

0.30 0.38

0.09 0.31

3.38 2.65

275.6 102.5

103.0 24.3

0.81 0.29

1.08 0.35

0.80 0.35

0.50

ND ND ND ND ND ND 11.3 11.6 27.0 24.7 22.3

0.15 0.06 0.10

1.05

"Values are expressed as nmoYmidmg of mitochondrial protein. bValue was significantly ( p < 0.01) reduced, using one-tailed Student's t test for independent mean probability. 'Value was significantly ( p < 0.05) reduced, using one-tailed Student's t test for independent mean probability.

+

I 111 = NADH-cytochrome c reductase; I1 + 111 = succinate-cytochrome c reductase; IV = cytochrome c oxidase; NADH = reduced nicotinamide adenine dinucleotide; ND = not detectable; SD = standard deviation.

Table 3. State 3 and State 4 Rates of Oxygen Consumption" and Respiratory Control and ADPIO Ratios in Complex I Deficiency

Respiratory Substrates 5 mM Pyruvate and 5 mM Malate Added Patient No. Multisystemic form 7 10 12 13 Myopathic form 14 15 Control(N = 14) Mean 2 SD

Respiratory Substrate 5 mM Succinate Added

State 3

State 4'

RCR

ADPIO Ratio

State 3

State4

RCR

ADP/O Ratio

ND ND ND ND

25.7 33.4 42.3 48.0

ND ND ND ND

ND ND ND ND

313.2 185.9 395.2 238.1

137.4 72.9 175.6 84.6

2.25 2.00 2.25 2.80

1.99 2.65 1.98 2.28

ND ND

22.2

29.7

ND ND

ND ND

382.3 298.6

160.3 88.9

2.38 3.36

1.93 3.34

170.3 92.4

63.1 24.2

3.02 0.76

3.36 0.75

355.6 172.4

128.1 68.7

3.19 1.41

2.28 0.34

"Values are expressed as nmol of oxygedmidmg of mitochondrial protein. bWhen nicotinamide adenine dinucleotide-dependent substrates (pyruvate and malate) were used as respiratory substrates, we could not recognize the distinction between state 3 and state 4 oxygen consumption after the addition of 0.189 pmol of ADP. Oxygen consumption rates of state 4 were significantly lower than the control value.

RCR = respiratory control ratio; ADPIO = adenosine diphosphate/oxygen; ND = not detectable; SD = standard deviation.

Koga et al: Muscle in Complex I Deficiency 755

This work was partially supported by a Grant-in- Aid for Scientific Research o n Priority Areas (No. 63617002) froni the Ministry of Education, Science a i d Culture of Japan. The authors wish t o express their cortiial thanks t o Professor S. M. Sumi (University of Washington, Seattle, W A j for his helpful suggestions and ‘idvice on this work and Ms R. Oketa (National Insticute of Neuroscience, Kodaira, Japan) for her technical assistance.

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15. van Erven PMM, Gdbreels FJM, Ruirenbeek W, et al. Mitochondrial encephalomyopathy associated with an N A D H dchydrugenlue deficiency. Arch Neurol 198’,44:7?5-7’8 16. Morgan-Hughes J A , Hayes DJ, Cooper M, Clark JB. Mito-

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