Fatal reducing body myopathy. ultrastructural and immnunohistochemical observations

JOVRNAL

OF THE

NEUROLOGICAL SCIENCES

ELSEVIER

Journal

of the Neurological

Sciences

128 (1995)

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Fatal reducing body myopathy. ultrastructural and immnunohistochemical observations Beatriz H. Kiyomoto a,ey*, Nobuyuki Murakami a, Yoko Kobayashi b, Kenji Nihei b, Tomoko Tanaka ‘, Kenzo Takeshita ‘, Ikuya Nonaka asd a Department

’ Division

of Ultrastncctural Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan b Department of Neurology, National Children Hospital, Setagaya, Japan ’ Department of Neuropediatrics, Brain Research Institute, Tottori University Medical School, Yonago, Japan of Laboratory Medicine, National Center Hospital for Mental, Nervous and Muscular Disorders, NCNP, Kodaira, Tokyo e Department of Neurology, Escola Paul&a de Medicina, SlIo Paula, Brazil Received

24 March

1994; revised

1 August

1994; accepted

11 August

187, Japan

1994

Abstract Two female infants who developed normally during infancy began to have progressive muscle hypotonia and weakness from 2 years 10 months and 2 years 3 months of ages, respectively. Both patients had rapidly progressivemuscleweaknesswith death from respiratory failure at 4 years 11 months and 3 years 9 months, respectively. In addition to mild inflammation in their muscle biopsies, the most striking finding was the presence of numerous reducing bodies (RB) in almost all degenerating fibers. By electron microscopy, these bodies consisted of fine granular material, usually located around the degenerating nucleus. These bodies showed no immunohistochemical reaction to antibodies against structural, cytoskeletal and membrane proteins and a histone-specific antibody against nuclei and chromosomes. They were occasionally positively stained with a ubiquitin antibody. Although the origin of these bodies remains unknown, they appeared to be related to active myofibrillar degeneration, probably resulting from primary nuclear degeneration. Keywords:

Congenital

myopathy;

Reducing

body myopathy;

Immunohistochemistry;

1. Introduction Reducing body myopathy was first described by Brooke and Neville in 1972 in two unrelated girls with a severe fatal progressive muscle weakness from early infancy. In their muscle biopsies, there were intracytoplasmic inclusion bodies which stained dark with menadione-linked cr-glycerophosphate dehydrogenase (MAG). Since the bodies reduced nitroblue tetrazolium (NBT) and were still positively stained without the substrate of cY-glycerophosphate, the term “reducing body” (RB) was coined.

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Rimmed

vacuole

On reviewing the literature, reducing body myopathy can be classified clinically into three forms: (1) severe infantile form in which the disease starts during the first 4 years of life with rapid progression and fatal outcome (Brooke and Neville 1972; Dubowitz 1978); (2) congenital myopathic form characterized by a delay in developmental milestones with relatively benign course mimicking nemaline myopathy and central core disease (Tome and Fardeau 1975; Oh et al. 1983; Carpenter

et al. 1985); (3) late onset form (Hubner

and

Pongratz 1981; Hubner and Pongratz 1982; Bertini et al. 1994) and a heterogeneous group in which RB formation may be incidental (Jay et al. 1992). We report 2 patients with the severe infantile form with progressive muscle weaknessleading to early death from

* Corresponding 46-1749.

Ultrastructure;

probable

infectious

or postinfectious

degenerative

process. To characterize the RB, we performed histochemical, ultrastructural and immunohistochemical examinations on the biopsied muscles.

B.H. Kiyomoto

2. Patients

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of the Neurological

and methods

Patient 1 A 3 year and 4 month-old girl (TN57-5672) was born after a normal pregnancy and delivery. Early developmental milestones were normal: she obtained head control at 3 months, sat alone at 7 months, spoke single words at 10 months and learned to walk at 1 year of age. She was able to run, jump and climb stairs. At the age of 2 years and 10 months she began to drag her left leg and fell frequently. Three months later, she already had difficulty in keeping her head erect. Subsequently she had difficulty in standing up from a sitting position without pushing with her hands on her knees. She had no difficulty in breathing or swallowing. On examination at the age of 3 years and 4 months she was a very slender girl with normal intelligence. She had generalized moderate proximal muscle weakness involving deltoid, trapezius, triceps, biceps brachii, iliopsoas, quadriceps and hamstring muscles. Her severe neck flexor muscle weakness and marked generalized hypotonia were strikingly impressive features. She stood up from the floor utilizing the Cowers’ maneuver and had difficulty in elevating her arms against gravity. Deep tendon reflexes were absent. No cranial nerve deficits were present. Electroencephalogram, electrocardiogram, serum electrolytes, urinalysis and complete blood cell count were within normal limits.

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Creatine kinase (CK) was 289 III/l (normal: 50-150). Except for anti-Coxsackie B16 virus titre of 1:16, no other viral titres examined were elevated significantly. Electromyography (EMG) showed myopathic changes. She did not improve on corticosteroid treatment. The first muscle biopsy was performed on the left biceps brachii muscle at the age of 3 years 5 months and the second on the same muscle on the right side at 3 years 11 months. The disease progressed rapidly and she died from respiratory failure at the age of 4 years and 11 months.

2.1. Patient 2 Since the clinical and pathologic findings of this patient have already been described elsewhere (Kobayashi et al. 1992), only a brief summary is given. This 2 year and 7 month-old girl was born following a normal pregnancy and delivery. No developmental delay or muscle symptom was noticed until the age of 2 years and 3 months when she began to have frequent falls. During the following 4 months, her muscle weakness progressed rapidly; she developed difficulty in raising the upper arms and lifting up her head from the supine position. She could not climb stairs. On examination she also had predominantly proximal muscle weakness, involving the neck, proximal arm and leg muscles. She had a waddling gait. The deep tendon reflexes were normal. There were

Fig. 1. Patient 1: in the first biopsy (A) the lesions are limited to some fascicles (the left half of figure), sparing most of the adjacent fascicle (the right half). In addition to a variation in fiber size there are reducing bodies (arrowhead), enlarged nuclei and rimmed vacuoles (arrow). The second biopsy (B) shows far advanced, more widespread changes involving all fascicles. There are marked variations in fiber size, rimmed vacuoles and reducing bodies. The RB (arrow) are strongly stained with menadione-linked a-glycerophosphate dehydrogenase (MAG) with (C) and without (D) the substrate, a-glycerophosphate. A,B: modified Gomori trichrome, X 220. C,D (serial frozen sections): X 360.

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no other abnormalities on neurological and general physical examination. Serum CK was 739 IU/I (normal:50-150). EMG showed a myopathic pattern. A biopsy of the left biceps brachii was performed at the age of 2 years and 7 months. Her muscle weakness progressed rapidly leading to dysarthria and dysphagia. She died at the age of 3 years and 9 months from respiratory failure.

Methods

Biopsy specimens were frozen in isopentane cooled in liquid nitrogen. Serial 6-lo-pm cryostat sections were stained with a battery of histological and histochemical techniques. Acridine orange (AO) staining was performed as described previously (Miike et al. 1983). For electron microscopy, the muscle specimens were fixed in 2.5% glutaraldehyde in phosphate buffer; ultrathin sections were double stained with uranyl acetate and lead citrate. For immunohistochemical analyses, we utilized immunoperoxidase or immunofluorescence techniques, using antibodies to desmin (D33, 1:200, monoclonal, Dakopats), vimentin (V9, l:lOO, monoclonal, Amersham), ubiquitin (1500, monoclonal, Chemicon), dystrophin (1:200, monoclonal), human nuclei and chromosomes (l:lOO, monoclonal, Chemicon), spectrin (PSN, 1:800, polyclonal, Transformation Research), laminin (PLN, 1:500, polyclonal, E-Y Laboratories) and cu-actinin (1:300, polyclonal).

Fig. 2. In patient 2 again a large variation can be seen in fiber around one blood vessel. Modified Gomori trichrome, X 280.

size, rimmed

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3. Results Light microscopy

In the first biopsy in patient 1 the pathologic changes were limited to only some fascicles where there was variation in muscle fiber size. Scattered fibers had reducing bodies (Fig. 1A). Such fibers had increased acid phosphatase activity. Rest of the fascicles showed only mild myopathic changes with normal fiber type distribution. The second biopsy showed more advanced changes involving all the fascicles (Fig. 1B): there was marked variation in fiber size measuring from 10 to 90 pm in diameter with occasional internal nuclei. The nuclei in many of the fibers were enlarged and sometimes vacuolated. The intermyofibrillar network was disorganized resulting in a moth-eaten appearance. An area of mild inflammatory cell infiltration was present in the interstitium (Fig. 2). The overall pathologic picture in patient 2 was similar to that in the second biopsy in patient 1. There was a marked variation in fiber size, measuring from 5 to 70 pm in diameter. Internal nuclei were noted in an approximately 5% of fibers, and the nuclei were occasionally enlarged. The intermyofibrillar network was markedly disorganized so that some fibers had a whorled appearance. Mild inflammatory cell infiltration around one blood vessel was noted. The rimmed

vacuoles,

reducing

bodies

(arrow)

and inflammatory

cell infiltration

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vacuoles were present in approximately 30% of fibers in the second biopsy from patient 1 and 5-6% in patient 2. The most striking abnormality in both patients was the presence of intracytoplasmic amorphous inclusion bodies in 30-40% of the muscle fibers. They were shown to be RB by their strong reaction to menadione-linked a-glycerophosphate dehydrogenase (MAG) (Fig. 1C) and menadione-NBT solution only (M-NBT) without the substrate, a-glycerophosphate (Fig. 1D). They were best demonstrated with the MNBT reaction. The RB stained bright red with hematoxylin and eosin (H&E), purple with modified Gomori trichrome (mGt), pink with periodic acid-Schiff (PAS), dark blue with MAG and M-NBT. They did not stain with adenosine triphosphatase (ATPase), NADH-tetrazolium reductase (NADH), succinate dehydrogenase (SDH), cytochrome c oxidase (CCO) and acridine orange (AO). The fibers with RB had high acid phosphatase activity. Electron microscopy

The ultrastructural findings were not significantly different in the two patients. The RB were located at the periphery of the sarcoplasm and almost always close to the nuclei in the degenerating fibers (Fig. 3).

Fig. 3. Nucleus (Nu) nuclear degeneration.

completely x 10000.

surrounded

by reducing

bodies

(arrow)

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They were round to oval in shape and frequently surrounded the nucleus forming a ring around the nuclear membrane. They occurred either singly or in aggregates. The RB were not membrane bound and consisted of fine granular material with slightly greater electron density than the chromatin granules. Sometimes intermingled among them were clear spaces containing glycogen particles. The individual granules were not filamentous and difficult to resolve completely but measured approximately from 15 to 25 nm in diameter. Mitochondria or triads were not present within the RB. In the severely degenerated muscle fibers with disorganized myofibrils, the RB were dispersed throughout the cytoplasm. The nuclei were also abnormal and occasionally pyknotic with markedly indented nuclear membrane (Fig. 4). Some nuclei were pale with decreased amount of chromatin granules. Although the nuclear membrane in the vicinity of RB appeared to be lost, no morphologic evidence of direct connection between the RB and nucleoplasm was seen. In muscle fibers containing numerous RB, the myofibrils were markedly decreased and degenerated with disorganized cross-striations. In these fibers, numerous autophagic vacuoles scavenging cytoplasmic debris and glycogen particles, and concentric lamellar bodies resembling myelin were present (Fig. 5).

suggesting

a close

relationship

between

reducing

body

formation

and

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Zmmunohistochemistry RB did not mentin, human trophin, spectrin, imately 10% of stained positively

stain with antibodies to desmin, vianti-nuclei and chromosomes, dyslaminin and alpha actinin. In approxthe muscle fibers with RB, the RB with anti-ubiquitin antibody (Fig. 6D).

4. Discussion Our 2 patients had similar clinical picture with early onset muscle weakness around 2 years of age with rapid progression leading to death. Muscle weakness with hypotonia was generalized but most severely affected in the proximal limb and neck muscles. Since their early development was normal and the early pathologic lesions were localized only to some fascicles, as seen in patient 1, this disease appears to be different

Fig. 4. F’yknotic nucleus with marked disorganized myofibrils with nemaline

indentation (Nu) in severely bodies (arrow) and autophagic

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from the congenital myopathic form (Tome and Fardeau 1975; Oh et al. 1983; Carpenter et al. 1985). From the rapid clinical course and mild inflammatory cellular infiltration in the muscle, we initially thought that this disease was some type of an inflammatory disease, but none of the viral titers tested for was elevated, and steroid treatment had no effect. However, there still remains a possibility that an unknown infectious or post infectious immunodeficiency process may have induced the muscle degeneration. In both patients, the most striking muscle pathology was the presence of RB in many muscle fibers, and our diagnosis was reducing body myopathy of unknown cause. The frequency of the RB in reducing body myopathy has varied from patient to patient, ranging from only 0.2 to 0.5% of muscle fibers (Tome and Fardeau 1975; Oh et al. 1983) to large numbers (Brooke and Neville 1972). The amount of the RB seems to correlate with

degenerated vacuoles.

X

muscle 10000.

fiber.

There

are scattered

reducing

bodies

(asterisk),

B.H. Kiyomoto

et al. /Journal

of the Neurological

the severity of the disease. Among various intracytoplasmic inclusions associated with myofibrillar degeneration including spheroid and cytoplasmic bodies, the RB seems to be a rather rare morphologic abnormality. On reviewing 3500 muscle biopsies in our laboratory, we observed the RB in only 3 biopsies including these 2 patients. The RB were found in rimmed vacuoles in a patient with distal myopathy with rimmed vacuole formation. The exact origin of the RB is so far unknown. Brooke and Neville suggested a viral or ribosomal origin. Others suggested that the granules forming the RB resulted from a myofibrillar component (Tome and Fardeau 1975; Oh et al. 1983). In our patients the RB were always present in fibers with degenerating myofibrils and nuclei. Since the RB were located almost always around the myonuclei and had nearly similar electron density to the chromatin granules, we first speculated that the RB were derived or enucleated from degenerating nuclei. However, acridine orange stain and immunostaining with anti-nuclear antibody failed to demonstrate the any relationship between the RB and the nucleus. The RB did not cross-react with

Fig. 5. Muscle x 7500.

fibers

with

severely

degenerated

myofibrils

in which

numerous

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antibodies against structural, cytoskeletal and membrane antibodies. Rimmed vacuole formation was another striking feature in our patients and this feature has already been described by Carpenter et a1.(1985). The rimmed vacuole itself is not a disease specific abnormality, but is frequently seen in oculopharyngeal dystrophy, distal myopathy (Nonaka et al. 1981) and inclusion body myositis (Carpenter et al. 1978). The rimmed vacuole formation reflects an active autophagic phenomenon because they are associated with high acid phosphatase and cathepsin activities (Ii et al. 1986) and numerous autophagic vacuoles on electron microscopy. Ubiquitin is a 74 amino acid protein found in all eukaryotic cell, whose synthesis increases in response to various metabolic disturbances. The presence of ubiquitin-positive inclusions also may not be a disease specific finding, because ubiquitin positivity is seen in a variety of diseases, especially these with rimmed vacuole formation, including inclusion body myositis (Askanas et al. 19911, oculopharyngeal dystrophy (Leclerc et al. 1993) and distal myopathy with rimmed vacuole formation (Satoyoshi et al. 1993).

reducing

bodies

(arrows),

and autophagic

vacuoles

(AV)

are seen.

.

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Fig. 6. The reducing bodies (arrow) (A,C) are not reactive to anti-desmin antibody antibody (D). A,C: MAG without a substrate; B: DAB; D: DAB and hematoxylin.

In conclusion, there is little doubt that nuclear degeneration plays a role in RB formation, and induces myofibrillar degeneration with active autophagic phenomenon, i.e., rimmed vacuole formation in our patients. Because of the paucity of the regenerating process despite active myofibrillar degeneration, this disease progressed rapidly leading to death within a few years.

Acknowledgement

Dr. B.H. Kiyomoto was a research fellow sponsored by the Ministry of Education, Science and Culture, Japan.

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(Bl. Some of them are positively A,B (serial sections): x320. C,D

stained with anti-ubiqc (serial sections): x320.

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