Acta neurol. scandinav. 57, 216-222, 1978

Department of Neurology, University of Milan, Milan, Italy

Pompe’s Disease: Ultrastructural Alterations of Muscle Tissue in Parents

GIULIO PELLEGRINI, GIANPIETRO MIOSCA i& CESARE CERRI

A histological, histochemical and ultrastructural study of muscle tissue was performed in the parents of a patient affected by a infantile form of acid maltase deficiency (Pompe’s disease).

In both parents the clinical examination was normal, but serum levels of creatine kinase (CK) and aldolase were high.

Histological and histochemical examination of muscle did not reveal any abnormality. Ultrastructural study showed an excess of glycogen granules below the sarcdemmal sheat and between myofibrils, often associated with clusters of mitochondria. There was no glycogen trapped in lysosomal vescicles.

The mechanism of glycogen storage in Pompe’s disease seems to involve an enzymatic deficiency other than acid maltase.

INTRODUCTION

The mechanism of accumulation of glycogen in muscular tissue is an unsolved problem of Pompe’s disease. Large amounts of this substance are present in muscle fibres, both inside lysosomes and in the cytoplasm between myofibrils (Hug et al. 1966, CardifJ 1966, Smith et al. 1967, Garancis et al. 1968).

The basis of the disease is supposed to be an hereditary deficiency of acid maltase or 1,4-~-gIucosidase (Hers et al. 1963), a lysosomal enzyme that hydrolyzes glycogen trapped in lysosomes after normal endocytosis (Hers 1973). The urigin of the extralysosomal glycogen is obscure. If the disease were due solely to the lack of acid maltase, one should expect storage of glycogen only in the lysosomal compartment of the muscle fibre.

We have performed a histological, histochemical and ultrastructural study of the skeletal muscle in the parents of a patient who was affected by infantile form of acid maltase deficiency. The parents were clinically normal but they had increased levels of serum creatine kinase (CK) and aldolase.

CLINICAL AND LABORATORY DATA, RESULTS

The patient affected by Pompe’s disease (D.G.A.) was a 2-year-old infant admitted for generalized weakness and slow development. She walked at 17 months and was unable

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to rise from a supine position without assistance. On examination there was severe, ge- neralised muscular weakness with hypotrophy and flaccidity especially of the proximal part of the upper and the lower extremities. Tendon jerks were weak. The liver was enlarged. The electrocardiogram showed left ventricular hypertrophy, the heart area being within normal limits. C.K. was 480 mU/ml (n.v. 0-50 mU/ml); aldolase: 17 mWml (n.v. 0.5-3.1 mU/ml); LDH 1280 mU/ml (n.v. 0-190 mU/ml). The electromyogram in both tibialis anterior muscles showed a shortened duration of motor unit potentials and an increased incidence of polyphasic potentials of low amplitude indicating myopathy. The motor nerve conduction velocity in the right ulnar nerve and right popliteal nerve were normal. Biopsy of the brachial biceps muscle showed many fibres with small vacuoles containing large amounts of PAS positive material, digested by diastase. In addition some fibres contained intracytoplasmic deposits of globular material that de- veloped "gamma" metachromasia with toludine blue. The concentration of glycogen in muscle tissue was gr. 12.4 % (n.v. gr. 0.4-1.1 %) (Montgomery 1957); acid maltase was absent (Hudgson et al. 1968); phosphorylase a and b were markedly reduced (respec- tively 48 and 54 P/mg/15'/37" C: n.v.: 383 f 11.9 and 481 2 10.8 (Leonard & Wimsatt 1959).

The parents D.G.F. (8) and D.G.A. (9) were 33 and 28 yars old. There was no consanguinity and no history of previous disease. The neurological examination and electromyography of the right deltoid and right quadriceps were normal. In both there was a slight elevation of serum CK and andolase. In the father CK was 125 mU/ml, aldolase 5.8 mU/ml; in the mother CK was 115 mUJml and aldolase 5.2 mU/ml (upper limits of normal 50 mU/ml for CK and 3.1 mU/ml for aldolase). a-glucosidase (lyso-

Figure 1 . Father of the putient. Accumulation of free glycogen particles between myofibrils. Electron micrograph. X 16200.

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soma1 acid maltase) (E.C. 3.2.1.20) in muscle tissue was below normal: 0.8 nM/mg non collagenous proteidmin in the father and 0.7 nM/mg non collagenous proteidmin in the mother, compared with 1.7 and 1.5 nM/mg non-collagenous protein/min. in two normal controls (Hudgson et al. 1968). Muscular glycogen in the father was gr. 1.56 % compared with 0.68 and 0.78 gr. % af two controls (n.v. gr. 0.4-1.1 Yo); (Montgomery 1957).

Muscle biopsy. Cryostat sections 10 ,u thick were obtained from biopsy specimens of the brachial biceps muscles in both parents. A battery of histological and histochemical reactions were performed (haematoxylin and eosin, modified Goniori trichrome (Engel & Cunnigham 1963), nicotinamide adenin nucleotide dehydrogenase (NAHD) (E.C. 1.6.99.1.) (Novikofl et al. 1961), succinicodehydrogenase (SDH) (E.C. 1.3.99.1.) (Nachlas et al. 1957), ATP ase pH 9.4-4.6-4.3 (E.C. 2.7.4.3.) (Pudykula & Herman 1955), phos- phorylase (E.C. 2.4.1.1.) (Takeuchi & Kuriaki 1955), PAS (Mowry & Millican 1952), acid phosphatase (E.C. 3.1.3.2.) (Barka & Anderson 1962), Oil red 0).

A sample of muscle tissue was fixed for electron microscopy in 0.6 % glutaraldehyde buffered with cacodylate at pH 7.4 for 20 h and post-fixed in 2 % buffered osmium tetroxyde. The material was embedded in Spurr resin and thin sections were stained with uranyl acetate and lead citrate.

Histopathology

The histological examination as well as histochemistry were normal; the only abnornia-

Figure 2 . Mother of the patient. Collection of free glycogen granules between rnyofibrils. Note the disruption and the reduction in size of some myofibrils. Electron micrograph.

X 16200.

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lity was a slight increase in granular acid phosphatase activity in sarcolemmal regions of some fibres.

The ultrastructural examination showed in the most muscle fibres of both parents an excess of glycogen, freely dispersed throughout the sarcoplasm, appearing as dark gran- ules in the sections stained by lead citrate. The glycogen granules were situated both in subsarcolemmal regions and between myofibrils (Figure 1). Aggregates of glycogen granules trapped in lysosomal vescicles were not observed. The myofibrils separated by glycogen granules were often disrupted or reduced in size (Figure 2). Many fibres contained clusters of proliferated mitochondria under the sarcolemmal membrane (Fig- ure 3) or between myofibrils. The mitochondria were irregular in shape and size but did not exhibit structural alterations of the matrix or the cristae. Some fibres had an increase in fat droplets (Figure 4).

DISCUSSION

The ultrastructural alterations in the muscles of both parents of a patient with Pompe’s disease are compatible with the existence of a heterozygous state. These changes are similar to those observed in the homozygous patients except for a lesser amount of glycogen stored in muscular tissue and for the absence of lysosomes filled with glycogen particles.

Moreover mitochondria1 abnormalities are generally absent in Pompe’s

Figure 3. Father of the patient. Aggregates of mitocondria and lipid droplets beneath the sarcolemmal sheath. Electron micrograph. X 26600.

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disease, but Engel & Dale (1968) reported a case with alterations in the number, shape and structure of these organelles.

The increased amount of glycogen lying free in the sarcoplasm between the myofibrils and also between myofilaments is not explained by the de- creased activity of acid maltase we found biochemically, in agreement with the data of Engel & Gomez (1970).

These findings are consistent with the hypothesis of H u g et al. (1966), Cardig (1966) and Hudgson et al. (1968) that also in homozygotes, at least in the early stages of the disease, the glycogen accumulates between the myo- fibrils and penetrates the lysosomes later on in the disease. This suggests other factors in the pathogenesis of glycogen storage of Pompe’s disease other than the simple deficiency of acid maltase. If glycogenolysis is impaired glycogen woluld primarily accumulate in the section of the cell where the defective enzyme is normally present, i.e. in the lysosomes.

Therefore one must assume a deficiency of another enzyme such as phos- phorylase or neutral maltase located in the soluble or microsomal fraction of the muscle fibre.

Neutral maltase decreased in the infantile form of the disease (Angelini & Engel 1972, Hers 1973, Mehler & Di Mauro 1977) and Mehler & Di

Figure 4 . Mother of the patient. Collection of large lipid droplets between myofibrils of n muscle fibre. Electron micrograph. X 16200.

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Mauro (1977) found decreased neutral maltase activity also1 in muscle tissue of an heterozygous subject and in leucocytes both of heterozygotes and pa- tients with late onset acid maltase deficiency. It is then possible that neutral maltase plays a role in the mechanism of accumulation of extralysosomal glycogen both in homozygous and in heterozygous patients with Pompe’s disease.

REFERENCES

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Engel, A. G. (& M. R. Gomez (1970): Acid maltase levels in heterozygous acid maltase deficiency and non-weak and neuromuscular disease controls. J. Neurol. Neurosurg. Psychiat. 33, 801-804.

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Received October 25, 1977, accepted January 22, 1978 Giulio Pellegrini, M.D.

Istituto di Neurologia Universitk di Milano Via F. Sforza 35 20122 Milano Italy