Adult glycogenosis type II (Pompe’s disease): morphological abnormalities in muscle and skin biopsies compared with acid alpha-glucosidase activity

Introduction
Glycogen storage disease type II (GSD II) is an autosomal recessive lysosomal disorder, caused by deficiency of acid alpha-1,4-glucosidase (GAA). Deficiency of GAA can be caused by about 150 mutations found in the GAA gene located on human chromosome 17 (17q25.2-q25.3) [6].
This storage disease was first reported by Pompe and Putschar in 1932 [14]. Following the identification of lysosomes, it was found out that Pompe’s disease occurs due to the absence of glycogen degrading lysosomal enzyme GAA. Excess glycogen is present in cardiac muscle fibres, smooth and skeletal muscles, as well as in vascular endothelium, Schwann cells, perineurium and astrocytes in the cerebral cortex [8,13,14,18]. In the infantile and juvenile forms of GSD II, the storage of glycogen is also visible in neurons in anterior horn cells, motor cranial nerve nuclei, basal ganglia and gastrointestinal tract plexuses [7]. Therefore, intra-lysosomal accumulation of glycogen is present in almost all body tissues [6].
Three forms of this storage disease (infantile, juvenile and adult onset types) have been described based on its severity and the age that onset of clinical symptoms is observed. It is supposed that the severity of the disease depends on the degree of GAA deficiency [5,14].

Material and Methods
Biopsies were performed on the biceps muscle and skin in each patient. Biopsy specimens of muscle and skin were fixed in 4% paraformaldehyde in 0.1 M phosphorane buffer saline, pH 7.4 for light microscope examination, and in 2.5% glutaraldehyde and postfixed in 2% osmium tetroxide for ultrastructure analysis. All biopsy samples were stained histologically (haematoxylin-eosin, PAS, PAS-dimedon), and immunohistologically with ubiquitine (DAKO 1:200).
Ultrastructural analysis was carried out on ultrathin sections of skin and muscles after staining with uranyl acetate and lead citrate, and examined with a transmission electron microscope (Opton DPS109).
Lysosomal acid alpha-glucosidase activity was measured fluorometrically in isolated peripheral blood leukocytes (L) and dried blood spots (DBS) with 4-methylumbelliferyl-alpha-D-glucopyranoside (4MU-alphaGlc) as substrate according to previously described methods with slight modifications [2,10].
Maltase-glucoamylase activity was inhibited with 30 µmol/L acarbose. For each patient, GAA activity was assayed in the presence and absence of acarbose at pH3.8. The results are expressed as the ratio of GAA activities with and without acarbose.

Clinical data/case reports
Patient 1
A 19-year-old woman was admitted to hospital because of rapid progressive respiratory failure. Her first symptoms began 7 months before admission. Initially, she complained about occasional exercise dyspnoea occurring during running and walking. Within two months exercise dyspnoea progressed and occasional resting dyspnoea occurred. The symptoms were accompanied by severe morning headaches and progressive muscle weakness. Due to menstrual cycle disturbances, the patient was referred to the endocrinological department, where hyperprolactinaemia and hypothyroidism were diagnosed. During hospitalisation, the patient was consulted neurologically and subjected to MRI scanning, which revealed diffuse T2 weighted changes in the brain. After multiple sclerosis was diagnosed, the patient underwent steroid treatment, which failed to be effective. Symptoms dramatically progressed. Exercise dyspnoea on every effort, obstructive sleep apnoeas and severe morning headaches were present daily. The patient was bedridden and finally transferred to the emergency department with severe respiratory failure and acidosis. She was admitted to the intensive care unit, put on the respirator, and subjected to IVIg treatment (intravenous immunoglobulin in the dose of 1 g per kilogram of body weight on 5 consecutive days), which also appeared to be ineffective. The neurological examination revealed flaccid tetraparesis, proximal muscle atrophy and respiratory failure. Electromyography (EMG) showed myopathic changes. A muscle and skin biopsy was taken and acid maltase activity was determined and the diagnosis made.

Patient 2
A 29-year-old woman was admitted to the hospital because of progressive nocturnal dyspnoea, muscle weakness and occasional spontaneous dyspnoea occurring during the day for two months.
Symptoms were more pronounced in the evening and showed a fluctuating intensity. Her history revealed the diagnosis of juvenile myoclonic epilepsy successfully treated with valproates. She had been free of attacks for the last two years. On neurological examination she revealed no abnormalities. EMG showed mild myopathic changes present seen only in the brachial biceps muscle. The muscle biopsy and activity of acid maltase confirmed the diagnosis.

Patient 3
A 31-year-old woman, sister of patient 2, complained about muscle weakness, occasional dyspnoea, poor exercise tolerance and exercise dyspnoea for 6 years. She was diagnosed for hepatitis after laboratory findings indicating elevated levels of serum aminotransferases and creatinine kinase. The patient underwent hepar biopsy, which revealed no significant changes. Symptoms had progressed during the last year. On neurological examination the patient revealed proximal muscle weakness, Gower’s phenomenon, and lack of deep tendon reflexes from biceps brachii, as well as lack of the patellar reflex. EMG showed advanced myopathic changes in all tested muscles. The biopsy of brachial muscle biceps and activity of acid maltase confirmed the diagnosis.

Results
Alpha-glucosidase activity at pH 3.8 was assayed in the presence and absence of acarbose in DBS and L. The assay revealed its reduced activity in all three patients (Table I).
The ratio of GAA activities in leukocytes were reduced to 0.07 in patients 1 and 2, while in patient 3 it was 0.09; in DBS the ratio was reduced to 0.12 in patients 1 and 3, but to 0.05 in patient 2. On electron microscope, the patients’ biopsies showed analogous degenerative changes.
Similar ultrastructural changes were visible in the majority of muscle fibrils in patients 1 and 2. Myofibrils were almost completely destroyed by accumulation of free glycogen granules, as well as in vacuoles of various size and in lysosomes (Fig. 4). In patient 1, these pathological changes were more intensive than in patients 2 and 3 (Fig. 5). In patient 3, some muscle fibrils showed intralysosomal glycogen and only a few glycogen granules in the sarcoplasm. Autophagic vacuoles were visible occasionally (Fig. 6). In the wall of blood vessels of muscle and skin biopsies the depositions of glycogen were also visible. In the cytoplasm of endothelial cells, pericytes and smooth muscle cells, free glycogen granules as well as those accumulated in lysosomes were frequently observed (Fig. 7A, B).

Discussion
Clinical manifestations, respiratory failure and muscle weakness were similar in all investigated patients. Similar clinical symptoms have been described by numerous authors in patients with deficiency of acid maltase [15,17]. The character and intensity of clinical manifestations differed between patients. The respiratory failure was more pronounced in patient 1 (aged 19) than in patients 2 (aged 31) or 3 (aged 29). The former (patient 1) showed generalised muscular weakness with more severe involvement of the limbs and respiratory muscles. The course of the disease was acute (patient 1), subacute (patient 2), and mild (patient 3). Clinical pictures did not correlate with the ratio of GAA activity in leukocytes and DBS (Table I) assayed in vitro with artificial substrate.
Morphological examination of the muscle and skin biopsies displayed vacuolar degeneration of muscles, abundant glycogen inside of muscles and in the walls of capillaries. Glycogen deposits were observed in lysosomes and as free glycogen deposits or in vacuoles of various size. Similar abnormalities were observed in several types of glycogenosis, but only in Pompe disease and Danon disease was glycogen visible in lysosomes [3,4,11,12,15-17].
The intensity of glycogen storage, vacuolar degeneration and general severity of the disease were reported to be dependent on the degree of acid alpha-1,4-glucosidase deficiency [5,14]. In our observation, neither the intensity of clinical symptoms nor storage of glycogen and vacuolar degeneration were related to the degree of the enzyme deficiency. The lowest ratio of GAA activity in DBS and L (0.05 and 0.07, respectively) was recorded in patient 2 (Table I; Fig. 2), while histopathological and clinical abnormalities were most strongly manifested in patient 1 (0.12 and 0.07, respectively). It is known that there is a spectrum of cellular glucosidases with optimum pH at different values; therefore the measurement of GAA activity at pH 3.8 and in the presence of maltase-glucoamylase (MGA) inhibitor (e.g. acarbose) is of essential importance in the diagnosis of GSD type II. Since we did not assay the activity of all enzymes related to glycogenolysis and glycogenesis, the combined deficiency of GAA and another enzyme could not be ruled out. On the other hand, the function of GAA cannot be limited only to lysosomal degradation of glycogen. Orth and Mundegar [11] suggested that GSDs cause lysosomal proliferation, as well as affect endosomes, the trans-Golgi network and vesicle population linked to lysosomes. They showed that the intensity of anti-LAMP-1 (lysosomal associated membrane protein-1) staining corresponded with the degree of vacuolation and glycogen storage. A link between the lysosomal membrane function and GAA activity seems to be possible. What is more, GAA plays an important role in the regulation of lysosomal autophagy [1]. An intensive reaction of ubiquitin around vacuoles in muscle biopsies taken from our patients seems to confirm the damage of membrane proteins.
GAA activity does not appear to be the only factor that determines the severity of GSD II.

References
1. Bursch W, Ellinger A. Autophagy – a basic mechanism and a potential role for neurodegeneration. Folia Neuropathol 2005; 43: 297-310.
2. Chamoles NA, Niizawa G, Blanco M, Gaggioli D, Casentini C. Glycogen storage disease type II: enzymatic screening in dried blood spots on filter paper. Clin Chim Acta 2004; 347: 97-102.
3. Fernández R, Fernandez JM, Cervera C, Teijeira S, Teijero A, Dominguez C, Navarro C. Adult glycogenosis II with paracrystalline mitochondrial inclusions and Hirano bodies in skeletal muscle. Neuromuscul Disord 1999; 9: 136-143.
4. Finsterer J, Stöllberger C, Blazek G. Neuromuscular implications in left ventricular hypertrabeculation/noncompaction. Int J Cardiol 2006; 110: 288-300.
5. Kamphoven JH, de Ruiter MM, Winkel LP, Van den Hout HM, Bijman J, De Zeeuw CI, Hoeve HL, Van Zanten BA, Van der Ploeg AT, Reuser AJ. Hearing loss in infantile Pompe’s disease and determination of underlying pathology in the knockout mouse. Neurobiol Dis 2004; 16: 14-20.
6. Lu IL, Lin CY, Lin SB, Chen ST, Yeh LY, Yang FY, Au LC. Correction/mutation of acid alpha-D-glucosidase gene by modified single-stranded oligonucleotides: in vitro and in vivo studies. Gene Ther 2003; 10: 1910-1916.
7. Mancall EL, Aponte GE, Berry RG. Pompe’s disease (diffuse glycogenosis) with neuronal storage. J Neuropathol Exp Neurol 1965; 24: 85-96.
8. Montalvo AL, Cariati R, Deganuto M, Guerci V, Garcia R, Ciana G, Bembi B, Pittis MG. Glycogenosis type II: identification and expression of three novel mutations in the acid alpha-glucosidase gene causing the infantile form of the disease. Mol Genet Metab 2004; 81: 203-208.
9. Nishino I, Fu J, Tanji K, Yamada T, Shimojo S, Koori T, Mora M, Riggs JE, Oh SJ, Koga Y, Sue CM, Yamamoto A, Murakami N, Shanske S, Byrne E, Bonilla E, Nonaka I, DiMauro S, Hirano M. Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature 2000; 406: 906-910.
10. Okumiya T, Keulemans JL, Kroos MA, Van der Beek NM, Boer MA, Takeuchi H, Van Diggelen OP, Reuser AJ. A new diagnostic assay for glycogen storage disease type II in mixed leukocytes. Mol Genet Metab 2006; 88: 22-28.
11. Orth M, Mundegar RR. Effect of acid maltase deficiency on the endosomal/lysosomal system and glucose transporter 4. Neuromuscul Disord 2003; 13: 49-54.
12. Premasiri MK, Lee YS. The myopathology of floppy and hypotonic infants in Singapore. Pathology 2003; 35: 409-413.
13. Raben N, Fukuda T, Gilbert AL, de Jong D, Thurberg BL, Mattaliano RJ, Meikle P, Hopwood JJ, Nagashima K, Nagaraju K, Plotz PH. Replacing acid alpha-glucosidase in Pompe disease: recombinant and transgenic enzymes are equipotent, but neither completely clears glycogen from type II muscle fibers. Mol Ther 2004; 11: 48-56.
14. Raben N, Plotz P, Byrne BJ. Acid alpha-glucosidase deficiency (glycogenosis type II, Pompe disease). Curr Mol Med 2002; 2: 145-166.
15. Sharma MC, Schultze C, von Moers A, Stoltenburg-Didinger G, Shin YS, Podskarbi T, Isenhardt K, Tews DS, Goebel HH. Delayed or late-onset type II glycogenosis with globular inclusions. Acta Neuropathol (Berl) 2005; 110: 151-157.
16. Slonim AE, Bulone L, Ritz S, Goldberg T, Chen A, Martiniuk F. Identification of two subtypes of infantile acid maltase deficiency. J Pediatr 2000; 137: 283-285
17. Tanaka K, Shimazu S, Oya N, Tomisawa M, Kusunoki T, Soyama K, Ono E. Muscular form of glycogenosis type II (Pompe’s disease). Pediatrics 1979; 63: 124-129.
18. Winkel LP, Kamphoven JH, van den Hout HJ, Severijnen LA, van Doorn PA, Reuser AJ, van der Ploeg AT. Morphological changes in muscle tissue of patients with infantile Pompe’s disease receiving enzyme replacement therapy. Muscle Nerve 2003; 27: 743-751.