168: 397403 (1992)
JOURNAL OF PATHOLOGY, VOL.
DOMINANTLY INHERITED TUBULAR AGGREGATE MYOPATHY c. H.
STUART CAMERON*, INGR~Dv.
ALL EN^, VICTOR PATTERSON$ AND
MARIA A. A V A R I A ~
Neuropathologyf and Electron Microscopy* Laboratories, School of Clinical Medicine, Queens University and The Muscle Clinic$, City Hospital, Belfast, Northern Ireland, U.K. Received I5 April 1992 Accepted 22 June 1992
SUMMARY We report an unusual familial myopathy characterized morphologically by the presence of large tubular aggregates in all fibre types. Two patients, a father and daughter, presented with slowly progressive proximal weakness, limitation of eye movement, and Achilles tendon contractures. Serum creatine kinase was 5-10 times normal. Light microscopy revealed type I fibre predominance. Basophilic accumulations, which stained intensely with the NADH-TR reaction, were present in both fibre types. Electron microscopy revealed that these consisted of tightly packed parallel tubular arrays. These varied somewhat in their ultrastructural appearance and were classified accordingly as type I, 11, and I11 tubular structures. The tubular aggregates appear to be derived from the sarcoplasmic reticulum. This report further supports the evidence of a distinct clinico-pathological entity of genetic origin. KEY WORDS-Familial
myopathy, proximal weakness, tubular aggregates, electron microscopy.
INTRODUCTION
climbing stairs. Since early childhood he had walked on his toes and had difficulty running and keeping Tubular aggregates (TAs) in muscle have been up with his peers, although he was able to play rugby reported in two situations. In the first, they occur as at 13. He had been diagnosed as having infectious a minor feature in well-establishedmuscle disease of hepatitis at the age of 19 and because his transoften known cause such as in periodic paralysis,'" aminases remained raised, a liver biopsy was myasthenia gravis? facioscapulohumeraldystrophy performed which revealed no abnormality. with aminoaciduria,'.' inflammatorymyopathie~?'~ He had previously been investigated because of myotonia," and alcohol and drug into~ication.'~-'~his weakness at 31 years of age. He had difficulty In the second situation, they are the major patho- rising from a lying position and was found to have logical feature. The best known occurs in some MRC grade 3 weakness of his scapular and pelvic patients with exercise-induced pain and stiffness in girdle muscles with lesser involvement of his ankle which TAs are found in type I1 muscle fibre^.^,'^''^ dorsiflexors. He gave a history of heavy alcohol In contrast, two families have been reported in intake for several years, having stopped 9 months which progressive muscle weakness was associated prior to consultation. A muscle biopsy (formalinwith widespread TAs in both types of muscle fixed) from his quadriceps was obtained at that time fibres."-I9 We have encountered a third family and a diagnosis of limb-girdle muscular dystrophy with this condition and suggest that it represents a was made. distinct clinico-pathologicalentity. At the age of 39 years, he underwent lengthening of his Achilles tendons. His parents and his brother CASE REPORTS and sister had no complaints of weakness or other neuromuscular symptoms. There was no history Case 1 This patient was seen at age44yearswithprogress- of muscle pain. Of his two daughters, one was ive weakness which was disablingfrom the age of 27, asymptomatic while the other is reported as case 2. His condition continued to deteriorate slowly, although from the age of 20 he had some difficulty in with increasing difficulty going upstairs and rising *Address for correspondence. from a chair. He developed a tendency to fall, 0022-341 7/92/I 20397-07 $08.50 0 1992 by John Wiley & Sons, Ltd.
398
C. H.S. CAMERON ETAL.
finding it very difficult to get up by himself and was referred for reassessment at 44 years. He had increased lordosis and walked with a waddling gait. Bilateral restriction of eye adduction and upward gaze was found. There was no facial weakness. Neck flexors were MRC grade 3; biceps and triceps grade 4.In his lower limbs, hip flexors and knee extensors were grade 3 and ankle dorsiflexors grade 5 . No loss of sensation or coordination was found and he had no muscle stiffness or cramps. Tendon reflexes were preserved and there was no sensory loss. Intravenous edrophonium chloride produced no improvement in strength. A muscle biopsy was taken from the left biceps. During a 6-year follow-up, he has developed some increase in weakness (biceps has gone from MRC grade 4 to 4 - and knee extensors from grade 3 to 3-) and intermittent features of a depressive illness for which he has been treated with tricyclic antidepressants.A therapeutic trial with Dantrolene had to be discontinued without benefit at a dose of 50 mg daily because of drowsiness. Case 2 This patient, the daughter of case 1, had been examined for the first time at 7 years of age, because of her father’s progressive weakness and was considered at that time to be normal. Her parents thought she was clumsy and she had difficulty in hopping and playing games. During the next 2 years she developed progressive difficulty in rising from the floor and climbing stairs and had pain in her limbs after exercise. Physical examination at that time revealed weakness which spared the facial muscles but affected the proximal muscles of both upper and lower limbs. She had increased lordosis and a waddling gait. Deep tendon jerks were normal. A muscle biopsy was performed on her left biceps. She was seen again at the age of 16 years, when she showed mild restriction of upward gaze; neck flexors were MRC grade 3 - , biceps and hip flexors grade 4 - .A needle biopsy from her quadriceps was performed. Shehas continued to show a very slow progression of muscle weakness with intermittent features of depression.Hip flexor strength has gone from MRC grade 4- to MRC grade 3 over 7 years. Special investigations Serum creatine kinase (CK) was elevated in both patients; lSSOp/l in case 1 and 700p/1 in case 2 (normal 30-140 p/l).
Electromyogramswere performed on the deltoid, biceps and quadriceps of both patients and no spontaneous activity was recorded. In case 1, motor unit potentials (MUPs) were found to be of normal amplitude and duration but with some increase in polyphasic activity. Nerve conduction velocity in the median nerve was 60m/s for both motor and sensory components. Case 2 showed a predominantly myopathic pattern on electromyographyand nerve conduction was within normal limits. MATERIAL AND METHODS In case 1, an open biopsy was performed at 44 years on the left biceps and muscle was obtained for light and electron microscopy. Tissue was snap-frozen and several cryostat sections were stained by an appropriate battery of histological and histochemical techniques. Fibre quantitation studies were undertaken on ATPasestained sections using a Leitz TAS Plus image analysis system. For electron microscopy, the muscle was fixed in 3 per cent phosphate-buffered glutaraldehyde (pH 7.2) post-osmicated and embedded in Polarbed 812 epoxy resin. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined using a Hitachi H-600electron microscope. In case 2, a needle biopsy was performed at 16 years on the left quadriceps and processed entirely for electron microscopy. The previous biopsies from both patients taken at 3 1 and 8 years, respectively,which had been fixed in formalin and glutaraldehyde for routine histology and electron microscopy, were also reviewed. RESULTS Light microscopy On routine cryostat sectioning, there was a marked variation in the range of fibre diameters involving both fibre types. There was a predominance of type I fibres, atrophy of both fibre types (type I type 11). Thirty-eight per cent of all fibres contained central nuclei and there was some fibre splitting. Basophilic areas were apparent within many fibres, particularly the larger ones. These areas stained intensely with the NADH-TR reaction (Fig. l), bright red with Gomori’s trichrome stain and were occasionally PAS positive.
TUBULAR AGGREGATE MYOPATHY
399
400 nm in diameter and were commonly found in subsarcolemmalaggregates. Type 111: Dilated parallel tubules mostly between 130 and 400nm in diameter, but each containing a number of smaller, 25-40 nm diameter, microtubule-like structures (Fig. 5). Central nuclei were frequent and usually associated with the TAs. Away from the large aggregates, many of the smaller tubular arrays appeared to arise as proliferations of the sarcoplasmicreticulum (Fig. 6). Mitochondria were generally unaffected and did not appear to be involved in the production of the honeycomb structures. Particulate glycogen was diffusely scattered between the tightly packed tubules. Lipid droplets were uncommon, whereas lipofuscin granules were occasionally encountered within some of the large TAs. Endomysial collagen was not increased. The previous muscle biopsy from both patients Fig. 1-A cryostat section showing strong NADH-TR staining was reviewed. Large TAs were predominant in all of the tubular aggregates fibres in case 1, whereas in case 2 they were much smaller, measuring betwen 1 and 12pm on their With the ATPase reaction, they appeared as pale longest dimension and less frequent than those in the present biopsy. Only type I1 tubular structures unstained areas in both fibre types. were present and these were also the most prevalent On review, similar abnormalities could be identstructures in case 1. Type I11 tubular structures were ified in the previous paraffin-embedded biopsy from not identified in either of these biopsies. case 1, while the histology in case 2 showed a relatively normal appearance with occasional central nuclei. DISCUSSION Involvement of the current cases with any of the other disorders related to TAs in skeletalmuscle was dismissed on the basis of their clinical and microscopical findings. The distribution of weakness with no facial involvement was not consistent with that seen in facioscapular humeral dystrophy. There was no evidence of periodic paralysis or myotonia. No drugs were involved at the time of biopsy. The possibility of an alcoholic myopathy in case 1 as the source of the TAs was unlikely as his daughter had similar features. Both patients presented with a slowly progressive Closely packed parallel tubules approxi- weakness affecting their proximal muscles but mately 70 nm in diameter. Each tubule sparing the face. Weakness became evident at about contained a smaller 40nm diameter 20 and 9 years, respectively, although with case 1 central tubule, or a microtubule-like there is evidence of early impairment during his school years, probably related to ankle contractures. structure (Fig. 3). Tubular structures containing a moder- In the two previous reports of this ~ondition,””~ ately dense, flocculent material instead onset was between 10 and 40 years. Subtle involveof a central tubule (Fig. 4). They were ment of the extra occular muscles was present in often dilated, measuringbetween 70 and both of our cases but has not been described in the
Electron microscopy Muscle from both the father and daughter had a similar ultrastructural appearance. Tubular aggregates were present in all muscle fibres. They were located both within the sacroplasm and adjacent to the sarcolemma. The aggregates measured from < 1 ,um to >7O,um in diameter (Fig. 2) and consisted of parallel tubular arrays (honeycomb-like structures). These varied somewhat in their ultrastructural appearance and were therefore classified accordingly:
Type I:
Type 11:
400
C. H.S. CAMERON ET AL.
Fig. 2-An
array of closely packed type I tubular structures. The bar equals 0.5 pm
Fig. 3-Type I tubular structures (arrow-heads) are seen in transverse section to contain an inner tubule (arrow). The bar equals 50 nm
Fig. &Type I1 tubular structures, most of which are dilated, contain a moderately dense flocculent material. The bar equals 250 nm
other two families. Another peculiar aspect not previously described was the presence of contractures, which were severe in case 1. By contrast, facial weakness does not appear to be a common feature,
having been described in only 1 of the 12 patients reported to date. Muscle pain does not appear to be uniformly associated with tubular aggregate myopathy. In the
TUBULAR AGGREGATE MYOPATHY
Fig. 5-A portion of a tubular aggregate consisting mostly of type I11 tubular structures. Several of the dilated tubules (arrows) are seen to contain a number of smaller tubules (inset). The bars equal 1 pm and 125 nm (inset)
Fig. &A small clusterof tubular structures is in contiguity with the sarcoplasmicreticulum (arrow).The bar equals 300 nm
40 1
402
C. H. S. CAMERON ET AL.
family described by Rohkamm et al.,I7 the main clinical features were of proximal weakness without wasting or pain. In the report by Rerobon-Bormioli et UZ.,'~ pain was the prominent symptom in four out of the five cases. While there is no history of muscle pain, cramps, or stiffness in case 1, case 2 had mild symptomology with some pain in her limbs following exercise. It is therefore suggested by these findings that muscle pain is not an essential feature of this condition, although it is a frequent symptom. In addition, elevated CK appears to be a similarly variable finding in the three families reported to date. Electromyographicfindingsin previously reported patients have ranged from normal to a slightly myopathic pattern with brief small action potentials (BSAPs). In some patients, long duration MUPs of 12-24 m/s were recorded. Case 1 had MUPs of normal amplitude and duration, but increased polyphasity and less than complete interference pattern on full effort, suggesting a neurogenic disorder. The occurrenceof positivesharp waves in a muscle has been reported in only one patientI7 and nerve conductionvelocities,when reported, have beennormal. Neuromuscular transmission has been tested either electrophysiologically or pharmacologically, as in our case 1, with no evidence of involvement. The biopsy findings in both of our patients are very similar to those reported by Rohkamm et ~ 1 . and Pierobon-Bormioliet all8 All have shown subsarcolemmal or centrally placed basophilic accumulations in haematoxylin and eosin sections, which stain bright red with Gomori's trichrome and dark blue/black with NADH-TR reaction. They do not react with ATPase reactions. Such structures have been found in most fibres, ranging from 50 per cent to almost 100 per cent of the fibres examined, being apparently more numerous in older patients. As with the present study, TAs are not restricted to type I1 fibres as with a variety of other disorders, but involve both fibre types. Features of degeneration or inflammation were not seen in Rohkamm et al.3 family,'7 but internal nuclei, fibre splitting and variation in fibre size were found in varying degrees in both of the previous studies. In case 1, there were some features of degeneration with slight mononuclear cell infiltration and a moderate increase in connective tissue. In common with the earlier studies, there was a marked variation in fibre size and central nuclei were numerous. Type I predominance with atrophy more marked in type I1 fibres and hypertrophy of both fibre types were present. Type I predominance
has been a feature in five out of seven biopsies reported p r e v i ~ u s l y . ' ~ There ~ ' ~ were no clear differences on clinical or laboratory grounds between patients with either fibre type predominance; therefore the significanceof this remains uncertain. Type I predominance was also found in the patients with myasthenic features and TAs.~,~' These patients also showed TAs in both fibre types, a feature not previously reported and shared by all the patients with the autosomal dominantly inherited myopathy. Tubular aggregates have often been described as 'double-walled t ~ b u l e s ' . ~ ~However, ' ~ , ' ~ ~ ~ our ' study together with those of En el et Meyers et a1.,5 and Morgan-Hughes et al!shows that theTAs have a more variable appearance consisting of an outer tubule, or vesicle, containing either amorphous material or one or more microtubule-likestructures. More commonly, they are best described as a tubule within a tubule. We have attempted to categorize the TAs into type I, I1 and I11 structures depending on their morphological appearances. These are somewhat similar to the various forms described by Morgan-Hughes et u I . , ~ who ~ together with Engel et al.? Dobkin and Verity6and Pierobon-Bormioli et aZ.'* have shown that the TAs are in contiguity with various aspects of the sacroplasmicreticulum. The previous suggestion of a mitochondria1 ' ~origin, based on the localization of certain mitochondrial e m me^,^^ has not been supported by other studies? TAs have not exhibited any structural association with mitochondria, which appear largely unaffected, and have consistently shown a lack of staining for succinic dehydrogenase and a-glycerophosphate dekiydrogena~e.~,'~~'~'~~~ Alternatively, experimental induction of TAs in rat skeletal muscleZ5has indicated that they are derived from the sarcoplasmicreticulum and not from mitochondria or T-tubules. Furthermore, lanthanum staining of the T-system'8i26 failed to show any connectionwith theTAs, whichdidnotstain.Al1 this has led to the assumption that TAs are a proliferation of the sarcoplasmicreticulum, a view supported by the present study. As the internal microtubular structures are not a normal feature of the sarcoplasmic reticulum, their origin and presence in TAs is less well understood. Despite the variability in diameter, they closely resemble the microtubules normally present in the cytoplasm of most cells and could therefore impart the same function within TAs by permitting the rapid transport of electrolytes. However, this seems highly unlikely and unnecessary in such an open
TUBULAR AGGREGATE MYOPATHY
tubular network. More likely, the internal microtubular structures could represent a polymerization of the amorphous material seen within type I1 structures and would appear to be analogous to the intracisternal tubular aggregates occasionally associated with amelanotic melanomas and soft tissue t u r n o ~ r s . ~ ~ , ~ ~ The overall significance of TAs is therefore unclear. It has been proposed that in some instances they ma be a hypertrophic response to drugs and Y or to enhance the uptake and transfer of toxins,4> ca1ci~rn.l~ Recently, there has been some immunohistochemical evidence to suggest the involvement of heat shock protein in the formation of TAs.’~ Whatever their cause, the occurrence of TAs as the main finding in the muscle of patients with slowly progressive weakness can be regarded as a distinct clinico-pathologicalentity of genetic origin. ACKNOWLEDGEMENTS
We are grateful to Mr Tom McLoughlin and Mr John Gilliland for technical assistance. REFERENCES 1. Gruner JE. Anomalies due reticulum sarcoplasmique et proliferation de tubules dans le muscle d’une paralysie peridique familiale. C R Soc
Biol Paris 1966; 160: 193-195. 2. MacDonald RD, Rewcastle NB, Humphrey JG. The myopathy of hyperkalemic periodic paralysis. An electron microscopic study. Arch Neurol1968; 1 9 274-283. 3 . Bradley MG. Ultrastructural changes in adnyamica episodica heriditaria and normokalaemic familial periodic paralysis. Bruin 1969;92: 379-390. 4. Engel WK, BishopDW, Cunningham GG. Tubularaggregatesin type I1 muscle fibres: ultrastructural and histochemical correlation. J Ultrastruct Res 1970; 31: 507-525. 5. Meyers KR, Gilden DH, Rinaldi CF, Hansen JL. Periodic muscle weakness, normokalemia, and tubular aggregates. Neurology 1972; 22: 269-219. 6. Dobkin BH, Verity MA. Familial neuromusclar disease with type I fiber hypoplasia, tubular aggregates, cardiomyopathy, and myasthenaicfeatures. Neurology 1978;2 8 1135-1 140. 7 . Hurwitz LJ, Carson NAJ, Allen IV, Fannin TF, Lyttle JA, Neil DW. Clinical, biochemical and histopathological findings in a family with muscular dystrophy. Bruin 1967;40:799-816.
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8. Sipila I, Simell 0,Rapola J, Sainio K, Leena T. Gyrate atrophy of the choroid and retina with hypernithinema: tubular aggregates and type 2 fiber atrophy in muscle. Neurology 1979;2 9 9961005. 9 , Rosenberg NL, Neville NE, Ringel SP. Tubular aggregates. Their association with neuromuscular diseases, including the syndrome of myalgia/cramps. Arch Neurol1985; 42: 973-976. 10 Grunnet ML, Abeles M, Hofbauer H. Polymyositis with tubular aggregates. J Rheumutol1988; 15: 1288-1290. I 1 Schroder JM, Becker PE. Anomalien des T-systems und des sarhoplasmatischen reticulums bei der myotonic, paramyotonic und adynamie. Virchows Arch 1972; 375: 31lL344. 12. Chui LA, Neustein H, Munstat TL. Tubular aggregates in subclinical alcoholic rnyopathy. Neurology 1975; 25: 4 0 5 4 1 2. 13. del Villar Negro A, Angulo JM, Pomar JMR, Errasti CA. Tubular aggregates in skeletal muscle of chronic alcoholic patients. Acta Neuropathol Berlin 1982;56: 25lL254. 14. Doriguzzi C, Monngini T, Jeantet A, Monga G. Tubular aggregatesin a case of osteomalacia myopathy due to anticonvulsant drugs. Clin Neuropathol1984; 34 2 4 5 . 15. Lazaro RP, Fenichel GM, Kilroy AW, Siato, Fleischer S. Cramps, muscle pain and tubular aggregates. Arch NeurolI980 37: 71 5-717. 16. Niakan E, Harati Y, Danon MJ. Tubular aggregates: their association with myalgia. J Neurol Neurosurg Psychiatry 1985; 48:882-886. 11. Rohkamm R, Boxler K, Ricker K, Jerusalem F. A dominantly inherited myopathy with excessive tubular aggregates. Neurology 1983; 33:331-336. 18. Pierobon-Bormioli S , Arani M, Ringel SP, el ul. Familial neuromuscular disease with tubular aggregates. Muscle Nerve 1985;8 29 1-298. 19. Salviati G, Pierobon-Bormioli S, Betto R, el al. Tubular aggregates: sarcoplasmic reticulum origin, calcium storage ability, and functional implications. Muscle Nerve 1985;8 299-306. 20. Bergman RA, Johns RJ, Afifi A. Ultrastructural alterations in muscle from patients with myasthenia gravis and Eaton-Lambert syndrome. Ann NYAcadSci 1971;18388-120. 21. Mastaglia FL, Walton J. Skeletal Muscle Pathology. Vol. 26. New York: Churchill Livingstone, 1982: 81-86. 22. Morgan-Hughes JA, Lecky BR, Landon DN, Murray NM. Alterations in the number of affinity of junctional acetylcholine receptors in a myopathy with tubular aggregates. A newly recognized receptor defect. Brain 1981; 104: 279-295. 23. LewisPD, Pallis C, Pearse AGE. ‘Myopathy’ with tubular aggregates. JNeurolSci 1971; 13381-388. 24. Meijer AEFH. Histochemical features of tubular aggregates in diseased human skeletal muscle fibres. J Neurol Sei 1988;86: 73-82. 25 Schiaffino S , Severin E, Cantini M, Sartore S. Tubular aggregates induced by anoxia in isolated rat skeletal muscle. Lab Invest 1977; 37: 223-228. 26 Takizawa D, Ishihara T, Shinohara Y. A case of hypokalaemic periodic paralysis with tubular aggregates in type 2A fibres and type 2B fibres. Clin Neurol1986; 2 6 81-86. 27 Bockus D, Remington F, Friedman S, Hammar S. Electron microscopy What Izdts. Ultrastruct Patholl985; 9: 1-30. 28 Cameron CHS, Kenny BD, Clements WDB, Toner PG. Unusual extraskeletal myxoid chondrosarcoma. Ultrustruct Pathol1992; 161: 239-244. 29 Martin JE, Mather K, Swash M, Gray A. Expression of heat shock protein epitopes in tubular aggregates. Muscle Nerve 1991; 14: 219-225.
JOURNAL OF PATHOLOGY, VOL.
DOMINANTLY INHERITED TUBULAR AGGREGATE MYOPATHY c. H.
STUART CAMERON*, INGR~Dv.
ALL EN^, VICTOR PATTERSON$ AND
MARIA A. A V A R I A ~
Neuropathologyf and Electron Microscopy* Laboratories, School of Clinical Medicine, Queens University and The Muscle Clinic$, City Hospital, Belfast, Northern Ireland, U.K. Received I5 April 1992 Accepted 22 June 1992
SUMMARY We report an unusual familial myopathy characterized morphologically by the presence of large tubular aggregates in all fibre types. Two patients, a father and daughter, presented with slowly progressive proximal weakness, limitation of eye movement, and Achilles tendon contractures. Serum creatine kinase was 5-10 times normal. Light microscopy revealed type I fibre predominance. Basophilic accumulations, which stained intensely with the NADH-TR reaction, were present in both fibre types. Electron microscopy revealed that these consisted of tightly packed parallel tubular arrays. These varied somewhat in their ultrastructural appearance and were classified accordingly as type I, 11, and I11 tubular structures. The tubular aggregates appear to be derived from the sarcoplasmic reticulum. This report further supports the evidence of a distinct clinico-pathological entity of genetic origin. KEY WORDS-Familial
myopathy, proximal weakness, tubular aggregates, electron microscopy.
INTRODUCTION
climbing stairs. Since early childhood he had walked on his toes and had difficulty running and keeping Tubular aggregates (TAs) in muscle have been up with his peers, although he was able to play rugby reported in two situations. In the first, they occur as at 13. He had been diagnosed as having infectious a minor feature in well-establishedmuscle disease of hepatitis at the age of 19 and because his transoften known cause such as in periodic paralysis,'" aminases remained raised, a liver biopsy was myasthenia gravis? facioscapulohumeraldystrophy performed which revealed no abnormality. with aminoaciduria,'.' inflammatorymyopathie~?'~ He had previously been investigated because of myotonia," and alcohol and drug into~ication.'~-'~his weakness at 31 years of age. He had difficulty In the second situation, they are the major patho- rising from a lying position and was found to have logical feature. The best known occurs in some MRC grade 3 weakness of his scapular and pelvic patients with exercise-induced pain and stiffness in girdle muscles with lesser involvement of his ankle which TAs are found in type I1 muscle fibre^.^,'^''^ dorsiflexors. He gave a history of heavy alcohol In contrast, two families have been reported in intake for several years, having stopped 9 months which progressive muscle weakness was associated prior to consultation. A muscle biopsy (formalinwith widespread TAs in both types of muscle fixed) from his quadriceps was obtained at that time fibres."-I9 We have encountered a third family and a diagnosis of limb-girdle muscular dystrophy with this condition and suggest that it represents a was made. distinct clinico-pathologicalentity. At the age of 39 years, he underwent lengthening of his Achilles tendons. His parents and his brother CASE REPORTS and sister had no complaints of weakness or other neuromuscular symptoms. There was no history Case 1 This patient was seen at age44yearswithprogress- of muscle pain. Of his two daughters, one was ive weakness which was disablingfrom the age of 27, asymptomatic while the other is reported as case 2. His condition continued to deteriorate slowly, although from the age of 20 he had some difficulty in with increasing difficulty going upstairs and rising *Address for correspondence. from a chair. He developed a tendency to fall, 0022-341 7/92/I 20397-07 $08.50 0 1992 by John Wiley & Sons, Ltd.
398
C. H.S. CAMERON ETAL.
finding it very difficult to get up by himself and was referred for reassessment at 44 years. He had increased lordosis and walked with a waddling gait. Bilateral restriction of eye adduction and upward gaze was found. There was no facial weakness. Neck flexors were MRC grade 3; biceps and triceps grade 4.In his lower limbs, hip flexors and knee extensors were grade 3 and ankle dorsiflexors grade 5 . No loss of sensation or coordination was found and he had no muscle stiffness or cramps. Tendon reflexes were preserved and there was no sensory loss. Intravenous edrophonium chloride produced no improvement in strength. A muscle biopsy was taken from the left biceps. During a 6-year follow-up, he has developed some increase in weakness (biceps has gone from MRC grade 4 to 4 - and knee extensors from grade 3 to 3-) and intermittent features of a depressive illness for which he has been treated with tricyclic antidepressants.A therapeutic trial with Dantrolene had to be discontinued without benefit at a dose of 50 mg daily because of drowsiness. Case 2 This patient, the daughter of case 1, had been examined for the first time at 7 years of age, because of her father’s progressive weakness and was considered at that time to be normal. Her parents thought she was clumsy and she had difficulty in hopping and playing games. During the next 2 years she developed progressive difficulty in rising from the floor and climbing stairs and had pain in her limbs after exercise. Physical examination at that time revealed weakness which spared the facial muscles but affected the proximal muscles of both upper and lower limbs. She had increased lordosis and a waddling gait. Deep tendon jerks were normal. A muscle biopsy was performed on her left biceps. She was seen again at the age of 16 years, when she showed mild restriction of upward gaze; neck flexors were MRC grade 3 - , biceps and hip flexors grade 4 - .A needle biopsy from her quadriceps was performed. Shehas continued to show a very slow progression of muscle weakness with intermittent features of depression.Hip flexor strength has gone from MRC grade 4- to MRC grade 3 over 7 years. Special investigations Serum creatine kinase (CK) was elevated in both patients; lSSOp/l in case 1 and 700p/1 in case 2 (normal 30-140 p/l).
Electromyogramswere performed on the deltoid, biceps and quadriceps of both patients and no spontaneous activity was recorded. In case 1, motor unit potentials (MUPs) were found to be of normal amplitude and duration but with some increase in polyphasic activity. Nerve conduction velocity in the median nerve was 60m/s for both motor and sensory components. Case 2 showed a predominantly myopathic pattern on electromyographyand nerve conduction was within normal limits. MATERIAL AND METHODS In case 1, an open biopsy was performed at 44 years on the left biceps and muscle was obtained for light and electron microscopy. Tissue was snap-frozen and several cryostat sections were stained by an appropriate battery of histological and histochemical techniques. Fibre quantitation studies were undertaken on ATPasestained sections using a Leitz TAS Plus image analysis system. For electron microscopy, the muscle was fixed in 3 per cent phosphate-buffered glutaraldehyde (pH 7.2) post-osmicated and embedded in Polarbed 812 epoxy resin. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined using a Hitachi H-600electron microscope. In case 2, a needle biopsy was performed at 16 years on the left quadriceps and processed entirely for electron microscopy. The previous biopsies from both patients taken at 3 1 and 8 years, respectively,which had been fixed in formalin and glutaraldehyde for routine histology and electron microscopy, were also reviewed. RESULTS Light microscopy On routine cryostat sectioning, there was a marked variation in the range of fibre diameters involving both fibre types. There was a predominance of type I fibres, atrophy of both fibre types (type I type 11). Thirty-eight per cent of all fibres contained central nuclei and there was some fibre splitting. Basophilic areas were apparent within many fibres, particularly the larger ones. These areas stained intensely with the NADH-TR reaction (Fig. l), bright red with Gomori’s trichrome stain and were occasionally PAS positive.
TUBULAR AGGREGATE MYOPATHY
399
400 nm in diameter and were commonly found in subsarcolemmalaggregates. Type 111: Dilated parallel tubules mostly between 130 and 400nm in diameter, but each containing a number of smaller, 25-40 nm diameter, microtubule-like structures (Fig. 5). Central nuclei were frequent and usually associated with the TAs. Away from the large aggregates, many of the smaller tubular arrays appeared to arise as proliferations of the sarcoplasmicreticulum (Fig. 6). Mitochondria were generally unaffected and did not appear to be involved in the production of the honeycomb structures. Particulate glycogen was diffusely scattered between the tightly packed tubules. Lipid droplets were uncommon, whereas lipofuscin granules were occasionally encountered within some of the large TAs. Endomysial collagen was not increased. The previous muscle biopsy from both patients Fig. 1-A cryostat section showing strong NADH-TR staining was reviewed. Large TAs were predominant in all of the tubular aggregates fibres in case 1, whereas in case 2 they were much smaller, measuring betwen 1 and 12pm on their With the ATPase reaction, they appeared as pale longest dimension and less frequent than those in the present biopsy. Only type I1 tubular structures unstained areas in both fibre types. were present and these were also the most prevalent On review, similar abnormalities could be identstructures in case 1. Type I11 tubular structures were ified in the previous paraffin-embedded biopsy from not identified in either of these biopsies. case 1, while the histology in case 2 showed a relatively normal appearance with occasional central nuclei. DISCUSSION Involvement of the current cases with any of the other disorders related to TAs in skeletalmuscle was dismissed on the basis of their clinical and microscopical findings. The distribution of weakness with no facial involvement was not consistent with that seen in facioscapular humeral dystrophy. There was no evidence of periodic paralysis or myotonia. No drugs were involved at the time of biopsy. The possibility of an alcoholic myopathy in case 1 as the source of the TAs was unlikely as his daughter had similar features. Both patients presented with a slowly progressive Closely packed parallel tubules approxi- weakness affecting their proximal muscles but mately 70 nm in diameter. Each tubule sparing the face. Weakness became evident at about contained a smaller 40nm diameter 20 and 9 years, respectively, although with case 1 central tubule, or a microtubule-like there is evidence of early impairment during his school years, probably related to ankle contractures. structure (Fig. 3). Tubular structures containing a moder- In the two previous reports of this ~ondition,””~ ately dense, flocculent material instead onset was between 10 and 40 years. Subtle involveof a central tubule (Fig. 4). They were ment of the extra occular muscles was present in often dilated, measuringbetween 70 and both of our cases but has not been described in the
Electron microscopy Muscle from both the father and daughter had a similar ultrastructural appearance. Tubular aggregates were present in all muscle fibres. They were located both within the sacroplasm and adjacent to the sarcolemma. The aggregates measured from < 1 ,um to >7O,um in diameter (Fig. 2) and consisted of parallel tubular arrays (honeycomb-like structures). These varied somewhat in their ultrastructural appearance and were therefore classified accordingly:
Type I:
Type 11:
400
C. H.S. CAMERON ET AL.
Fig. 2-An
array of closely packed type I tubular structures. The bar equals 0.5 pm
Fig. 3-Type I tubular structures (arrow-heads) are seen in transverse section to contain an inner tubule (arrow). The bar equals 50 nm
Fig. &Type I1 tubular structures, most of which are dilated, contain a moderately dense flocculent material. The bar equals 250 nm
other two families. Another peculiar aspect not previously described was the presence of contractures, which were severe in case 1. By contrast, facial weakness does not appear to be a common feature,
having been described in only 1 of the 12 patients reported to date. Muscle pain does not appear to be uniformly associated with tubular aggregate myopathy. In the
TUBULAR AGGREGATE MYOPATHY
Fig. 5-A portion of a tubular aggregate consisting mostly of type I11 tubular structures. Several of the dilated tubules (arrows) are seen to contain a number of smaller tubules (inset). The bars equal 1 pm and 125 nm (inset)
Fig. &A small clusterof tubular structures is in contiguity with the sarcoplasmicreticulum (arrow).The bar equals 300 nm
40 1
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C. H. S. CAMERON ET AL.
family described by Rohkamm et al.,I7 the main clinical features were of proximal weakness without wasting or pain. In the report by Rerobon-Bormioli et UZ.,'~ pain was the prominent symptom in four out of the five cases. While there is no history of muscle pain, cramps, or stiffness in case 1, case 2 had mild symptomology with some pain in her limbs following exercise. It is therefore suggested by these findings that muscle pain is not an essential feature of this condition, although it is a frequent symptom. In addition, elevated CK appears to be a similarly variable finding in the three families reported to date. Electromyographicfindingsin previously reported patients have ranged from normal to a slightly myopathic pattern with brief small action potentials (BSAPs). In some patients, long duration MUPs of 12-24 m/s were recorded. Case 1 had MUPs of normal amplitude and duration, but increased polyphasity and less than complete interference pattern on full effort, suggesting a neurogenic disorder. The occurrenceof positivesharp waves in a muscle has been reported in only one patientI7 and nerve conductionvelocities,when reported, have beennormal. Neuromuscular transmission has been tested either electrophysiologically or pharmacologically, as in our case 1, with no evidence of involvement. The biopsy findings in both of our patients are very similar to those reported by Rohkamm et ~ 1 . and Pierobon-Bormioliet all8 All have shown subsarcolemmal or centrally placed basophilic accumulations in haematoxylin and eosin sections, which stain bright red with Gomori's trichrome and dark blue/black with NADH-TR reaction. They do not react with ATPase reactions. Such structures have been found in most fibres, ranging from 50 per cent to almost 100 per cent of the fibres examined, being apparently more numerous in older patients. As with the present study, TAs are not restricted to type I1 fibres as with a variety of other disorders, but involve both fibre types. Features of degeneration or inflammation were not seen in Rohkamm et al.3 family,'7 but internal nuclei, fibre splitting and variation in fibre size were found in varying degrees in both of the previous studies. In case 1, there were some features of degeneration with slight mononuclear cell infiltration and a moderate increase in connective tissue. In common with the earlier studies, there was a marked variation in fibre size and central nuclei were numerous. Type I predominance with atrophy more marked in type I1 fibres and hypertrophy of both fibre types were present. Type I predominance
has been a feature in five out of seven biopsies reported p r e v i ~ u s l y . ' ~ There ~ ' ~ were no clear differences on clinical or laboratory grounds between patients with either fibre type predominance; therefore the significanceof this remains uncertain. Type I predominance was also found in the patients with myasthenic features and TAs.~,~' These patients also showed TAs in both fibre types, a feature not previously reported and shared by all the patients with the autosomal dominantly inherited myopathy. Tubular aggregates have often been described as 'double-walled t ~ b u l e s ' . ~ ~However, ' ~ , ' ~ ~ ~ our ' study together with those of En el et Meyers et a1.,5 and Morgan-Hughes et al!shows that theTAs have a more variable appearance consisting of an outer tubule, or vesicle, containing either amorphous material or one or more microtubule-likestructures. More commonly, they are best described as a tubule within a tubule. We have attempted to categorize the TAs into type I, I1 and I11 structures depending on their morphological appearances. These are somewhat similar to the various forms described by Morgan-Hughes et u I . , ~ who ~ together with Engel et al.? Dobkin and Verity6and Pierobon-Bormioli et aZ.'* have shown that the TAs are in contiguity with various aspects of the sacroplasmicreticulum. The previous suggestion of a mitochondria1 ' ~origin, based on the localization of certain mitochondrial e m me^,^^ has not been supported by other studies? TAs have not exhibited any structural association with mitochondria, which appear largely unaffected, and have consistently shown a lack of staining for succinic dehydrogenase and a-glycerophosphate dekiydrogena~e.~,'~~'~'~~~ Alternatively, experimental induction of TAs in rat skeletal muscleZ5has indicated that they are derived from the sarcoplasmicreticulum and not from mitochondria or T-tubules. Furthermore, lanthanum staining of the T-system'8i26 failed to show any connectionwith theTAs, whichdidnotstain.Al1 this has led to the assumption that TAs are a proliferation of the sarcoplasmicreticulum, a view supported by the present study. As the internal microtubular structures are not a normal feature of the sarcoplasmic reticulum, their origin and presence in TAs is less well understood. Despite the variability in diameter, they closely resemble the microtubules normally present in the cytoplasm of most cells and could therefore impart the same function within TAs by permitting the rapid transport of electrolytes. However, this seems highly unlikely and unnecessary in such an open
TUBULAR AGGREGATE MYOPATHY
tubular network. More likely, the internal microtubular structures could represent a polymerization of the amorphous material seen within type I1 structures and would appear to be analogous to the intracisternal tubular aggregates occasionally associated with amelanotic melanomas and soft tissue t u r n o ~ r s . ~ ~ , ~ ~ The overall significance of TAs is therefore unclear. It has been proposed that in some instances they ma be a hypertrophic response to drugs and Y or to enhance the uptake and transfer of toxins,4> ca1ci~rn.l~ Recently, there has been some immunohistochemical evidence to suggest the involvement of heat shock protein in the formation of TAs.’~ Whatever their cause, the occurrence of TAs as the main finding in the muscle of patients with slowly progressive weakness can be regarded as a distinct clinico-pathologicalentity of genetic origin. ACKNOWLEDGEMENTS
We are grateful to Mr Tom McLoughlin and Mr John Gilliland for technical assistance. REFERENCES 1. Gruner JE. Anomalies due reticulum sarcoplasmique et proliferation de tubules dans le muscle d’une paralysie peridique familiale. C R Soc
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