Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 58, Num. 5, 2010, pp. 747-751

Neurology India, Vol. 58, No. 5, September-October, 2010, pp. 747-751

Brief Report

Tubular aggregate myopathy: A phenotypic spectrum and morphological study

Amrita Ghosh¹, Gayathri Narayanappa¹, Arun B Taly², Yasha T Chickbasavaiya¹, Anita Mahadevan¹, Santosh Vani¹, Nalini Atchayaram², Ishani Mohapatra¹, Krishna Shankar Susarala¹

¹ Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, India
² Department of Neurology, National Institute of Mental Health and Neurosciences, Bangalore, India

Correspondence Address:
Gayathri Narayanappa
Department of Neuropathology, National Institute of Mental Health and Neurosciences, Hosur Road, Bangalore - 560 029
India
gayathrin12@rediffmail.com

Date of Acceptance: 08-Jul-2010

Code Number: ni10204

PMID: 21045502

DOI: 10.4103/0028-3886.72166

Abstract

Keywords: Cramps, familial dementia, limb girdle myasthenia, lower motor neuron syndrome, myalgia, noncompressive myelopathy, periodic paralysis, tubular aggregates, ultrastructure

Introduction

Tubular aggregates (TAs), inclusions in skeletal muscle derived from sarcoplasmic reticulum, are ultrastructurally characterized by accumulation of densely packed membranous tubules. They are uncommon but an important indicator of myopathies. Initially described by Engel ^[1] in a case of hypokalemic periodic paralysis and myotonia congenita, TAs are presently known to occur in a wide variety of conditions, ^{[2],[3],[4],[5]} and in male inbred mice during aging ^[6] and can be induced experimentally. ^[7] However, in several cases, TAs constitute the cardinal structural pathology indicating myopathy with TAs as a distinct entity. ^[8] In the present study, 18 cases with TAs seen over a period of 10 years were reviewed to determine the spectrum of clinical phenotypes associated with these distinctive inclusions and morphological types of TAs encountered. An attempt was made to correlate clinical phenotypes, duration of illness and ultrastructural types of TAs.

Materials and Methods

This is a retrospective analysis of 18 cases with TAs diagnosed during the period from January 2001 to April 2009. Case records were reviewed for clinical features, including onset of illness, pattern and distribution of weakness, presence of myalgia, cramps, myotonia; consanguinity; and family history. Biochemical, electrophysiological and radiological findings were also noted. Skeletal muscle biopsy was carried out with informed consent [biceps (n=13), quadriceps (n=5)]. Fresh frozen sections were stained for hematoxylin and eosin (H and E), modified Gomori trichrome (MGT) and enzyme stains [succinic dehydrogenase (SDH), succinic dehydrogenase with cytochrome c oxidase (SDH-COX), nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), menadione- α glycerophosphate dehydrogenase (MAG), menadione- nitro BT (M-NBT), adenosine triphosphatase (ATPase) pH 9.4 and 4.6, acid phosphatase, myophosphorylase and periodic acid Schiff's (PAS)]. Tiny bits of muscle fixed in 3% buffered gluteraldehyde were processed for electron microscopy. Ultrathin sections contrasted with uranyl acetate and lead citrate were observed under FEI Tecnai electron microscope. Immunoperoxidase staining was carried out using monoclonal antibodies against ubiquitin, tau and S-100 in all the cases.

Results

Clinical

Of the 7,441 muscle biopsies received (January 2001 to April 2009), 18 (0.24%) cases were diagnosed as having TAs based on morphological findings. The salient clinical features are provided in [Table - 1]. Eighteen patients (15 men and 3 women) aged between 2 and 55 years, had onset of symptoms between1.5 and 50 years of age and duration of illness ranging from 6 months to 19 years. Weakness was the presenting symptom in the majority (n=15) of patients; while in three patients, myoclonus (n= 1) and stroke-like episode (n=2) were noted. Weakness was episodic in six,, progressive in another six and associated with hypotonia in two patients. An adult patient diagnosed to have noncompressive myelopathy of unknown etiology presented with tightness and weakness. Myalgia and cramps were present in two patients, while myalgia or cramps in isolation were noted in two patients each. Mental subnormality was present in three patients. There was history of seizure in three patients, one of whom was on antiepileptic drugs for 20 years. Consanguinity was recorded in four patients and a family history of similar illness in five instances.

Examination revealed lower limb weakness (n=7), bilateral symmetrical weakness of upper and lower limbs (n=4), wasting (n=3), waddling gait (n=4), impaired tandem gait (n=1) and normal in the rest. Ptosis was present in two patients with congenital myopathy; and in one patient, with myasthenia. There was no evidence of myotonia either clinically or electrophysiologically in any of the patients. Radiological investigations [computed tomography (CT) or magnetic resonance imaging (MRI)] carried out in 11 patients revealed cerebral atrophy (n=2), deep white matter hyperintensities with cystic component (n=1), bilateral middle cerebellar artery MCA infarct (n=1) and were normal in the rest. Thymic hyperplasia was noted in one patient with myasthenia. Electro myography (EMG) was myopathic (n=4), neurogenic (n=1) or normal (n=3). Serum creatine kinase (CK) values ranged from 187 to 11,012 IU/L (normal range, 20-170 IU/L), with highest level recorded in a case of polymyositis (11,012 IU/L). Serum potassium was low in two cases and elevated in one patients. Serum sodium was within normal range in all the patients.

On the basis of clinical, electrophysiological and biochemical findings, the clinical diagnoses were: muscle cramp disease (n=2), congenital myopathy (n=3), dyskalemia [hypokalemic (n=2), hyperkalemic (n=1)], limb girdle myasthenia (n=2), recurrent stroke (n=2), polymyositis (n=1), metabolic myopathy (n=1), lower motor neuron syndrome (n=1), noncompressive myelopathy of unknown etiology (n=1), familial autosomal dominant dementia with myoclonus (n=1) and mitochondrial cytopathy (n=1).

Morphology

Light microscopy

Haematoxylin-eosin (H and E) sections revealed polygonal fibers with mild variation in diameter and basophilic granular material in subsarcolemmal region [Figure - 1]a in all the biopsies. The percentage of fibers involved ranged from 2% to 80% [Table - 2]. The material appeared bright red on MGT [Figure - 1]b, stained intensely with NADH-TR [Figure - 1]c, and remained unstained on SDH [Figure - 1]d, MAG and ATPase reaction. In addition, type II fiber predominance (n=5), type II fiber grouping (n=1) and COX-deficient fibers (n=2) were noted. The other histological features included presence of atrophic fibers with clumped nuclei (n=1), myophagocytosis (n= 3), regenerating fibers and perivascular inflammation with transmural lymphocytic infiltration (n=1). The deposits were seen in type-II fibers in all the biopsies and also in type-I fibers in nine biopsies.. Immuostaining to monoclonal antibodies against ubiquitin showed subsarcolemmal positive labeling in ten biopsies, while antibodies against S-100 and tau did not label the TAs.

Electron microscopy

Ultrastructural studies in 17 patients revealed large aggregates of tubules predominantly in the subsarcolemmal regions and to a lesser extent in the intermyofibrillar zones. The following morphologic types of tubular aggregates were encountered in decreasing order of frequency: (1) Vesicular membrane collections (VMC) (n=15): Clusters of closely set round-to-oval dilated vesicular structures bound by single membrane containing moderately dense flocculent material. The diameter of these vesicular structures varied greatly (60-300 nm) within the cluster [Figure - 2]a; (2) Single-walled tubules (SWT) (n=10): Bundles of parallel dilated single-walled tubules traversing in different directions without any particular orientation to the long axis of the myofibers. These tubules were placed in regular hexagonal arrangement, well defined in both longitudinal and transverse plane. The diameter (60 nm) was fairly constant and uniform throughout the length. The individual tubules were either empty or contained pale to moderately dense granular or flocculent material [Figure - 2]b; (3) Double-walled tubules (DWT) (n=7): Large assemblies of tightly packed long membranous double-walled straight tubules arranged in parallel bundles, which on transverse plane appeared as closely packed hexagonal arrays. The outer tubule had a diameter of 60 nm, with a single smaller inner tubule of diameter 30 nm separated by a clear space. The inner core either contained electron-dense material or was empty [Figure - 2]c; (4) Dilated tubules with inner tubules (DTT) (n=2): These had much wider and shorter tubules containing numerous smaller inner tubules regularly spaced, giving a sieve-like appearance [Figure - 2]d.

The types of TAs present in various disease entities and the frequency of occurrence are presented in [Table - 2]. All four types of TAs were present in one case of congenital myopathy. One case (Case 17), in addition, showed central crisscross arrangement of thin filaments embedded in granular material. These areas on MGT appeared greenish and showed intense reaction to NADH-TR. The nature of these filaments is unclear. There was no correlation between morphologic type of TAs, duration of illness and clinical phenotype.

Discussion

TAs, a distinctive histochemical feature, have been the major, if not the sole, pathological abnormality in skeletal muscle of patients, with four rare but distinctive clinical conditions, which occur either sporadically or may show evidence of genetic transmission, referred to as myopathy with TAs. Several families with autosomal dominant and autosomal recessive inheritance have been described in Caucasian population. ^[9],[10] The four major clinical phenotypes primarily associated with TAs are: (i) weakness with exercise-induced cramps, pain and stiffness; (ii) isolated slowly progressive proximal muscles weakness; (iii) limb girdle myasthenia (LGM); (iv) gyrate atrophy of retina and choroids.

In the present series, TAs were noted in the commonly described disease entities, in addition to their presence in the following unusual clinical phenotype: familial dementia with myoclonus, lower motor neuron syndrome, noncompressive myelopathy of unknown etiology and suspected case of mitochondrial cytopathy, thus expanding the spectrum of clinical conditions with TAs. This study describes a large series from south India. Jain et al.,^[11] from north India, describe four patients (muscle pain and cramps, secondary to alcoholism and /or hyperparathyroidism and pituitary adenoma) with TAs and ultrastructure evidence of only DWT.

The common morphological abnormality seen in all these diverse neuromuscular disorders was the presence of tubular aggregates reacting intensely to NADH-Tr predominantly in type-II fibers. In addition, its presence in type-I fibers was seen in nine patients. It has been reported that in periodic paralysis and the disorder characterized by muscle cramps and stiffness, the changes are confined to type-II fibers; while in inherited myopathies, they occur in both fiber types. ^[2],[12] However, in our series, TAs were observed in both fiber types in familial (familial myopathy with tubular aggregates and familial LGM) and nonfamilial forms (motor neuron syndrome, recurrent ischemic stroke, noncompressive myelopathy of unknown etiology and 1 case of muscle cramp disease).

Ultrastructurally, the various morphological forms were classified as described previously. ^[13] All four morphological forms (VMC, SWT, DWT and DDTA) were encountered. However, a definite pattern of association between clinical phenotypes, duration of illness and type of TAs could not be elucidated. Coexistence of tubular aggregates and tubulofilamentous inclusions of inclusion body myositis (IBM) type in a case of congenital myopathy with progressive proximal muscle weakness, lumbar hyperlordosis and bilateral Achilles tendon contractures has been reported. ^[14] Mass of intermediate filaments organized in a crisscross pattern was noted in the center of the fiber in one of our patients who presented with tightness and weakness of lower limb (Case 17). The filaments were negative for desmin, ubiquitin, tau and S-100. The nature of these filaments and its association with disease process are unclear.

The occurrence of TAs in such diverse and unrelated conditions has made it difficult to evaluate and predict their functional significance and identify the factors involved in their evolution. On the other hand, remarkable consistency of their ultrastructural features suggests their origin from a single subcellular organelle. In their study of skinned muscle fibers from affected members of a family with a dominantly inherited tubular aggregate myopathy, Salviati et al.^[15] demonstrated presence of TAs potentiated the calcium-loading capacity of slow muscle fibers, and hypothesized that TAs are functional equivalents to the hypertrophied terminal cisterns of the sarcoplasmic reticulum; while rearrangement of preexisting sarcoplasmic reticulum (SR) or neo-formation from the SR due to various insults to skeletal muscle fiber as an adaptive response to increased calcium influx was suggested by Chevessier et al.^[13] There is prevailing and widely accepted evidence for the origin of TAs from terminal cisterns of SR, ^[13],[16] as seen ultrastructurally in our cases. Presence of SR proteins like RyR, triadin, calsequestrin, sarcalumenin and sarcoplasmic/ endoplasmic calcium ATPase (SERCA1, 2) necessary for storage and release of calcium by the SR; and dihydropyridine receptor (DHPR), which is specifically located in the T-tubule, suggests that TAs represent a tubular arrangement of all endoplasmic and sarcoplasmic reticulum proteins and are functionally competent. ^[13] Up-regulation of glucose-regulated proteins (GRPs), necessary for tertiary folding of proteins and epitopes of heat shock proteins found in the area of TAs, suggests their role in modulation of the tertiary structure of proteins. ^[17]

The pathophysiological process that is widely accepted is a disturbance in sarcoplasmic calcium homeostasis. TAs identical to those seen in human diseases are noted in congenic mice (MRL +/+ sub-strain), ^[18] which are age related and gender specific. Castration of male mutants early in life, before TAs have developed, completely abolishes their occurrence, suggesting hormonal and gonadal influences, in addition to genetic factors involved in pathogenesis.

Acknowledgment

References

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