Abnormalities in the expression of nebulin in chromosome-2 linked nemaline myopathy

Nemaline myopathy is clinically and genetically heterogeneous. The most common autosomal recessive form affecting infants (NEM2) links to chromosome 2q, and is caused by mutations in the gene for nebulin. We have examined the immunocytochemical expression of nebulin in skeletal muscle in 11 cases of nemaline myopathy, from ten families, with linkage compatible to chromosome 2q.22, the locus for nebulin. Mutations in the gene for nebulin have been found in eight of these cases. Immunolabelling with polyclonal antibodies to C-terminal regions of nebulin was compared with antibodies to fibre-type-specific myofibrillar proteins, including myosin heavy chain isoforms and alpha-actinin isoforms. No cases showed a complete absence of C-terminal nebulin, and no enhancement of labelling of the rods was seen with conventional fluorescence microscopy. In control muscle an antibody to the M176-181 repeat region of nebulin showed higher expression in fibres with slow myosin, while ones to the serine-rich domain and to the SH3 domain showed uniform expression. In some cases of nemaline myopathy differences in these patterns were observed. Two siblings with a homozygous mutation in exon 185, that produces a stop codon, showed an absence of labelling only with the SH3 antibody, and other cases showed uneven labelling with this antibody or some fibres devoid of label. Fibre type correlations also showed differences from controls, as some fibres had a fast isoform of one protein but a slow isoform of another. These results indicate that analysis of nebulin expression may detect abnormalities in some cases linked to the corresponding locus and may help to direct molecular analysis. In addition, they may also be relevant to studies of fibre type plasticity and diversity in nemaline myopathy.     

Mitochondrial changes in acute myopathy after treatment of respiratory failure with mechanical ventilation (acute relaxant-steroid myopathy)

A case of acute myopathy was observed in the course of treatment of respiratory failure with mechanical ventilation combined with prolonged neuromuscular blockade and administration of corticosteroids. A muscle biopsy revealed degeneration of muscle fibres. Electron microscopy showed loss of thick filaments as well as nemaline rods, vacuoles and cytoplasmic bodies. The mitochondria were increased in number, many harbouring paracrystalline inclusions, which were hitherto unknown in this condition.     

Idiopathic adult‐onset nemaline myopathy presenting with isolated respiratory failure

Idiopathic adult‐onset nemaline myopathy is a rare condition of unknown etiology that usually presents with proximal weakness. This case study reports a 60‐year‐old woman who presented with isolated type 2 respiratory failure secondary to bilateral hemidiaphragm weakness. A left vastus medialis muscle biopsy examined under light microscopy revealed appearances typical of nemaline myopathy. Electron microscopy confirmed the presence of nemaline rods in most muscle fibers, thus establishing idiopathic adult‐onset nemaline myopathy as the cause of her respiratory failure. Our patient's presentation highlights the importance of considering neuromuscular weakness as a cause of respiratory failure. Unless appropriate tests are performed—including a muscle biopsy, if indicated—specific neuromuscular diseases are easily missed. This can lead to inappropriate counseling and treatment. Muscle Nerve 39: 406–408, 2009     

Infantile centronuclear myopathy. Evidence suggesting incomplete innervation

A male case of centronuclear myopathy is reported, with severe weakness at birth and death at 7 weeks. In all the muscles studied the fibres, despite their immature appearances, showed normal histochemical differentiation into type I and type II moieties. In contrast to the extrafusal fibres, the intrafusal fibres seemed to be normal in their development. Although the small centrally-nucleated muscle fibres were equipped with motor end-plates, the EMG revealed profuse fibrillation activity. The conflicting findings are postulated to arise from the presence of inexcitable neuromuscular junctions which nevertheless permitted a neurotrophic influence to be exerted on the muscle fibres.     

Myalgia as the revealing symptom of multicore disease and fibre type disproportion myopathy

BACKGROUND: Multicore disease and congenital fibre type disproportion myopathy are diseases assigned to the heterogeneous group of congenital myopathies. Although hypotonia and muscle weakness appearing in early life are the commonest manifestations of these diseases, distinct phenotypes and late onset cases have been described. OBJECTIVE: To report the occurrence of myalgia as the revealing symptom of multicore disease and fibre type disproportion myopathy. METHODS: The clinical cases of three patients with fibre type disproportion myopathy and one with multicore disease are described. Skeletal muscle biopsies were processed for routine histological and histochemical studies. RESULTS: The clinical picture was unusual in that the symptoms were of late onset and the predominant complaint was muscle pain exacerbated by exercise. Muscle weakness was found in only a single patient, the mother of a patient with fibre type disproportion myopathy. Physical examination was unremarkable in the other patients. Muscle biopsies from patients 1 and 2 contained type I fibres that were considerably smaller than the type II fibres, supporting the diagnosis of fibre type disproportion myopathy. Skeletal muscle of patient 4 showed multiple areas, predominantly but not exclusively in the type I fibres, from which oxidative enzyme activities were absent, as seen in multicore disease. CONCLUSIONS: Muscle pain was the main clinical manifestation in our patients. Recognition of the broader clinical expression of these myopathies is important for prognostic reasons and for genetic counselling of the family members.     

Congenital myopathy with myasthenic features and congenital cataract in two siblings

Summary Two siblings with congenital myopathy showing myasthenic manifestations together with congenital cataract are reported. Their muscle weakness fluctuated and was alleviated by edrophonium chloride. Their serum creatine kinase activity was elevated, and the waning phenomenon was observed on repetitive nerve stimulation. Biopsied muscle showed degenerative changes with type 1 fibre predominance and abnormal morphology of neuromuscular junctions.     

A case of congenital neuromuscular disease with uniform type I fibers, abnormal mitochondrial network and jagged Z-line

Histological, histochemical and ultrastructural studies of muscle biopsy in a case of congenital neuromuscular disease revealed unusual findings consisting of muscle fibers uniformity which were all type I and of small diameter, jagged Z-line and abnormally developed transverse network of mitochondria. E.M.G. examination demonstrated a myopathic pattern, but mitochondrial changes are quite different from those reported in mitochondrial myopathies and jagged Z-line seems poorly correlated with Z-line streaming present in denervation atrophy, target fibers, core-like lesions or other Z-line abnormalities of the nemaline myopathy. On the other hand type I histochemical uniformity seems more likely related to some dysfunction of the neuronal mechanisms that control both the fiber type differentiation and other trophic influences. It also suggests that myogenic E.M.G. pattern might actually be pseudo-myopathic and due to a reduction of the cross sectional area of the individual muscle fibers composing the motor unit.     

Congenital nemaline myopathy with mitochondrial abnormalities. An adult case report]

We report a 42-year-old male suffering from congenital nemaline myopathy accompanied with mitochondrial abnormalities in his muscle biopsy. He had a dysmorphic face with a high-arched and narrow palate and slowly progressive generalized muscle weakness. He was still able to walk with a cane. CT showed symmetrical muscle atrophy and low densities in the thigh muscles, especially in the posterior compartment, and in the soleus muscles. Preferential posterior thigh involvement was unusual in congenital nemaline myopathy. The lumbar quadrate and paravertebral muscles were relatively well preserved; these muscles were reported to be severely involved in adult-onset nemaline myopathy patients. Muscle biopsy findings were consistent with nemaline myopathy; nemaline rods in approximately 10% of fibers, type 1 fiber atrophy, and type 2B fiber deficiency. In addition, ragged-red fibers were scattered and focal cytochrome c oxidase (CCO) deficiency was present. Formazan granules were large on succinate dehydrogenase stain. Many fibers with nemaline rods showed focal CCO deficiency. On electron microscopy, large (megaconial) mitochondria were lined regularly between Z lines. PCR and Southern blot analysis of muscle mitochondrial DNA revealed multiple deletions. It remains to be clarified whether mitochondrial abnormalities are primarily related to nemaline myopathy or secondarily induced phenomenon after a long-standing disease process.     

Two adult cases of nemaline myopathy presenting different clinical symptoms and CT findings]

We report herein two cases of nemaline myopathy which showed peculiar muscle involvement and clinical symptoms. Case 1: A 44-year-old woman had developed gradual woresening of muscle weakness. Neurologically, only flexion of her neck was found to be weak. Her muscular CT revealed mild atrophy of four extremities and the sternocleidomastoid muscles. Histological examinations with Gomori-trichrome staining revealed tiny structures whose form was compatible with nemaline rods. Moreover, electronmicroscopic examination demonstrated the lattice pattern of electron-dense structures, and they also appeared to possess structural continuity with the Z-band. Case 2: A 56-year-old woman visited our department because of neck pulsations. Neurological examinations revealed bilateral hearing disturbance, marked atrophy of neck muscles, muscle weakness in four extremities and hypoactive deep tendon reflexes. She also exhibited steppage and waddling gaits. Her muscular CT demonstrated degenerative processes in the neck muscles(splenius muscles and semispinal muscles), trapezius muscles, para-spinal muscles, deltoid muscles and gluteal muscles. Among them, the para-spinal muscles and extensor muscles of the lower limbs showed marked degeneration and had been partly replaced by fatty tissues. A muscle biopsy was performed, and the presence of nemaline rods was confirmed by Gomori-trichrome staining. Although these two cases could be diagnosed as nemaline myopathy, the clinical symptoms and muscular CT findings were not quite the same. Whether these differences might simply indicate different clinical phases during the disease progression or be of further pathogenic significance still remains unclear. Additionally, since long-term follow-up studies of nemaline myopathy are quite rare, further follow-up examinations of these cases are necessary in order to understand the clinical and pathological alterations of nemaline myopathy.     

Clinical and pathological studies on nemaline myopathy in adulthood]

We examined 22 biopsied muscles from adult patients who had the histopathological characteristics of nemaline myopathy. In the first group, 13 patients had muscle weakness and/or skeletal abnormalities, such as high-arched palate, pes cavus and scoliosis which are often accompanied with the congenital nemaline myopathy. Their appropriate diagnosis had never been made until muscle biopsy was done, because of benign clinical course. In the second group, the symptoms of nine patients became manifest in adulthood and failed to show typical skeletal abnormalities. However, six muscle biopsies showed the histopathologic characteristics of congenital nemaline myopathy; abnormal fiber type distribution including type 1 fiber predominancy, type 1 fiber atrophy and type 2B fiber deficiency. Three patients remained in good health until adulthood when they developed muscle weakness with pathologic findings of nemaline myopathy. Accordingly, nemaline myopathy in adulthood can be categorized into three forms; the first two forms have clinical and pathologic evidence of the congenital benign form, whereas the symptoms are too mild to be noticed. The third form is not a hereditary disorder which may result from autoimmune pathophysiology.     

Nemaline myopathy appearing in adults as cardiomyopathy. A clinicopathologic study

We examined a 29-year-old woman with nemaline myopathy that appeared as cardiomyopathy. Clinical examination showed dilated cardiomyopathy, but no neuromuscular abnormalities of the skeletal muscles. Electromyography showed neither neurogenic nor myopathic abnormalities. A biopsy specimen from the quadripecs muscle showed typical nemaline bodies in about 50% of the muscle fibers. The patient died six months later of biventricular heart insufficiency. Autopsy revealed nemaline bodies in the working and conducting tissues of the myocardium. Earlier, the patient's mother and one of her sisters died unexplained, sudden deaths at the ages of 47 and 37 years, respectively. Sections of the myocardium taken from the sister at autopsy were available, and also disclosed nemaline bodies after restaining with trichrome.     

Implications of longitudinal muscle fibre splitting in neurogenic and myopathic disorders

Histological and electromyographic studies indicate that longitudinal muscle fibre splitting is a common finding in neuromuscular disorders. Separated fragments derived by splitting may undergo degeneration or enlarge to become separate, innervated fibres, thus leading to an increased number of fibres within motor units. Splitting may, therefore, lead to the formation of clusters of fibres of uniform histochemical type, but of variable diameter and length, both in neurogenic and in myopathic disorders. Fibre splitting is thus a factor leading to functional compensation in these disorders.     

Homozygosity for a nonsense mutation in the alpha-tropomyosin slow gene TPM3 in a patient with severe infantile nemaline myopathy

The nemaline myopathies are muscle disorders of variable severity and age of onset, with characteristic nemaline bodies in the sarcoplasm. Genes for dominant (NEM1) and recessive (NEM2A) nemaline myopathy have been localised to chromosomes one and two, respectively. A missense mutation in the alpha-tropomyosin gene (TPM3) has been associated with NEM1 in one family. Probands from 76 other nemaline myopathy families have now been screened for TPM3 mutations. One proband, who was not noted to have any weakness neonatally, but who died at 21 months of age, was shown to be homozygous for a single strand conformation polymorphism (SSCP) in skeletal-muscle-specific exon 1 of TPM3. Sequencing revealed homozygosity for a nonsense mutation at codon 31 (CAG to TAG). The patient should have no functioning alpha-tropomyosin slow protein. The nemaline bodies in this patient were exclusively in type one fibres, consistent with the expression of TPM3 only in type one fibres.     

Properties of slow- and fast-twitch muscle fibres in a mouse model of amyotrophic lateral sclerosis

This investigation was undertaken to determine if there are altered histological, pathological and contractile properties in presymptomatic or endstage diseased muscle fibres from representative slow-twitch and fast-twitch muscles of SOD1 G93A mice in comparison to wildtype mice. In presymptomatic SOD1 G93A mice, there was no detectable peripheral dysfunction, providing evidence that muscle pathology is secondary to motor neuronal dysfunction. At disease endstage however, single muscle fibre contractile analysis demonstrated that fast-twitch muscle fibres and neuromuscular junctions are preferentially affected by amyotrophic lateral sclerosis-induced denervation, being unable to produce the same levels of force when activated by calcium as muscle fibres from their age-matched controls. The levels of transgenic SOD1 expression, aggregation state and activity were also examined in these muscles but there no was no preference for muscle fibre type. Hence, there is no simple correlation between SOD1 protein expression/activity, and muscle fibre type vulnerability in SOD1 G93A mice.     

Adult-onset nemaline myopathy and monoclonal gammopathy: a case report

A 47-year-old female developed proximal limb weakness after hysterectomy for uterine fibromatosis. Muscle strength slowly improved, but relapse occurred at age 52. She presented with progressive gait difficulty, proximal limb weakness, and painful calves. Family history was not contributory. Cranial nerves, deep tendon reflexes, and sensation were normal. Serum creatine kinase was normal. An IgG kappa monoclonal protein was found. Nerve conduction studies were normal, but EMG showed brief small polyphasic motor unit action potentials with early recruitment in proximal muscles. Muscle biopsy showed abundant rods, atrophic muscle fibres, and type 1 fibre predominance. The sarcolemma was immunoreactive for IgG kappa. Plasmapheresis was unsuccessful, but methylprednisolone and azathioprine led to moderate improvement of muscle strength, associated with reduced monoclonal protein levels. This is the third case report, describing the association of monoclonal gammopathy and late-onset nemaline myopathy. Presence of a monoclonal protein at the sarcolemma and responsiveness to immunosuppressive treatment are suggestive of a dys-immune origin.     

REGENERATION AND INNERVATION OF NORMAL AND DYSTROPHIC MUSCLE CULTURED WITH NORMAL AND DYSTROPHIC SPINAL CORD

Teased strips of normal or dystrophic adult mouse muscle were cultured with embryonic normal or 'dystrophic' mouse spinal cord explants, in order to determine whether muscular dystrophy is of primary myopathic or neuropathic origin. When cultured with normal spinal cord, both normal and dystrophic muscle regenerated to form new cross-striated muscle fibres with peripherally located nuclei and showing spontaneous, synchronized contractions. Silver impregnation and acetyl-cholinesterase activity showed that neuromuscular junctions were formed. In marked contrast, both normal and dystrophic muscle cultured with spinal cord from dystrophic animals failed to show such functional regeneration, and neuromuscular junctions could not be identified. Only a few myotubes were formed, and these were rarely cross-striated, had internal nuclei, and only some showed asynchronous fibrillatory contractions. These results strongly suggest that murine muscular dystrophy has a neural pathogenesis. Histochemical reactions were carried out on serial frozen sections of the regenerated muscle fibres, and also of embryonic mouse muscle cultured in cord-myotome explants, to determine whether 'fibre types' were formed. All the fibres gave uniformly strong or medium reactions to phosphorylase, NADH-TR, myofibrillar ATPase, ATPase after acid preincubation, and the PAS reaction for glycogen, except a few fibres in an embryonic muscle which gave weak reactions for all enzymes. Histochemical fibre types were not formed. Two studies of explants of human muscle in association with normal embryonic mouse spinal cord resulted in the establishment of functional neuromuscular contact and the production of well striated myotubes, with peripherally placed nuclei and spontaneous regular contractions.     

Muscle fibre type composition in distal myopathy (Welander). An analysis with enzyme- and immuno-histochemical, gel-electrophoretic and ultrastructural techniques

The myopathic muscle of distal myopathy (Welander's disease), the dominantly inherited neuromuscular disorder which occurs frequently in Sweden, has been characterized by electron microscopy, enzyme- and immuno-histochemistry (using antibodies against embryonic, neonatal, fast and slow myosin, and against the muscle-specific intermediate filament protein, desmin), and with gel electrophoretic techniques. Of special interest is the fact that the ultrastructural appearance of the fibres with regard to M- and Z-band structures does not fit the proposed classification criteria for ultrastructural fibre typing of normal human muscle. Furthermore, contrary to previous results, we conclusively demonstrate that the predominating fibres are of a slow-twitch type. Unexpectedly, we also observed that embryonic and neonatal myosin was expressed in some residual fibres. This emphasises the importance of supplementing stains to demonstrate activity of ATPase with myosin immuno-histochemistry in order to improve understanding of fibre type characteristics in myopathic muscles. The origin of the myopathic muscle fibres in distal myopathy could not be definitely determined, but it is suggested that neurogenic disturbances play an important part in the pathophysiology of Welander's disease.     

The syndrome of continuous muscle fibre activity: light and electron microscopic studies in muscle and nerve biopsies

Summary Histological and ultrastructural studies were performed on nerve and muscle biopsy specimens from two patients with the syndrome of continuous muscle fibre activity. The characteristics of muscle biopsies were as follows. By light microscopy, internal nuclei were present in many of the fibres. By electron microscopy many fibres contained filamentous bodies and subsarcolemmal aggregates of mitochondria embedded in the peripheral zone of cytoplasm, and occasional mitochondria with disorganized or branched cristae were larger than normal. Biopsies of sural nerves revealed a decreased number of myelinated fibres, clusters of small myelinated fibres, and evidence of active axonal degeneration such as disintegrated myelin segments and degenerated axon components, as well as loss of axonal contents. With the present biopsy findings, it is suggested that the pathological process of this syndrome affects peripheral nerves as well as muscles.