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Dysferlin, the protein product of the dysferlin gene (DYSF), has been shown to have a role in calcium-induced membrane fusion and repair. Dysferlin is absent or drastically reduced in patients with the following autosomal recessive disorders: limb-girdle muscular dystrophy type 2B (LGMD-2B), Miyoshi myopathy (MM) and distal anterior compartment myopathy. To date, less than 45 mutations have been described in DYSF and a wide inter- and intra-familial variation in clinical phenotype has been associated with the same mutation. This observation underlines the relevance of any new report describing genotype/phenotype correlations in dysferlinopathic patient and families. Here we present the results of clinical, biochemical and genetic analysis performed on one MM and three LGMD Italian families. By screening the entire coding region of DYSF, we identified three novel mutations (two missense substitutions and one frame shift microdeletion). The possible existence of a founder effect for the Arg959Trp mutation in the Italian population is discussed.

Limb-girdle myasthenia is an uncommon disease and includes familial and autoimmune forms. Patients present proximal muscle weakness and wasting, and sometimes fatigability, without cranial nerve involvement and fluctuations. We observed, during a 15-year period, nine subjects with limb-girdle myasthenia, (24-55 years; 8 males, 1 female) who constituted 3.2% of 281 myasthenic patients attending our department. All had previously received a diagnosis different from myasthenia. Diagnosis of limb-girdle myasthenia was established by clinical, muscle biopsy and electrophysiological assessment including repetitive nerve stimulation and single fiber electromyography. Five patients had the familial form with tubular aggregates in skeletal muscle; four patients had the autoimmune form. Patients with the familial form had a good response to acetylcholinesterase inhibitors, and the patients with the autoimmune form responded to immunotherapy. Our findings reinforce the opportunity to suspect limb-girdle myasthenia in unclassifiable proximal myopathies and to differentiate familial from autoimmune cases, especially for therapeutic implications.

DNA fragmentation and apoptosis-related proteins have been investigated in thyroid cells and there is evidence that Fas-mediated apoptosis is inhibited by thyroid stimulating hormone (TSH). We investigated DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and Bcl-2 and Fas antigen expression by immunocytochemistry in skeletal muscles from 12 patients with hypothyroid myopathy and 5 patients with hyperthyroid myopathy. The finding of very few TUNEL-positive muscle fibers in both conditions suggests that apoptosis does not play a role in the pathogenesis of thyroid myopathies. Bcl-2 expression increased significantly in hypothyroid myopathy, correlating with high serum TSH levels, and not with either triiodothyronine (T3) or thyroxine (T4) serum levels. By contrast, Fas antigen was overexpressed in hyperthyroid myopathy, correlating with low TSH levels. These findings suggest an anti-apoptotic role for TSH itself in skeletal muscle., (Copyright 2002 Wiley Periodicals, Inc.)

Motor neuropathy with multifocal conduction blocks represents a recently identified autoimmune disorder of the peripheral nerve myelin. Association of motor neuropathies or neuronopathies with thyroid disorders, such as hyperthyroidism, hypothyroidism or thyroid neoplasms has been rarely described. We studied a 61-year-old man with a 2-year-history of slowly progressive weakness of the left limbs with atrophy and fasciculations. Nerve conduction velocity studies revealed multifocal motor conduction blocks. Serum IgM titer of antibodies against GM1 was elevated (1:1280; n.v. up to 1:640). Thyroid studies were compatible with Hashimoto's thyroiditis. Therapy with high dose intravenous immunoglobulins was followed by a prompt clinical recovery. Then the disease assumed an intravenous immunoglobulins dependent course with a full clinical, but transient, recovery. This is the first observation of an association of multifocal motor neuropathy with high titers of GM1 and Hashimoto's thyroiditis and reinforces the multifocal motor neuropathy autoimmune origin as well as the repeated clinical recoveries after intravenous immunoglobulins. This case also suggests to deeply investigate the thyroid function in patients with multifocal motor neuropathy.

Sensory loss and ulcero-mutilating features have been observed in hereditary sensory neuropathy type I and in hereditary motor and sensory neuropathy type IIB, also referred as Charcot-Marie-Tooth disease type 2B. To date two loci associated with ulcero-mutilating neuropathy have been described: CMT2B at 3q13-q22 and HSN I at 9q22.1-q22.3. We performed linkage analysis with chromosomal markers representing the hereditary sensory neuropathy type I and Charcot-Marie-Tooth disease type 2B loci on an Italian family with a severe distal sensory loss leading to an ulcero-mutilating peripheral neuropathy. Negative likelihood-of-odds scores excluded any evidence of linkage to both chromosome 3q13 and chromosome 9q22 markers, confirming the genetic heterogeneity of this clinical entity and the presence of a third locus responsible for ulcero-mutilating neuropathies.

Although autonomic symptoms are not prominent in dystrophinopathies, a reduced vagal activity and an enhanced sympathetic tone have been found in Duchenne muscular dystrophy. Twenty patients with Becker muscular dystrophy (BMD) were investigated by a battery of six cardiovascular autonomic tests (beat-to-beat variability during quiet breathing and deep breathing, heart rate responses to Valsalva maneuver and standing, blood pressure responses to standing and sustained handgrip) and power spectral analysis (PSA) of heart rate variability. Although 11 patients revealed abnormal findings at some cardiovascular tests, none of them had a definite autonomic damage, as indicated by two or more abnormal tests. The mean results of the single tests did not differ from normal controls, except for the beat-to-beat variability during quiet breathing, which was significantly higher in BMD (p<0.05). Such finding was confirmed by a significantly higher total variance (p<0.05), indicating an increased parasympathetic activity. Spectral components were not significantly different from normal controls. PSA values were not influenced by age, functional ability score or presence of heart abnormalities. Our data suggest that autonomic involvement does not represent a major finding in BMD.

The authors report a case of multiple system atrophy (MSA) with an onset as a peripheral nerve involvement. Their patient, a 55-year-old man, had a 3-year history of distal weakness and atrophy in upper limbs with dysesthesia in the feet. Other identifiable causes of peripheral neuropathy were ruled out. The authors postulate that peripheral nervous system impairment can anticipate the typical appearance of MSA, and they suggest that, in peripheral neuropathies with autonomic system dysfunction, after excluding main causes of autonomic neuropathy, MSA may need to be suspected.

Although disorders of thyroid function may cause a wide range of muscle disturbances, an overt myopathy has been rarely reported as an isolated clinical presentation of hypothyroidism. We observed 10 patients (5 males and 5 females) who had been referred to the department of neurology because of muscular fatigability, myalgia, cramps, or proximal weakness. Laboratory investigation showed that all patients had hypothyroidism due to Hashimoto's thyroiditis (atrophic variant in 9/10). Classic symptoms/signs of hypothyroidism such as lethargy, constipation, cold intolerance, myxedematous facies, and/or bradycardia were absent, as assessed independently by the three coauthoring thyroidologists. Muscular complaints improved greatly and then disappeared after substitutive levothyroxine treatment. Muscle biopsy revealed nonspecific changes. Nicotinamide adenine dinucleotide reductase (NADH-TR)-hyporeactive cores were present in two patients (10% and 90% of type 1 fibers). On electron microscopy, the core areas showed disorganized myofibrils, Z-band streaming, rod formation, and paucity of mitochondria and glycogen granules. Desmin intermediate filaments were overexpressed only in some cores. The similarity of the pattern of desmin expression between hypothyroid cores and target lesions of denervated fibers supports the hypothesis that, at least in some of our patients, myopathy was the result of an impaired nerve-mediated action of thyroid hormones on skeletal muscle. Our observations suggest that an isolated myopathy as the sole manifestation of hypothyroidism is not a rare event. We postulate that our cases may constitute a peculiar subgroup of Hashimoto's thyroiditis patients: (1) the strikingly abnormal F/M ratio of 1:1; (2) the relatively younger age; (3) the rarity of the goitrous variant; (4) the unusual finding of antithyroglobulin (Tg-Ab) > antithyroid peroxidase (TPO-Ab). Thorough evaluation of thyroid function is appropriate in patients with myopathy of uncertain origin.

The time course of the expression of Dp116, talin, vinculin and vimentin in rat sciatic nerve was investigated after experimental transection. Dp116 was still found at 5 days after experiment in some degenerating myelinated fibers of both proximal and distal stumps. The findings are consistent with the known preservation of electrical excitability of the distal nerve in the first days after injury. Some regenerating nerve fibers into the neuroma also expressed Dp116 at 25 and 40 days after nerve transection. Talin and vinculin markedly and diffusely immunostained the neuroma. Talin in the distal stump and vimentin in both proximal and distal stumps were found decreased during the time course of the experiment. Vinculin binding increased in the distal stump, due to a real overexpression or simply to a cross-reaction to degeneration products.

We studied the immunolocalization of Dp116 (a 116 kDa protein product of the dystrophin gene), vinculin, talin, vimentin, desmin, spectrin and titin in the sural nerve biopsies of 25 patients with peripheral neuropathies of different origin. 4 patients presented with HMSN type 1, 4 with HMSN type 2, 2 with HNPP, 4 with CIDP, 5 with chronic axonal neuropathy of unknown origin, 3 with vasculitic neuropathy, 3 with diabetic neuropathy. Expression and localization of Dp116, vinculin, vimentin, desmin, spectrin and titin did not differ from normal control cases. Spectrin and titin immunoreactivities were absent and desmin was occasionally found in few epineurial vessels. A thin rim of Dp116 binding surrounded the outermost layer of myelin sheaths. Perineurium and epineurial vessels stained deeply for vinculin. Vimentin immunoreactivity was seen in all endoneurial, perineurial and epineurial cells. Immunoreactivity for talin was normally found at endoneurial and epineurial vessel walls, perineurial cells and epineurial fibroblasts in all the sural nerves except diabetic nerves. In the latter, whereas talin binding was normal in the vessel walls and epineurial fibroblasts, it was markedly reduced in the perineurium. On immunoblot, two bands at 235 and 190 kDa were found in the sural nerves with the antibody anti-talin, and both were reduced only in the patients with diabetic neuropathy. We postulate that decreased perineurium talin in diabetic polyneuropathy may be related to the known alterations of the tight junctions of the perineurial cells, which have been proposed to be a contributory factor to impaired permeability barrier properties.

Peripheral neuropathy has been described in a number of cases of mitochondrial diseases. In these patients the onset of neuropathy varies from childhood to adulthood, whereas late onset is quite rare. We report here three males, ranging from 71 to 75 years with onset of peripheral neuropathy between 64 and 74 years of age. They complain of ataxic gait, muscle aches, weakness and mild muscle atrophy, sensory impairment with predominant glove and stocking distribution, reduced or absent deep tendon reflexes. Neurophysiological examinations and sural nerve biopsy studies showed a sensorimotor neuropathy with axonal degeneration in two cases and demyelination in one. Peroneus brevis muscle biopsy revealed, apart from frank neurogenic changes, presence of ragged-red fibers and cytochrome c oxidase negative fibers. Electron microscopy confirmed an abnormally increased presence of subsarcolemmal and intermyofibrillar mitochondria in muscle samples. These morphological features suggested a mitochondrial disease that was confirmed by biochemical investigations on muscle homogenate showing that the mitochondrial respiratory chain (MRC) enzyme activities were all reduced when compared to citrate synthase activity. In addition the presence of a partially inactive cytochrome c oxidase protein by ELISA was demonstrated in two cases. According to a recent "mitochondrial theory of aging", we think that a progressive decline of MRC function has affected either skeletal muscle or peripheral nerves in our patients. Being energy-requiring processes, muscle metabolism as well as active axonal transport may become progressively defective with age resulting in a late-onset neuropathy.

Impaired muscle function may be a predominant aspect of hypothyroidism and is virtually present in all patients with overt thyroid failure. Less common is the onset of hypothyroidism with clinical features mimicking a polymyositis. We have observed 3 patients, whose age ranged 63-68 years, presenting with muscle aches, cramps, proximal weakness and stiffness. Two patients had dysphagia. Serum creatine kinase (CK) and electromyography (EMC) were altered in two patients. Muscle biopsy showed type II atrophy, sporadic type I and type II grouping, "core-like" areas, and some myopathic changes such as central nuclei and muscle necrosis. No inflammatory changes were present. Immunohistochemistry of several muscle cytoskeletal proteins revealed increased desmin in "corelike" areas. Detection of serum thyroid hormone levels revealed very low triiodo-L-thyronine (T3) and thyroxine (T4), whereas thyroid-stimulating hormone (TSH) was greatly increased as well as serum anti-thyroglobulin, anti-peroxidase and anti-microsome antibodies. The patients were diagnosed having a hypothyroid myopathy due to Hashimoto thyroiditis. L-thyroxine treatment normalized clinical and hormone levels, but serum antibodies remained elevated. Muscle biopsy was fundamental to establish the correct diagnosis in our patients. Presence of over-expression of desmin in cores, as described in target lesions in neurogenic diseases, may suggest a nerve-mediated pathogenesis of hypothyroid myopathy.

Immunolocalization of spectrin, vinculin, talin, desmin and titin was investigated in human sural nerve. No binding for spectrin and titin was seen in any structure of the nerve. Antibody against desmin immunostained sporadic epineurial vessels only. Endoneurial and epineurial vessels were intensely positive for vinculin and talin. We found expression of vinculin and talin at the perineurial cells, using immunocytochemistry and gold immunoelectron microscopy. Since vinculin and talin are known to be involved in cell-cell and cell-extracellular matrix transmembrane connections, we propose that they, possibly together with other cytoskeletal proteins, may be implicated in the permeability barrier property of the perineurium. In pathological conditions, perineurium plays an as yet unknown role. Future studies are needed to investigate expression of vinculin and talin in neuropathies.

Despite advances in genetics the pathogenesis of central core disease (CCD) is still unknown. We studied muscles from 5 CCD patients by immunocytochemistry using monoclonal antibodies against various cytoskeletal proteins (dystrophin, spectrin, vinculin, desmin, vimentin, myosin heavy chain (MHC) of developmental, neonatal, adult slow and fast types). Dystrophin, spectrin and vinculin immunoreactivity was localized only at sarcolemma as in normal muscle. Vimentin was not present in myofibers. Only sporadic fibers were positive for developmental and neonatal MHC isoforms in adult CCD muscles. A 4-month-old patient had 5% of neonatal MHC-immunoreactive fibers, a finding similar to that of age-matched normal muscle. Desmin intermediate filaments were overexpressed in many core-fibers in extra-core regions, reduced or absent at cores, and greatly increased at the periphery of some cores. Moreover, irregular desmin-positive spots were seen within some cores. On the contrary, in neurogenic muscle atrophy patients, target lesions had increased desmin. These features indicate a possible role of desmin in the pathogenesis of cores, although we do not know if primary or secondary. In addition, they suggest that: (i) cores and targets may be manifestations of different processes; (ii) it is likely that core-fibers are not denervated fibers.