Exome sequencing detection of two untranslated GFPT1 mutations in a family with limb-girdle myasthenia.

The term 'limb-girdle myasthenia' (LGM) was first used to describe three siblings with proximal limb weakness without oculobulbar involvement, but with EMG decrement and responsiveness to anticholinesterase medication. We report here that exome sequencing in the proband of this family revealed several sequence variations in genes linked to proximal limb weakness. However, the only mutations that cosegregated with disease were an intronic IVS7-8A>G mutation and the previously reported 3'-UTR c.*22C>A mutation in GFPT1, a gene linked to LGM. A minigene assay showed that IVS7-8A>G activates an alternative splice acceptor that results in retention of the last seven nucleotides of intron 7 and a frameshift leading to a termination codon 13 nucleotides downstream from the new splice site. An anconeus muscle biopsy revealed mild reduction of the axon terminal size and postsynaptic fold simplification. The amplitudes of miniature endplate potentials and quantal release were also diminished. The DNA of the mildly affected father of the proband showed only the intronic mutation along with sequence variations in other genes potentially relevant to LGM. Thus, this study performed in the family originally described with LGM showed two GFPT1 untranslated mutations, which may cause disease by reducing GFPT1 expression and ultimately impairing protein glycosylation.

The L-coding region of the DA strain of Theiler's murine encephalomyelitis virus causes dysfunction and death of myelin-synthesizing cells.

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) induce an early transient subclinical neuronal disease followed by a chronic progressive inflammatory demyelination, with persistence of the virus in the central nervous system (CNS) for the life of the mouse. Although TMEV-induced demyelinating disease (TMEV-IDD) is thought to be immune mediated, there is also evidence that supports a role for the virus in directly inducing demyelination. In order to clarify the function of DA virus genes, we generated a transgenic mouse that had tamoxifen-inducible expression of the DA L-coding region in oligodendrocytes (and Schwann cells), a cell type in which the virus is known to persist. Tamoxifen-treated young transgenic mice usually developed an acute progressive fatal paralysis, with abnormalities of the oligodendrocytes and Schwann cells and demyelination, but without significant lymphocytic infiltration; later treatment led to transient weakness with demyelination and persistent expression of the recombined transgene. These findings demonstrate that a high level of expression of DA L can cause the death of myelin-synthesizing cells and death of the mouse, while a lower level of L expression (which can persist) can lead to cellular dysfunction with survival. The results suggest that expression of DA L plays an important role in the pathogenesis of TMEV-IDD. Virus-induced infection and death of oligodendrocytes may play a part in the demyelination of other diseases in which an immune-mediated mechanism has been stressed, including multiple sclerosis.

Congenital myasthenic syndrome associated with epidermolysis bullosa caused by homozygous mutations in PLEC1 and CHRNE.

Mutations in the plectin gene (PLEC1) cause epidermolysis bullosa simplex (EBS), which may associate with muscular dystrophy (EBS-MD) or pyloric atresia (EBS-PA). The association of EBS with congenital myasthenic syndrome (CMS) is also suspected to result from PLEC1 mutations. We report here a consanguineous patient with EBS and CMS for whom mutational analysis of PLEC1 revealed a homozygous 36 nucleotide insertion (1506_1507ins36) that results in a reduced expression of PLEC1 mRNA and plectin in the patient muscle. In addition, mutational analysis of CHRNE revealed a homozygous 1293insG, which is a well-known low-expressor receptor mutation. A skin biopsy revealed signs of EBS, and an anconeus muscle biopsy showed signs of a mild myopathy. Endplate studies showed fragmentation of endplates, postsynaptic simplification, and large collections of thread-like mitochondria. Amplitudes of miniature endplate potentials were diminished, but the endplate quantal content was actually increased. The complex phenotype presented here results from mutations in two separate genes. While the skin manifestations are because of the PLEC1 mutation, footprints of mutations in PLEC1 and CHRNE are present at the neuromuscular junction of the patient indicating that abnormalities in both genes contribute to the CMS phenotype.

Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome.

BACKGROUND: We describe a severe form of congenital myasthenic syndrome (CMS) associated with congenital nephrosis and ocular malformations caused by two truncating mutations in the gene encoding the laminin beta2 subunit (LAMB2). METHODS AND RESULTS: Mutational analysis in the affected patient, who has a history of a serious untoward reaction to treatment with acetylcholinesterase inhibition, revealed two frame-shifting heteroallelic mutations, a maternally inherited 1478delG and a paternally inherited 4804delC. An anconeus muscle biopsy demonstrated a profound distortion of the architecture and function of the neuromuscular junction, which was strikingly similar to that seen in mice lacking laminin beta2 subunit. The findings included: pronounced reduction of the axon terminal size with encasement of the nerve endings by Schwann cells, severe widening of the primary synaptic cleft and invasion of the synaptic space by the processes of Schwann cells, and moderate simplification of postsynaptic folds and intact expression of the endplate acetylcholinesterase. The endplate potential quantal content was notably reduced, while the frequencies and amplitudes of miniature endplate potentials were only moderately diminished and the decay phases of miniature endplate potentials were normal. Western blot analysis of muscle and kidney tissue and immunohistochemistry of kidney tissue showed no laminin beta2 expression. CONCLUSION: This case, which represents a new type of synaptic CMS, exemplifies the wide variability of phenotypes associated with LAMB2 mutations and underscores the fundamental role that laminin beta2 plays in the development of the human neuromuscular junction.

Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin.

gamma-Sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. The murine gamma-sarcoglycan gene was disrupted using homologous recombination. Mice lacking gamma-sarcoglycan showed pronounced dystrophic muscle changes in early life. By 20 wk of age, these mice developed cardiomyopathy and died prematurely. The loss of gamma-sarcoglycan produced secondary reduction of beta- and delta-sarcoglycan with partial retention of alpha- and epsilon-sarcoglycan, suggesting that beta-, gamma-, and delta-sarcoglycan function as a unit. Importantly, mice lacking gamma-sarco- glycan showed normal dystrophin content and local- ization, demonstrating that myofiber degeneration occurred independently of dystrophin alteration. Furthermore, beta-dystroglycan and laminin were left intact, implying that the dystrophin-dystroglycan-laminin mechanical link was unaffected by sarcoglycan deficiency. Apoptotic myonuclei were abundant in skeletal muscle lacking gamma-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking gamma-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. Our data demonstrate that sarcoglycan loss was sufficient, and that dystrophin loss was not necessary to cause membrane defects and apoptosis. As a common molecular feature in a variety of muscular dystrophies, sarcoglycan loss is a likely mediator of pathology.

The clinical significance of extracellular matrix in gangliogliomas.

Based on in vitro studies which demonstrate that collagen IV and laminin inhibit the proliferation and invasiveness of glioma cells, we investigated the clinical significance of these extracellular matrix proteins (ECM) in patients with gangliogliomas, tumors in which ECM is often a prominent feature. Our study compared the relative presence and deposition pattern of collagen IV and laminin in 19 gangliogliomas and in 18 gliomas without ganglion cell differentiation (8 low-grade astrocytomas, 7 anaplastic astrocytomas, and 3 anaplastic mixed gliomas). We also examined whether the presence of collagen IV and laminin correlated with other features often observed in gangliogliomas, including perivascular lymphocytic inflammation, granular bodies, microcalcification, and subarachnoid extension, and whether any of these features were associated with the patient's clinical course. Significant deposition of collagen IV and laminin was found in 9 gangliogliomas (47%), but in none of the other gliomas. The presence of these extracellular proteins in gangliogliomas correlated with both perivascular inflammation (P = 0.003), and involvement of the leptomeninges by tumor (P = 0.008). The duration of symptoms prior to surgical resection was significantly longer for patients whose tumors showed extracellular deposition of collagen IV and laminin than for patients whose tumors lacked deposition of these proteins (mean 13.7 vs 5.1 years; P = 0.02). In addition, the duration of symptoms was significantly longer for patients whose tumors exhibited perivascular inflammation than for patients whose tumors displayed little or no perivascular inflammation (mean 14.8 vs 4.8 years; P = 0.01). These findings suggests that collagen IV and laminin and perivascular inflammation are related to the indolent behavior of gangliogliomas.

Necrotizing myelopathy complicating Hodgkin's disease.

A case of necrotizing myelopathy is presented as a complication of Hodgkin's disease. The distinctive pathologic feature of this case was severe acute vasculitis involving only the vessels of the spinal cord that was considered to have been the cause of the segmental necrosis.

Amyotrophic lateral sclerosis with antecedent poliomyelitis.

Histopathological and virological studies were performed on autopsy tissue from a 47-year-old man who had a history of acute poliomyelitis at age 15 years and died after a three-year course of amyotrophic lateral sclerosis (ALS). The poliovirus serologic tests suggested prior infection with poliovirus type 3 but no ongoing poliovirus infection. The CNS showed typical features of ALS with no inclusion bodies or inflammatory cells. Attempts to isolate poliovirus in the CNS were unsuccessful and results of immunofluorescence studies for poliovirus antigen were negative. Molecular hybridization experiments using a DNA copy of the complete poliovirus genome failed to demonstrate poliovirus-related RNA or DNA sequences in the CNS. These studies, using sensitive techniques, indicate that there was no evidence of the continuing presence of poliovirus in this patient with ALS and antecedent poliomyelitis.

Inflammation at the neuromuscular junction in myasthenia gravis.

To better define the pathogenic mechanisms in the antibody-mediated autoimmune disease myasthenia gravis (MG), we analyzed the morphology and electrophysiology of the neuromuscular junction in anconeus muscle biopsy specimens from eight patients with MG and seven control subjects. There were inflammatory cells at the neuromuscular junction in seven of the eight biopsies from MG patients. The endplate index (length of the postsynaptic membrane divided by the length of the apposed presynaptic membrane) was abnormally reduced in all the MG patients, and fiber type grouping, suggestive of reinnervation, was present in six of the eight MG patients. Intracellular recording revealed diminished amplitude of miniature endplate potentials and miniature endplate currents in the MG patients compared with the controls. The time constant of decay of miniature endplate currents did not differ from that of controls, suggesting no change in mean channel open time of the acetylcholine receptor. The endplate receptor sensitivity to iontophoretically applied acetylcholine was also decreased in MG patients compared with controls. The quantal content of neurally evoked endplate potentials was reduced in six of the eight MG patients, demonstrating abnormal presynaptic function as well. The presence of inflammatory cells at the neuromuscular junctions of limb muscles in MG reconciles an apparent disparity between the animal model of MG, experimental autoimmune myasthenia gravis, and the human disease. This study also demonstrates a frequent presynaptic component to the abnormal neuromuscular transmission in MG.

Muscle acid protease activity in amyotrophic lateral sclerosis: correlation with clinical and pathologic features.

Acid protease activity was increased in skeletal muscle of patients with ALS. The highest levels of activity were found in individuals with the clinically and histologically most affected muscle. High levels of proteolytic activity correlated with the extent of muscle atrophy, the presence of target fibers and the overall severity of disease.

Immunologic and virologic studies of measles inclusion body encephalitis in an immunosuppressed host: the relationship to subacute sclerosing panencephalitis.

An immunosuppressed child with acute lymphoblastic leukemia in clinical remission developed measles inclusion body encephalitis (MIBE). Although measles antigen and nonbudding measles virus nucleocapsids were detected in brain tissue, no virus was isolated. Immune precipitation of measles virus proteins with the patient's serum showed no detectable antibody to virus M protein, a finding that has been reported in subacute sclerosing panencephalitis (SSPE). The virologic and immune precipitation studies suggest a similar virus mutation in MIBE and SSPE. The pathogenesis of the two diseases may also be similar.

Presynaptic congenital myasthenic syndrome due to quantal release deficiency.

OBJECTIVE: To provide clinical, electrophysiologic, and ultrastructural findings in three patients with a presynaptic congenital myasthenic syndrome (CMS). BACKGROUND: Familial infantile myasthenia and paucity of synaptic vesicles are the only two fully characterized CMS. We are describing here three patients with another form of presynaptic CMS characterized by deficiency of the action potential-dependent release without reduction of the spontaneous release of neurotransmitter from the nerve terminal. METHODS: The authors performed electromyography and anconeus muscle biopsies that included intracellular recordings and electron microscopy of the neuromuscular junction in three patients with presynaptic CMS. They also sequenced part of the P/Q-calcium alpha(1)-subunit gene (CACNA1A) and the acetylcholine receptor subunit (AChR) genes in these patients. RESULTS: In these patients there were additional neurologic findings including nystagmus and ataxia. In all three patients the end-plate potential quantal content (m) was markedly reduced but neither the amplitudes nor the frequencies of miniature end-plate potentials were diminished. Ultrastructurally, postsynaptic end-plate folds, nerve terminal size, and synaptic vesicle number were normal but double-membrane-bound sacs containing synaptic vesicles were present in the nerve terminal of all three patients. The screening of reported pathogenic mutations in the CACNA1A and a mutational analysis of AChR subunit genes were negative. CONCLUSION: This form of CMS appears to result only from a deficiency of the quantal release of neurotransmitter that may be due to an abnormal calcium mechanism or impaired endocytosis and recycling of synaptic vesicles.

Clinical and genetic studies of fatal familial insomnia.

We report a 42-year-old man who, for 8 months, had intermittent motor abnormalities and mild difficulty falling asleep. A diagnosis of fatal familial insomnia (FFI) became evident over the next 6 months when he developed progressive insomnia, myoclonus, sympathetic hyperactivity, and dementia. The amyloid or prion protein (PrP) genotype showed features typically seen in FFI, with a 178Asn mutation and a 129Met polymorphism. There was also a deletion of one octapeptide repeat, suggesting that the association of 178Asn mutation with the 129Met polymorphism is not due to "founder effect." Western immunoblot showed a trace of protease-resistant PrP in the thalamus--which had the most significant neuronal loss and gliosis--a moderate amount of PrP in the fronto-temporal area, and no detectable protein elsewhere in the brain. Endocrine studies showed that a circadian modulation of hormonal levels could be maintained despite a near-total absence of sleep. Administration of gamma-hydroxybutyrate induced a remarkable increase in slow-wave sleep.

Sympathectomy augments adoptively transferred experimental allergic encephalomyelitis.

Adoptively transferred experimental allergic encephalomyelitis (EAE) was significantly augmented in Lewis rats with ablated sympathetic nervous system. Sympathectomy was obtained by treatment of newborn rats with 6-hydroxydopamine. Sham-injected rats were used as a control. EAE was elicited in 7-8-week-old donor Lewis rats by immunization with a suspension of guinea pig (GP) brain and spinal cord in complete Freund's adjuvant. Successful transfer of EAE was accomplished with 50 x 10(6) lymph node cells (LNC)/rat, incubated for 72 h with GP myelin basic protein. LNC were obtained from draining lymph nodes, 9 days after immunization for EAE. The severity of passively transferred EAE was significantly augmented when donor LNC obtained from normal Lewis rats immunized for EAE were injected into sympathectomized rats as compared to sham-injected rats. When LNC were obtained from sympathectomized or sham-injected donors, the disease was significantly more severe in recipients of cells from sympathectomized animals. The severity of histological lesions in the brain and spinal cord was greater in rats with passively transferred EAE which received LNC from sympathectomized donors.

The beta 2-adrenergic agonist terbutaline suppresses experimental allergic neuritis in Lewis rats.

Treatment of rats with experimental allergic neuritis with the beta 2-adrenergic agonist terbutaline suppresses clinical symptoms, decreases demyelination and Wallerian degeneration in peripheral nerves and improves electrophysiological parameters. Treatment is highly effective when given from the time of immunization through the acute phase of illness, when given for the first 12 days after immunization and also when given after the onset of the disease.

The effect of short-term and chronic immunosuppression on Theiler's virus demyelination.

Theiler's virus (TV)-infected mice were treated with antithymocyte serum (ATS), cyclophosphamide or pepstatin (a protease inhibitor) to determine the effect on demyelination. When ATS and cyclophosphamide were begun at the time of infection there was significantly less demyelination at 2.5-3.5 weeks than in pepstatin or non-treated infected controls. When immunosuppression was continued for 5 weeks, or when it was not started until 5 weeks post-infection, no significant decrease in demyelination was seen compared to controls. The findings indicate that timing of immunosuppression is critical in determining the extent of TV demyelination. Such demyelination may occur by different mechanisms that are active at different times. The "bystander effect' may be important in early demyelination, but late demyelination may be due to other causes, such as oligodendrocyte lytic infection.

Striational autoantibodies in myasthenia gravis patients recognize I-band titin epitopes.

Myasthenia gravis (MG) patients develop autoantibodies primarily against the acetylcholine receptor in the motor endplate, but also against intracellular striated muscle proteins, notably titin, the giant elastic protein of the myofibrillar cytoskeleton. Titin antibodies have previously been shown to be directed against a single epitope on the molecule, located at the A-band/I-band junction and referred to as the main immunogenic region (MIR) of titin. By using immunofluorescence microscopy on stretched single myofibrils, we now report that approximately 40% of the sera from 18 MG/thymoma patients and 8 late-onset MG patients with thymus atrophy contain antibodies that bind to a more central I-band titin region. This region consists of homologous immunoglobulin domains and is known to be differentially spliced dependent on muscle type. All patients with I-band titin antibodies also had antibodies against the MIR. Although a statistically significant correlation between the occurrence of I-band titin antibodies and MG severity was not apparent, the results could hint at an initial immunoreactivity to titin's MIR, followed by reactivity along the titin molecule in the course of the disease.

Cellular neuropathology of tuberous sclerosis.

The study of cerebral lesions of TSC by special histologic methods suggests that two populations of neurons and glia occur in TSC brains. One is a population of normally differentiated cells that form a normally constituted cortical plate. The other is a group of cells that are poorly differentiated, fail to organize into a normal cortical architecture, and form a variety of abnormal cellular aggregates in cortex and in subcortical locations. The proportion of these abnormal cells varies greatly from patient to patient. In some the central nervous system appears to be entirely spared. In others, only one or a few islands of dysplastic cells occur, whereas in still others a large number, perhaps even a majority, of neuroectodermal cells in the forebrain may be affected. The proportion of total cells that undergo abnormal differentiation apparently is an important factor relative to cortical function in TSC. At present we have no explanation for this marked heterogeneity in expression of the TSC gene or genes, and it remains one of the many unsolved mysteries of this illness.

Function and ultrastructure of the neuromuscular junction in post-polio syndrome.

We performed a detailed morphological and electrophysiological analysis of the neuromuscular junction in muscle biopsies from 10 patients with post-polio syndrome (PPS). This was done to clarify the basis for the apparent neuromuscular transmission impairment in PPS. In six patients, intracellular microelectrode recordings demonstrated either reduction of amplitudes of miniature end-plate potentials (MEPPs) or decreased quantal content or both. In one patient, reduction of quantal content was only present with prolonged or high-frequency nerve stimulation. In three patients no significant abnormalities were found by the intracellular microelectrode studies. Histologically, atrophy of individual muscle fibers were present in 6 out of the 10 biopsies, but grouped atrophy was not seen. Fiber type grouping suggesting reinnervation was seen in 8 out of the 10 muscle biopsies. Fragmentation and dispersion of the end plate was present in three patients. In two of these patients dispersion of the end plate was associated with an apparent increase of the quantal content. Electron microscopy revealed either normal neuromuscular junctions or small axon termini apposed to normal postsynaptic folds. In summary, variable degrees and different types of failure of neuromuscular transmission were seen in association with histological signs of reinnervation in the muscle biopsies of affected patients. Functional and structural abnormalities of the neuromuscular junction, although very common, were not invariably present and, therefore, they do not appear to be a necessary condition to define the post-poliomyelitic syndrome.

Distribution of S-100 protein outside the central nervous system.

The distribution of S-100 outside the central nervous system in humans and rats was explored using antiserum to S-100 and the peroxidase anti-peroxidase method of Sternberger. In peripheral nerves the Schwann cells and the outermost part of the myelin sheaths were stained; axons were not. In dorsal root ganglia and ganglia of the autonomic nervous system only satellite cells were stained. In the adrenal medulla a considerable number of cells were stained. In all other organs studied Schwann cells and satellite cells of ganglia were the only elements that were stained. We conclude that S-100 could serve as a marker for Schwann cells in situ.