Anti-MuSK autoantibodies block binding of collagen Q to MuSK.

OBJECTIVE: Muscle-specific receptor tyrosine kinase (MuSK) antibody-positive myasthenia gravis (MG) accounts for 5%-15% of autoimmune MG. MuSK mediates the agrin-signaling pathway and also anchors the collagenic tail subunit (ColQ) of acetylcholinesterase (AChE). The exact molecular target of MuSK-immunoglobulin G (IgG), however, remains elusive. As acetylcholine receptor (AChR) deficiency is typically mild and as cholinesterase inhibitors are generally ineffective, we asked if MuSK-IgG interferes with binding of ColQ to MuSK. METHODS: We used 3 assays: in vitro overlay of the human ColQ-tailed AChE to muscle sections of Colq-/- mice; in vitro plate-binding assay to quantitate binding of MuSK to ColQ and to LRP4; and passive transfer of MuSK-IgG to mice. RESULTS: The in vitro overlay assay revealed that MuSK-IgG blocks binding of ColQ to the neuromuscular junction. The in vitro plate-binding assay showed that MuSK-IgG exerts a dose-dependent block of MuSK binding to ColQ by but not to LRP4. Passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the size and density of AChR and MuSK. CONCLUSIONS: As lack of ColQ compromises agrin-mediated AChR clustering in Colq-/- mice, a similar mechanism may lead to AChR deficiency in MuSK-MG patients. Our experiments also predict partial AChE deficiency in MuSK-MG patients, but AChE is not reduced in biopsied NMJs. In humans, binding of ColQ to MuSK may be dispensable for clustering ColQ, but is required for facilitating AChR clustering. Further studies will be required to elucidate the basis of this paradox.

Myasthenic syndrome caused by plectinopathy.

BACKGROUND: Plectin crosslinks intermediate filaments to their targets in different tissues. Defects in plectin cause epidermolysis bullosa simplex (EBS), muscular dystrophy (MD), and sometimes pyloric atresia. Association of EBS with a myasthenic syndrome (MyS) was documented in a single patient in 1999. OBJECTIVES: To analyze the clinical, structural, and genetic aspects of a second and fatal case of EBS associated with a MyS and search for the genetic basis of the disease in a previously reported patient with EBS-MD-MyS. METHODS: Clinical observations; histochemical, immunocytochemical, and electron microscopy studies of skeletal muscle and neuromuscular junction; and mutation analysis. RESULTS: An African American man had EBS since early infancy, and progressive muscle weakness, hyperCKemia, and myasthenic symptoms refractory to therapy since age 3 years. Eventually he became motionless and died at age 42 years. At age 15 years, he had a marked EMG decrement, and a reduced miniature endplate potential amplitude. The myopathy was associated with dislocated muscle fiber organelles, structurally abnormal nuclei, focal plasmalemmal defects, and focal calcium ingress into muscle fibers. The neuromuscular junctions showed destruction of the junctional folds, and remodeling. Mutation analysis demonstrated a known p.Arg2319X and a novel c.12043dupG mutation in PLEC1. The EBS-MD-MyS patient reported in 1999 also carried c.12043dupG and a novel p.Gln2057X mutation. The novel mutations were absent in 200 Caucasian and 100 African American subjects. CONCLUSIONS: The MyS in plectinopathy is attributed to destruction of the junctional folds and the myopathy to defective anchoring of muscle fiber organelles and defects in sarcolemmal integrity.

Myasthenic syndrome due to defects in rapsyn: Clinical and molecular findings in 39 patients.

BACKGROUND: Pathogenic mutations in rapsyn result in endplate acetylcholine receptor (AChR) deficiency and are a common cause of postsynaptic congenital myasthenic syndromes. METHODS: Clinical, electrophysiologic, pathologic, and molecular studies were done in 39 patients. RESULTS: In all but one patient, the disease presented in the first 2 years of life. In 9 patients, the myasthenic symptoms included constant or episodic ophthalmoparesis, and 1 patient had a pure limb-girdle phenotype. More than one-half of the patients experienced intermittent exacerbations. Long-term follow-up was available in 25 patients after start of cholinergic therapy: 21 became stable or were improved and 2 of these became asymptomatic; 3 had a progressive course; and 1 died in infancy. In 7 patients who had endplate studies, the average counts of AChR per endplate and the synaptic response to ACh were less reduced than in patients harboring low AChR expressor mutations. Eight patients were homozygous and 23 heterozygous for the common p.N88K mutation. Six mutations, comprising 3 missense mutations, an in-frame deletion, a splice-site mutation, and a nonsense mutation, are novel. Homozygosity for p.N88K was associated with varying grades of severity. No genotype-phenotype correlations were observed except in 8 Near-Eastern patients homozygous for the promoter mutation (c.-38A>G), who had a mild course. CONCLUSIONS: All but 1 patient presented early in life and most responded to cholinergic agonists. With early diagnosis and therapy, rapsyn deficiency has a benign course in most patients. There was no consistent phenotype-genotype correlation except for an E-box mutation associated with jaw deformities.

Novel congenital myasthenic syndromes associated with defects in quantal release.

BACKGROUND: Most congenital myasthenic syndromes are caused by defects in postsynaptic or synaptic basal lamina-associated proteins; congenital myasthenic syndromes (CMSs) associated with presynaptic defects are uncommon. Here, the authors describe clinical, electrophysiologic, and morphologic features of two novel and highly disabling CMSs, one determined by presynaptic and the other determined by combined presynaptic and postsynaptic defects. METHODS: Microelectrode, single channel patch clamp, immunocytochemical, [(125)I]alpha-bungarotoxin binding, and quantitative electron microscopy studies of endplates were performed. Candidate genes were directly sequenced. RESULTS: Patient 1, a 7-year-old boy, had severe myasthenic symptoms since infancy. Patient 2, a 48-year-old man, had delayed motor milestones and became progressively weaker after age 2 years. Both used wheelchairs and had a 30-50% EMG decrement on 2-Hz stimulation. Evoked quantal release was reduced to approximately 25% of normal in both. In Patient 2, the synaptic response to acetylcholine was further compromised by degeneration of the junctional folds with concomitant loss of the acetylcholine receptor (AChR). A search for mutations in components of the synaptic vesicle release complex and in other candidate proteins failed to identify the molecular basis of the two syndromes. CONCLUSIONS: Combined clinical, morphologic, and in vitro electrophysiologic findings define two novel congenital myasthenic syndromes. The molecular basis of these syndromes awaits discovery.

Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries.

BACKGROUND: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor epsilon subunit, cause congenital myasthenic syndromes. Only three of the eight intronic splice site mutations of CHRNE reported to date have had their splicing consequences characterised. METHODS: We analysed four previously reported and five novel splicing mutations in CHRNE by introducing the entire normal and mutant genomic CHRNEs into COS cells. RESULTS AND CONCLUSIONS: We found that short introns (82-109 nucleotides) favour intron retention, whereas medium to long introns (306-1210 nucleotides) flanking either or both sides of an exon favour exon skipping. Two mutations are of particular interest. Firstly, a G-->T substitution at the 3' end of exon 8 predicts an R286M missense mutation, but instead results in skipping of exon 8. In human genes, a mismatch of the last exonic nucleotide to U1 snRNP is frequently compensated by a matching nucleotide at intron position +6. CHRNE intron 8 has a mismatch at position +6, and accordingly fails to compensate for the exonic mutation at position -1. Secondly, a 16 bp duplication, giving rise to two 3' splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 3' splice site. This conforms to the scanning model of recognition of the 3' splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing.

Congenital endplate acetylcholinesterase deficiency responsive to ephedrine.

The authors describe two patients with congenital myasthenic syndrome (CMS) with end plate acetylcholinesterase (AChE) deficiency caused by mutations in the collagenic tail (ColQ) of AChE: a homozygous C-terminal Y230S mutation in Patient 1 and Y430S and a C-terminal splice-site mutation in Patient 2. In Patient 1, a Prostigmin (neostigmine bromide) test failed to distinguish between AChE deficiency and a slow-channel CMS. Both patients responded dramatically to ephedrine therapy.

[Differential congenital myasthenia syndrome diagnosis]. / Differenzialdiagnostik eines kongenitalen myasthenen Syndroms.

Among myopathies and disorders of neuromuscular transmission, the congenital myasthenic syndromes (CMS) are particularly rare. However, because of the available therapeutic options, it is still clinically important to achieve a correct diagnosis in these patients. We report an adult patient with ophthalmoplegia and nonfluctuating limb-girdle syndrome. For almost 20 years, a congenital myopathy or mitochondriopathy had been suspected before CMS was diagnosed caused by an epsilon subunit mutation of the acetylcholine receptor (epsilon1276delG).

Congenital myasthenic syndrome caused by low-expressor fast-channel AChR delta subunit mutation.

OBJECTIVE: To determine the molecular basis of a disabling congenital myasthenic syndrome (CMS) observed in two related and one unrelated Arab kinship. BACKGROUND: CMS can arise from defects in presynaptic, synaptic basal lamina-associated, or postsynaptic proteins. Most CMS are postsynaptic, and most reside in the AChR epsilon subunit; only two mutations have been reported in the AChR delta subunit to date. METHODS: Cytochemistry, electron microscopy, alpha-bungarotoxin binding studies, microelectrode and patch-clamp recordings, mutation analysis, mutagenesis, and expression studies in human embryonic kidney cells were employed. RESULTS: Endplate studies showed AChR deficiency, fast decaying, low-amplitude endplate currents, and abnormally brief channel opening events. Mutation analysis revealed a novel homozygous missense mutation (deltaP250Q) of the penultimate proline in the first transmembrane domain (TMD1) of the AChR delta subunit. Expression studies indicate that deltaP250Q (1) hinders delta/alpha subunit association during early AChR assembly; (2) hinders opening of the doubly occupied closed receptor (A(2)R); and (3) speeds the dissociation of acetylcholine from A(2)R. Mutagenesis studies indicate that deltaP250L also has fast-channel effects, whereas epsilon P245L and epsilon P245Q, identical mutations of the corresponding proline in the epsilon subunit, have mild slow-channel effects. CONCLUSIONS: deltaP250Q represents the third mutation observed in the AChR delta subunit. The severe phenotype caused by deltaP250Q is attributed to endplate AChR deficiency, fast decay of the synaptic response, and lack of compensatory factors. That the penultimate prolines in TMD1 of the delta and epsilon subunits exert a reciprocal regulatory effect on the length of the channel opening bursts reveals an unexpected functional asymmetry between the two subunits.

Congenital myasthenic syndrome associated with episodic apnea and sudden infant death.

The sudden infant death syndrome has multiple etiologies. Some congenital myasthenic syndromes can cause sudden infant death syndrome by apnea, but the frequency of this etiology is unknown. We report here a young patient with sudden respiratory crises culminating in apnea followed by recovery, against a background of no or variable myasthenic symptoms without dyspnea. One sib without myasthenic symptoms and one sib who only had mild ptosis died previously during febrile episodes. Studies reported by us elsewhere traced the proband's illness to mutations in choline acetyltransferase. Here, we describe in detail the morphologic investigations and electrophysiologic findings, which point to a presynaptic defect in acetylcholine resynthesis or vesicular filling, in the proband. Analysis of DNA from a sib who previously died of sudden infant death syndrome revealed the same choline acetyltransferase mutation. Thus, mutations in choline acetyltransferase may be a cause of sudden infant death syndrome as, theoretically, could other presynaptic myasthenic disorders.

Three novel COLQ mutations and variation of phenotypic expressivity due to G240X.

OBJECTIVE: To determine the molecular basis and consequences of endplate (EP) acetylcholinesterase (AChE) deficiency. BACKGROUND: The EP species AChE is an asymmetric enzyme consisting of a tail subunit composed of three collagenic strands (ColQ), each attached to a tetramer of catalytic subunits. The tail subunit is essential for insertion of AChE into the synaptic basal lamina. Human EP AChE deficiency is caused by mutations in COLQ. The authors report three novel COLQ mutations in eight kinships. METHODS: Immunocytochemistry, electron microscopy, microelectrode recordings, mutation analysis, and expression studies in COS cells were employed. RESULTS: Two mutations (275insC and Q211X) were heterozygous in one patient. EP studies in this patient revealed no EP AChE, small nerve terminals, reduced presynaptic membrane length, as well as abnormally low-evoked quantal release. The third mutation (G240X) was homozygous in six Palestinian Arab families of the same tribe and in an Iraqi Jewish patient. Expression studies of the three mutations in COS cells indicate that each abrogates formation of insertion competent asymmetric AChE. Although the three mutations have identical predicted consequences at the EP, their phenotypic expressivity varies as regards age at onset, rate of progression, and severity of symptoms. CONCLUSIONS: 1) After mutations in the AChR epsilon subunit, mutations in COLQ are emerging as second most common cause of congenital myasthenic syndromes. 2) A founder effect is likely for G240X in the Palestinian Arab families. 3) That mutations predicting total absence of AChE from the EP have variable phenotypic expressivity suggests that modifying genes or environmental factors can partially compensate for EP AChE deficiency.

The earliest pathologic alterations in dysferlinopathy.

BACKGROUND: Dysferlinopathies are associated with proximal or distal muscular dystrophy. Dysferlin immunolocalizes to the muscle fiber periphery but does not associate with the dystrophin--glycoprotein complex; its function in humans, and the mechanism by which it causes muscle fiber injury, are not known. The authors therefore searched for pathogenetic clues by examining early abnormalities in nonnecrotic muscle fibers in dysferlinopathy. Five dysferlin-deficient patients were investigated. Weakness was distal in two, proximal in one, and both proximal and distal in two. Patient 5 was only mildly affected. METHODS: Immunoblot analysis, membrane attack complex (MAC) immunolocalization, and quantitative electron microscopy. RESULTS: In Patients 1 through 4, but not in 5, part or the entire surface of isolated nonnecrotic muscle fibers immunostained for MAC. Quantitative electron microscopy of 175 nonnecrotic muscle fibers revealed one or more of the following: 1) small (0.11 to 1.8 microm) plasmalemmal defects in 64% of fibers; 2) thickened basal lamina over some defects; 3) replacement of the plasma membrane by one to multiple layers of small vesicles in 57% of fibers; 4) papillary projections, frequently disintegrating, in 24 to 36% of fibers in Patients 1 through 4 but absent in fibers of Patient 5; 5) small subsarcolemmal vacuoles, some undergoing exocytosis, in 57% of fibers; and 6) infrequent subsarcolemmal regions containing rough endoplasmic reticulum and abundant free ribosomes. CONCLUSIONS: Dysferlin is likely required for maintaining the structural integrity of the muscle fiber plasma membrane, and plasma membrane injury is an early event in the pathogenesis of dysferlinopathy. MAC activation can participate in but is not an initial or primary event causing muscle fiber injury.

Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans.

Choline acetyltransferase (ChAT; EC ) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist in Caenorhabditis elegans and Drosophila, but no CHAT mutations have been observed in humans to date. Here we report that mutations in CHAT cause a congenital myasthenic syndrome associated with frequently fatal episodes of apnea (CMS-EA). Studies of the neuromuscular junction in this disease show a stimulation-dependent decrease of the amplitude of the miniature endplate potential and no deficiency of the ACh receptor. These findings point to a defect in ACh resynthesis or vesicular filling and to CHAT as one of the candidate genes. Direct sequencing of CHAT reveals 10 recessive mutations in five patients with CMS-EA. One mutation (523insCC) is a frameshifting null mutation. Three mutations (I305T, R420C, and E441K) markedly reduce ChAT expression in COS cells. Kinetic studies of nine bacterially expressed ChAT mutants demonstrate that one mutant (E441K) lacks catalytic activity, and eight mutants (L210P, P211A, I305T, R420C, R482G, S498L, V506L, and R560H) have significantly impaired catalytic efficiencies.

Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome.

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation epsilonA411P in the amphipathic helix of the acetylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three unrelated families are either homozygous for epsilonA411P or are heterozygous and harbor a null mutation in the second epsilon allele, indicating that epsilonA411P is recessive. We expressed human AChRs containing wild-type or A411P epsilon subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by epsilonA411P. Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.

AlphaB-crystallin immunolocalization yields new insights into inclusion body myositis.

OBJECTIVE: To study the expression of the small heat shock protein, alphaB-crystallin (alphaBC), in inclusion body myositis (IBM). BACKGROUND: In humans, alphaBC is constitutively expressed in the eye lens, muscle, and heart, but not in lymphoid tissues. Induced expression of alphaBC occurs under metabolic stress, in virus-infected lymphocytes, and in degenerative brain lesions, including neurofibrillary tangles and senile plaques in AD. The previously reported pathologic similarities between AD and IBM prompted us to study alphaBC expression in IBM. METHODS: Immunolocalization of alphaBC in muscle of 11 patients with IBM, 50 patients with other muscle diseases, and 4 controls; and quantitative analysis of the frequency of fibers with 1) increased alphaBC expression in IBM and polymyositis and 2) structural abnormality (vacuolated, non-necrotic and invaded by mononuclear cells, Congo red-positive, SMI-31 positive, and ubiquitin positive) in IBM. RESULTS: We detected enhanced expression of alphaBC not only in all structurally abnormal IBM fibers, but also, and with severalfold higher frequency, in IBM fibers without significant structural abnormality (X fibers) (p values in paired t-tests < 0.001). We also found enhanced alphaBC in abnormal fibers in other diseases; X fibers, however, were extremely sparse or absent, except in two atypical cases of polymyositis refractory to immunotherapy. CONCLUSION: That the X fibers are much more frequent than the structurally abnormal fibers in IBM points to a pathogenic stressor acting upstream to the development of structural abnormalities. The identification of this stressor is now of paramount importance for deciphering the enigma of IBM.

The spectrum of mutations causing end-plate acetylcholinesterase deficiency.

The end-plate species of acetylcholinesterase (AChE) is an asymmetric enzyme consisting of a collagenic tail subunit composed of three collagenic strands (ColQ), each attached to a tetramer of the T isoform of the catalytic subunit (AChE(T)) via a proline-rich attachment domain. The principal function of the tail subunit is to anchor asymmetric AChE in the synaptic basal lamina. Human end-plate AChE deficiency was recently shown to be caused by mutations in COLQ. We here report nine novel COLQ mutations in 7 patients with end-plate AChE deficiency. We examine the effects of the mutations on the assembly of asymmetric AChE by coexpressing each genetically engineered COLQ mutant with ACHE(T) in COS cells. We classify the newly recognized and previously reported COLQ mutations into four classes according to their position in ColQ and their effect on AChE expression. We find that missense mutations in the proline-rich attachment domain abrogate attachment of catalytic subunits, that truncation mutations in the ColQ collagen domain prevent the assembly of asymmetric AChE, that hydrophobic missense residues in the C-terminal domain prevent triple helical assembly of the ColQ collagen domain, and that other mutations in the C-terminal region produce asymmetric species of AChE that are likely insertion incompetent.