Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect.

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.

Mutation analysis of CHRNA1, CHRNB1, CHRND, and RAPSN genes in multiple pterygium syndrome/fetal akinesia patients.

Multiple pterygium syndromes (MPS) comprise a group of multiple congenital anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). MPS are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. Previously, we and others reported that recessive mutations in the embryonal acetylcholine receptor g subunit (CHRNG) can cause both lethal and nonlethal MPS, thus demonstrating that pterygia resulted from fetal akinesia. We hypothesized that mutations in acetylcholine receptor-related genes might also result in a MPS/fetal akinesia phenotype and so we analyzed 15 cases of lethal MPS/fetal akinesia without CHRNG mutations for mutations in the CHRNA1, CHRNB1, CHRND, and rapsyn (RAPSN) genes. No CHRNA1, CHRNB1, or CHRND mutations were detected, but a homozygous RAPSN frameshift mutation, c.1177-1178delAA, was identified in a family with three children affected with lethal fetal akinesia sequence. Previously, RAPSN mutations have been reported in congenital myasthenia. Functional studies were consistent with the hypothesis that whereas incomplete loss of rapsyn function may cause congenital myasthenia, more severe loss of function can result in a lethal fetal akinesia phenotype.

IgG1 antibodies to acetylcholine receptors in 'seronegative' myasthenia gravis.

Only around 80% of patients with generalized myasthenia gravis (MG) have serum antibodies to acetylcholine receptor [AChR; acetylcholine receptor antibody positive myasthenia gravis (AChR-MG)] by the radioimmunoprecipitation assay used worldwide. Antibodies to muscle specific kinase [MuSK; MuSK antibody positive myasthenia gravis (MuSK-MG)] make up a variable proportion of the remaining 20%. The patients with neither AChR nor MuSK antibodies are often called seronegative (seronegative MG, SNMG). There is accumulating evidence that SNMG patients are similar to AChR-MG in clinical features and thymic pathology. We hypothesized that SNMG patients have low-affinity antibodies to AChR that cannot be detected in solution phase assays, but would be detected by binding to the AChRs on the cell membrane, particularly if they were clustered at the high density that is found at the neuromuscular junction. We expressed recombinant AChR subunits with the clustering protein, rapsyn, in human embryonic kidney cells and tested for binding of antibodies by immunofluorescence. To identify AChRs, we tagged either AChR or rapsyn with enhanced green fluorescence protein, and visualized human antibodies with Alexa Fluor-labelled secondary or tertiary antibodies, or by fluorescence-activated cell sorter (FACS). We correlated the results with the thymic pathology where available. We detected AChR antibodies to rapsyn-clustered AChR in 66% (25/38) of sera previously negative for binding to AChR in solution and confirmed the results with FACS. The antibodies were mainly IgG1 subclass and showed ability to activate complement. In addition, there was a correlation between serum binding to clustered AChR and complement deposition on myoid cells in patients' thymus tissue. A similar approach was used to demonstrate that MuSK antibodies, although mainly IgG4, were partially IgG1 subclass and capable of activating complement when bound to MuSK on the cell surface. These observations throw new light on different forms of MG paving the way for improved diagnosis and management, and the approaches used have applicability to other antibody-mediated conditions.

Clinical features of the DOK7 neuromuscular junction synaptopathy.

Mutations in DOK7 have recently been shown to underlie a recessive congenital myasthenic syndrome (CMS) associated with small simplified neuromuscular junctions ('synaptopathy') but normal acetylcholine receptor and acetylcholinesterase function. We identified DOK7 mutations in 27 patients from 24 kinships. Mutation 1124_1127dupTGCC was common, present in 20 out of 24 kinships. All patients were found to have at least one allele with a frameshift mutation in DOK7 exon 7, suggesting that loss of function(s) associated with the C-terminal region of Dok-7 underlies this disorder. In 15 patients, we were able to study the clinical features in detail. Clinical onset was usually characterized by difficulty in walking developing after normal motor milestones. Proximal muscles were usually more affected than distal, leading to a 'limb-girdle' pattern of weakness; although ptosis was often present from an early age, eye movements were rarely involved. Patients did not show long-term benefit from anticholinesterase medication and sometimes worsened, and where tried responded to ephedrine. The phenotype can be distinguished from 'limb-girdle' myasthenia associated with tubular aggregates, where DOK7 mutations were not detected and patients respond to anticholinesterase treatments. CMS due to DOK7 mutations are common within our UK cohort and is likely to be under-diagnosed; recognition of the phenotype will help clinical diagnosis, targeted genetic screening and appropriate management.

Effect of sera from AChR-antibody negative myasthenia gravis patients on AChR and MuSK in cell cultures.

A proportion of patients with myasthenia gravis (MG) do not have antibodies to the acetylcholine receptor (AChR). Some of these patients have antibodies to muscle specific kinase (MuSK), whereas others have neither antibody (seronegative MG, SNMG). Both MuSK antibody positive MG (MuSK-MG) and SNMG are antibody-mediated diseases but how they cause neuromuscular junction failure is not clear. One possibility is that they reduce the clustering and expression of AChRs. We looked at the effects of MuSK-MG and SNMG sera/IgG on surface AChR distribution and expression, and AChR subunit and MuSK mRNA by quantitative RT-PCR, in TE671 and C2C12 myotubes. In TE671 cells MuSK-MG sera reduced AChR expression by about 20%, but had no effect on AChR subunit or MuSK mRNA expression. In C2C12 myotubes, MuSK-MG sera caused a reduction in the number of agrin-induced clusters, but the clusters became larger and there was no significant effect on total surface AChR numbers or AChR subunit or MuSK mRNA. By contrast, SNMG sera not only reduced AChR numbers by about 20% in TE671 cells, but modestly upregulated AChR gamma subunit expression in TE671 cells and both AChR gamma subunit and MuSK expression in C2C12 myotubes. Thus, although these results have, disappointingly, demonstrated little effect of MuSK antibodies on AChR expression, they do imply that SNMG antibodies act on AChR-associated pathways.

CHRND mutation causes a congenital myasthenic syndrome by impairing co-clustering of the acetylcholine receptor with rapsyn.

The objective of this study was to analyse the mutations of the acetylcholine receptor (AChR) delta subunit gene (CHRND) in a patient with sporadic congenital myasthenic syndrome (CMS). Mutations in various genes encoding proteins expressed at the neuromuscular junction may cause CMS. Mutations of AChR subunit genes lead to end-plate AChR deficiency or to altered kinetic properties of the receptor. Mutations in the alpha, beta and delta subunits of the AChR are less frequent than mutations of the epsilon subunit; mutations in these subunits leading to AChR deficiency are often associated with a severe phenotype. A sporadic patient from Germany was studied, who presented with an early onset CMS associated with feeding difficulties, ptosis, a moderate general weakness responsive to anticholinesterase treatment and recurrent episodes of respiratory insufficiency provoked by infections. The CHRND gene was screened for mutations by RFLP, long-range PCR and sequence analysis. Subsequently, we conducted functional studies of AChR mutants co-transfected with rapsyn in HEK 293 cells. Heterozygously to a 2.2 kb microdeletion disrupting the CHRND gene, we identified a novel point mutation in the long cytoplasmic loop, CHRND E381K. The cytoplasmic loop of the AChR subunits is known to be essential for AChR-rapsyn co-clustering. We therefore studied the interaction of AChR containing the CHRND E381K mutation with rapsyn by evaluating expression and co-localization of rapsyn and mutated AChR subunits in co-transfected HEK 293 cells. Interestingly, the mutated receptor showed severely reduced cluster formation compared with the wild-type receptor. In contrast, the corresponding amino acid substitution in the cytoplasmic loop of the AChR epsilon (CHRNE E376K) as well as a recently reported CMS mutation affecting this domain (CHRNE N436del) had no impact on cluster formation. CHRND mutations are a rare cause for CMS but should be considered in patients with a severe, early onset disease form, clinically resembling a rapsyn phenotype with recurrent episodic apnoeas. Our results suggest that impairment of AChR-rapsyn co-clustering--a well-known molecular mechanism for rapsyn mutations--could also result from mutations in the delta subunit. Introduction of the same mutation in the epsilon subunit had no effect on AChR clustering indicating a special role of the delta subunit in AChR-rapsyn interactions.

Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations.

Congenital myasthenic syndromes are inherited disorders of neuromuscular transmission characterized by fatigable muscle weakness. Autosomal recessive acetylcholine receptor (AChR) deficiency syndromes, in which levels of this receptor at the neuromuscular junction are severely reduced, may be caused by mutations within genes encoding the AChR or the AChR-clustering protein, rapsyn. Most patients have mutations within the rapsyn coding region and are either homozygous for N88K or heteroallelic for N88K and a second mutation. In some cases the second allele carries a null mutation but in many the mutations are missense, and are located in different functional domains. Little is known about the functional effects of these mutations, but we hypothesize that they would have an effect on AChR clustering by a variety of mechanisms that might correlate with disease severity. Here we expressed RAPSN mutations A25V, N88K, R91L, L361R and K373del in TE671 cells and in rapsyn-/- myotubes to determine their pathogenic mechanisms. The A25Vmutation impaired colocalization of rapsyn with AChR and prevented agrin-induced AChR clusters in rapsyn-/- myotubes. In TE671 cells, R91L reduced the ability of rapsyn to self-associate, and K373del-rapsyn was significantly less stable than wild-type. The effects of mutations L361R and N88K were more subtle: in TE671 cells, in comparison with wild-type rapsyn, L361R-rapsyn showed reduced expression/stability, and both N88K-rapsyn and L361R-rapsyn showed significantly reduced co-localization with AChR. N88K-rapsyn and L361R-rapsyn could effectively mediate agrin-induced AChR clusters, but these were reduced in number and were less stable than with wild-type rapsyn. The disease severity of patients harbouring the compound allelic mutations was greater than that of patients with homozygous rapsyn mutation N88K, suggesting that the second mutant allele may largely determine severity.

IgG-specific cell-based assay detects potentially pathogenic MuSK-Abs in seronegative MG.

OBJECTIVE: To increase the detection of MuSK-Abs using a CBA and test their pathogenicity. METHODS: Sera from 69 MuSK-RIA-positive patients with myasthenia gravis (MG) (Definite MuSK-MG), 169 patients negative for MuSK-RIA and AChR-RIA (seronegative MG, SNMG), 35 healthy individuals (healthy controls, HCs), and 16 NMDA receptor-Ab-positive (NMDAR-Ab) disease controls were tested for binding to MuSK on a CBA using different secondary antibodies. RESULTS: Initially, in addition to 18% of SNMG sera, 11% of HC and 19% of NMDAR-Ab sera showed positive binding to MuSK-transfected cells; this low specificity was due to anti-IgG(H+L) detection of IgM bound nonspecifically to MuSK. Using an IgG Fc gamma-specific secondary antibody, MuSK-Abs were detected by CBA in 68/69 (99%) of Definite MuSK-MG, 0/35 HCs, 0/16 NMDAR-Ab, and 14/169 (8%) of SNMG sera, providing increased sensitivity with high specificity. The RIA-negative, CBA-positive MuSK-IgG sera, but not IgM-MuSK-binding sera, reduced agrin-induced AChR clustering in C2C12 myotubes, qualitatively similar to RIA-positive MuSK-Abs. CONCLUSIONS: An IgG-specific MuSK-CBA can reliably detect IgG MuSK-Abs and increase sensitivity. In the MuSK-CBA, IgG specificity is essential. The positive sera demonstrated pathogenic potential in the in vitro AChR-clustering assay, although less effective than Definite MuSK-MG sera, and the patients had less severe clinical disease. Use of IgG-specific secondary antibodies may improve the results of other antibody tests. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that an IgG-specific MuSK-CBA identifies patients with MG.

Dok-7 mutations underlie a neuromuscular junction synaptopathy.

Congenital myasthenic syndromes (CMSs) are a group of inherited disorders of neuromuscular transmission characterized by fatigable muscle weakness. One major subgroup of patients shows a characteristic "limb girdle" pattern of muscle weakness, in which the muscles have small, simplified neuromuscular junctions but normal acetylcholine receptor and acetylcholinesterase function. We showed that recessive inheritance of mutations in Dok-7, which result in a defective structure of the neuromuscular junction, is a cause of CMS with proximal muscle weakness.

Hes6 regulates myogenic differentiation.

Hes6 is a basic helix-loop-helix transcription factor homologous to Drosophila Enhancer of Split (EoS) proteins. It is known to promote neural differentiation and to bind to Hes1, a related protein that is part of the Notch signalling pathway, affecting Hes1-regulated transcription. We show that Hes6 is expressed in the murine embryonic myotome and is induced on C2C12 myoblast differentiation in vitro. Hes6 binds DNA containing the Enhancer of Split E box (ESE) motif, the preferred binding site of Drosophila EoS proteins, and represses transcription of an ESE box reporter. When overexpressed in C2C12 cells, Hes6 impairs normal differentiation, causing a decrease in the induction of the cyclin-dependent kinase inhibitor, p21(Cip1), and an increase in the number of cells that can be recruited back into the cell cycle after differentiation in culture. In Xenopus embryos, Hes6 is co-expressed with MyoD in early myogenic development. Microinjection of Hes6 RNA in vivo in Xenopus embryos results in an expansion of the myotome, but suppression of terminal muscle differentiation and disruption of somite formation at the tailbud stage. Analysis of Hes6 mutants indicates that the DNA-binding activity of Hes6 is not essential for its myogenic phenotype, but that protein-protein interactions are. Thus, we demonstrate a novel role for Hes6 in multiple stages of muscle formation.

A mouse model of AChR deficiency syndrome with a phenotype reflecting the human condition.

The two subtypes of mammalian muscle nicotinic acetylcholine receptors (AChR) are generated by the substitution of the epsilon (adult) subunit for the gamma (fetal) subunit within the AChR pentamer. Null mutations of the adult AChR epsilon-subunit gene are the most common cause of the AChR deficiency syndrome. This is a disorder of neuromuscular transmission characterized by non-progressive fatigable muscle weakness present throughout life. In contrast with the human disorder, mice with AChR epsilon-subunit null mutations die between 10 and 14 weeks of age. We generated transgenic mice that constitutively express the human AChR gamma-subunit in an AChR epsilon-subunit 'knock-out' background. These mice, in which neuromuscular transmission is mediated by fetal AChR, live well into adult life but show striking similarities to human AChR deficiency syndrome. They display fatigable muscle weakness, reduced miniature endplate potentials and endplate potentials, reduced motor endplate AChR number and altered endplate morphology. Our results illustrate how species differences in the control of ion-channel gene expression may affect disease phenotype, demonstrate that expression of adult AChR subtype is not essential for long-term survival, and suggest that in patients with AChR deficiency syndrome, up-regulation of the gamma-subunit could be a beneficial therapeutic strategy.