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INTRODUCTION Congenital myasthenic syndromes (CMS) usually present neonatally or in early childhood. When they present later, they may be mistaken for seronegative autoimmune myasthenia, and unnecessary immunosuppressive treatment may be administered. METHODS Patients who met criteria for seronegative generalized myasthenia without congenital or early childhood onset, but with an affected sibling were tested for CMS associated genes using exome and Sanger sequencing. RESULTS Four sibling pairs from nonconsanguineous families were identified. Three had mutations in the RAPSN gene, and 1 had a mutation in CHRNA1. One sibling of a pair with symptoms of fatigue but no convincing features of neuromuscular dysfunction tested negative on genetic studies. The definite CMS cases comprised 7 of 25 seronegative patients with definite generalized myasthenia in the clinic, and over half had been treated for autoimmune myasthenia. CONCLUSIONS CMS is probably underdiagnosed in seronegative myasthenic disorders and should be considered in the differential diagnosis. Muscle Nerve 54: 721-727, 2016.

bicoid mRNA localises to the Drosophila oocyte anterior from stage 9 of oogenesis onwards to provide a local source for Bicoid protein for embryonic patterning. Live imaging at stage 9 reveals that bicoid mRNA particles undergo rapid Dynein-dependent movements near the oocyte anterior, but with no directional bias. Furthermore, bicoid mRNA localises normally in shot2A2, which abolishes the polarised microtubule organisation. FRAP and photo-conversion experiments demonstrate that the RNA is stably anchored at the anterior, independently of microtubules. Thus, bicoid mRNA is localised by random active transport and anterior anchoring. Super-resolution imaging reveals that bicoid mRNA forms 110–120 nm particles with variable RNA content, but constant size. These particles appear to be well-defined structures that package the RNA for transport and anchoring. DOI: http://dx.doi.org/10.7554/eLife.17537.001, eLife digest Molecules of messenger RNA, or mRNA for short, contain the instructions needed to make proteins. Many mRNAs are only found in certain parts of the cell to ensure that the corresponding proteins are only produced where they are actually needed. The mRNAs are delivered to their final location in the cell by motor proteins that move along tracks made of filaments called microtubules. In female fruit flies, a mRNA called bicoid is transported to front end of a developing egg cell, while another mRNA called oskar is moved to the rear end. When the egg is fertilized, the region that contains bicoid mRNA develops into the head of the embryo, while the other end gives rise to the abdomen. A motor protein called kinesin-1 transports the oskar mRNA to the rear end, but how bicoid mRNA moves to the front end is not clear. Trovisco et al. used microscopy to study how bicoid mRNA moves. The experiments show that another motor protein called Dynein moves bicoid mRNA along microtubules. However, unlike oskar mRNA, bicoid mRNA moves along microtubules in all directions and is not biased towards the front end of the cell. Trovisco et al. hypothesized that when bicoid mRNA reaches the front end of the egg it is trapped there by other factors. Further experiments found that bicoid mRNA is indeed anchored at the front end of the cell. The mRNA does not seem to be trapped at the ends of the microtubules along which it is transported, nor does it form large clumps. Instead, it forms small, well-defined particles that remain the same size as the egg develops. The findings of Trovisco et al. raise the possibility that bicoid mRNA is packaged into these particles in order to be transported and anchored at the front end of the egg cell. Future work is needed to understand how particles containing bicoid mRNA are tethered at the front end of the egg cell and whether other mRNAs are also packaged in a similar manner. DOI: http://dx.doi.org/10.7554/eLife.17537.002

SummaryProtein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic ‘lipid-altered’ tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics forMycobacterium tuberculosis, enabling treatmentin vitro,in celluloandin vivothereby providing a promising new class of antimicrobial drug.HighlightsStructures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysisDPAGT1 mutants in patients with glycosylation disorders modulate DPAGT1 activityStructures, kinetics and biosynthesis reveal role of lipid in tunicamycinLipid-altered, tunicamycin analogues give non-toxic antibiotics against TB

Protein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic ‘lipid-altered’ tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo thereby providing a promising new class of antimicrobial drug.

Congenital myasthenic syndromes are associated with impairments in neuromuscular transmission. Belaya et al. show that mutations of the glycosylation pathway enzyme GMPPB, which has previously been implicated in muscular dystrophy dystroglycanopathy, also cause a congenital myasthenic syndrome. This differential diagnosis is important to ensure that affected individuals receive appropriate medication., Congenital myasthenic syndromes are inherited disorders that arise from impaired signal transmission at the neuromuscular junction. Mutations in at least 20 genes are known to lead to the onset of these conditions. Four of these, ALG2, ALG14, DPAGT1 and GFPT1, are involved in glycosylation. Here we identify a fifth glycosylation gene, GMPPB, where mutations cause congenital myasthenic syndrome. First, we identified recessive mutations in seven cases from five kinships defined as congenital myasthenic syndrome using decrement of compound muscle action potentials on repetitive nerve stimulation on electromyography. The mutations were present through the length of the GMPPB, and segregation, in silico analysis, exon trapping, cell transfection followed by western blots and immunostaining were used to determine pathogenicity. GMPPB congenital myasthenic syndrome cases show clinical features characteristic of congenital myasthenic syndrome subtypes that are due to defective glycosylation, with variable weakness of proximal limb muscle groups while facial and eye muscles are largely spared. However, patients with GMPPB congenital myasthenic syndrome had more prominent myopathic features that were detectable on muscle biopsies, electromyography, muscle magnetic resonance imaging, and through elevated serum creatine kinase levels. Mutations in GMPPB have recently been reported to lead to the onset of muscular dystrophy dystroglycanopathy. Analysis of four additional GMPPB-associated muscular dystrophy dystroglycanopathy cases by electromyography found that a defective neuromuscular junction component is not always present. Thus, we find mutations in GMPPB can lead to a wide spectrum of clinical features where deficit in neuromuscular transmission is the major component in a subset of cases. Clinical recognition of GMPPB-associated congenital myasthenic syndrome may be complicated by the presence of myopathic features, but correct diagnosis is important because affected individuals can respond to appropriate treatments.

Mutations in DPAGT1 are a newly recognised cause of congenital myasthenic syndrome. DPAGT1 encodes an early component of the N-linked glycosylation pathway. Initially mutations in DPAGT1 have been associated with the onset of the severe multisystem disorder – congenital disorder of glycosylation type 1J. However, recently it was established that certain mutations in this gene can cause symptoms restricted to muscle weakness resulting from defective neuromuscular transmission. We report four cases from a large Iranian pedigree with prominent limb-girdle weakness and minimal craniobulbar symptoms who harbour a novel mutation in DPAGT1, c.652C>T, p.Arg218Trp. This myasthenic syndrome may mimic myopathic disorders and is likely under-diagnosed.

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies targeting proteins expressed at the neuromuscular junction (NMJ). In most cases the targets are acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), or occasionally low-density lipoprotein receptor-related protein 4 (LRP4), but there is still a group of patients, often called seronegative MG (SNMG), with unknown antibody targets. One potential target is collagen Q (COLQ), which is restricted to the NMJ and is crucial for anchoring the NMJ-specific form of acetylcholinesterase (AChE). 415 serum samples with a clinical diagnosis of MG and 43 control samples were screened for the presence of COLQ autoantibodies using a cell-based assay (CBA) with HEK293 cells overexpressing COLQ at the cell surface. COLQ antibodies were detected in 12/415 MG sera and in one/43 control samples. Five of the COLQ-Ab + individuals were also positive for AChR-Abs and 2 for MuSK-Abs. Although the COLQ antibodies were only present at low frequency, and did not differ significantly from the small control cohort, further studies could address whether they modify the clinical presentation or the benefits of anti-cholinesterase therapy.

Background A newly defined congenital myasthenic syndrome (CMS) caused by DPAGT1 mutations has recently been reported. While many other CMS-associated proteins have discrete roles localised to the neuromuscular junction, DPAGT1 is ubiquitously expressed, modifying many proteins, and as such is an unexpected cause of isolated neuromuscular involvement. Methods We present detailed clinical characteristics of five patients with CMS caused by DPAGT1 mutations. Results Patients have prominent limb girdle weakness and minimal craniobulbar symptoms. Tubular aggregates on muscle biopsy are characteristic but may not be apparent on early biopsies. Typical myasthenic features such as pyridostigmine and 3, 4- diaminopyridine responsiveness, and decrement on repetitive nerve stimulation are present. Conclusions These patients mimic myopathic disorders and are likely to be under-diagnosed. The descriptions here should facilitate recognition of this disorder. In particular minimal craniobulbar involvement and tubular aggregates on muscle biopsy help to distinguish DPAGT1 CMS from the majority of other forms of CMS. Patients with DPAGT1 CMS share similar clinical features with patients who have CMS caused by mutations in GFPT1 , another recently identified CMS subtype.

Background Congenital myasthenic syndrome (CMS) due to mutations in GMPPB has recently been reported confirming the importance of glycosylation for the integrity of neuromuscular transmission. Methods Review of case notes of patients with mutations in GMPPB to identify the associated clinical, neurophysiological, pathological and laboratory features. In addition, serum creatine kinase (CK) levels within the Oxford CMS cohort were retrospectively analysed to assess its usefulness in the differential diagnosis of this new entity. Results All patients had prominent limb-girdle weakness with minimal or absent craniobulbar manifestations. Presentation was delayed beyond infancy with proximal muscle weakness and most patients recall poor performance in sports during childhood. Neurophysiology showed abnormal neuromuscular transmission only in the affected muscles and myopathic changes. Muscle biopsy showed dystrophic features and reduced α-dystroglycan glycosylation. In addition, myopathic changes were present on muscle MRI. CK was significantly increased in serum compared to other CMS subtypes. Patients were responsive to pyridostigimine alone or combined with 3,4-diaminopyridine and/or salbutamol. Conclusions Patients with GMPPB-CMS have phenotypic features aligned with CMS subtypes harbouring mutations within the early stages of the glycosylation pathway. Additional features shared with the dystroglycanopathies include myopathic features, raised CK levels and variable mild cognitive delay. This syndrome underlines that CMS can occur in the absence of classic myasthenic manifestations such as ptosis and ophthalmoplegia or facial weakness, and links myasthenic disorders with dystroglycanopathies. This report should facilitate the recognition of this disorder, which is likely to be underdiagnosed and can benefit from symptomatic treatment.

Congenital myasthenic syndromes (CMS) usually present neonatally or in early childhood. When they present later, they may be mistaken for seronegative autoimmune myasthenia, and unnecessary immunosuppressive treatment may be administered.Patients who met criteria for seronegative generalized myasthenia without congenital or early childhood onset, but with an affected sibling were tested for CMS associated genes using exome and Sanger sequencing.Four sibling pairs from nonconsanguineous families were identified. Three had mutations in the RAPSN gene, and 1 had a mutation in CHRNA1. One sibling of a pair with symptoms of fatigue but no convincing features of neuromuscular dysfunction tested negative on genetic studies. The definite CMS cases comprised 7 of 25 seronegative patients with definite generalized myasthenia in the clinic, and over half had been treated for autoimmune myasthenia.CMS is probably underdiagnosed in seronegative myasthenic disorders and should be considered in the differential diagnosis. Muscle Nerve 54: 721-727, 2016.

Around 80% of myasthenia gravis patients have antibodies against the acetylcholine receptor, and 0-60% of the remaining patients have antibodies against the muscle-specific tyrosine kinase, MuSK. Another recently identified antigen is low-density lipoprotein receptor-related protein 4 (Lrp4). To improve the existing assays and widen the search for new antigenic targets, we have employed cell-based assays in which candidate target proteins are expressed on the cell surface of transfected cells and probed with patient sera. These assays, combined with use of myotube cultures to explore the effects of the antibodies, enable us to begin to identify new antigenic targets and test antibody pathogenicity in vitro.

Congenital myasthenic syndromes (CMS) are a group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. This is a heterogenous group of disorders with 15 different genes implicated in the development of the disease. Using whole-exome sequencing we identified DPAGT1 as a new gene associated with CMS. DPAGT1 catalyses the first step of N-linked protein glycosylation. DPAGT1 patients are characterized by weakness of limb muscles, response to treatment with cholinesterase inhibitors, and the presence of tubular aggregates on muscle biopsy. We showed that DPAGT1 is required for glycosylation of acetylcholine receptor (AChR) subunits and efficient export of AChR to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1-associated CMS is reduced levels of AChRs at the endplate region. This finding demonstrates that impairment of the N-linked glycosylation pathway can lead to the development of CMS.

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed whole-exome sequencing to determine the underlying defect in a group of individuals with an inherited limb-girdle pattern of myasthenic weakness. We identify DPAGT1 as a gene in which mutations cause a congenital myasthenic syndrome. We describe seven different mutations found in five individuals with DPAGT1 mutations. The affected individuals share a number of common clinical features, including involvement of proximal limb muscles, response to treatment with cholinesterase inhibitors and 3,4-diaminopyridine, and the presence of tubular aggregates in muscle biopsies. Analyses of motor endplates from two of the individuals demonstrate a severe reduction of endplate acetylcholine receptors. DPAGT1 is an essential enzyme catalyzing the first committed step of N-linked protein glycosylation. Our findings underscore the importance of N-linked protein glycosylation for proper functioning of the neuromuscular junction. Using the DPAGT1-specific inhibitor tunicamycin, we show that DPAGT1 is required for efficient glycosylation of acetylcholine-receptor subunits and for efficient export of acetylcholine receptors to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1 mutations is reduced levels of acetylcholine receptors at the endplate region. These individuals share clinical features similar to those of congenital myasthenic syndrome due to GFPT1 mutations, and their disorder might be part of a larger subgroup comprising the congenital myasthenic syndromes that result from defects in the N-linked glycosylation pathway and that manifest through impaired neuromuscular transmission. © 2012 The American Society of Human Genetics.

Congenital myasthenic syndromes (CMS) are a group of inherited diseases that affect synaptic transmission at the neuromuscular junction and result in fatiguable muscle weakness. A subgroup of CMS patients have a recessively inherited limb-girdle pattern of weakness caused by mutations in DOK7. DOK7 encodes DOK7, an adaptor protein that is expressed in the skeletal muscle and heart and that is essential for the development and maintenance of the neuromuscular junction. We have screened the DOK7 gene for mutations by polymerase chain reaction amplification and bi-directional sequencing of exonic and promoter regions and performed acetylcholine receptor (AChR) clustering assays and used exon trapping to determine the pathogenicity of detected variants. Approximately 18% of genetically diagnosed CMSs in the UK have mutations in DOK7, with mutations in this gene identified in more than 60 kinships to date. Thirty-four different pathogenic mutations were identified as well as 27 variants likely to be non-pathogenic. An exon 7 frameshift duplication c.1124_1127dupTGCC is commonly found in at least one allele. We analyse the effect of the common frameshift c.1124_1127dupTGCC and show that 10/11 suspected missense mutations have a deleterious effect on AChR clustering. We identify for the first time homozygous or compound heterozygous mutations that are localized 5' to exon 7. In addition, three silent variants in the N-terminal half of DOK7 are predicted to alter the splicing of the DOK7 RNA transcript. The DOK7 gene is highly polymorphic, and within these many variants, we define a spectrum of mutations that can underlie DOK7 CMS that will inform in managing this disorder.

Around 80% of myasthenia gravis patients have antibodies against the acetylcholine receptor, and 0-60% of the remaining patients have antibodies against the muscle-specific tyrosine kinase, MuSK. Another recently identified antigen is low-density lipoprotein receptor-related protein 4 (Lrp4). To improve the existing assays and widen the search for new antigenic targets, we have employed cell-based assays in which candidate target proteins are expressed on the cell surface of transfected cells and probed with patient sera. These assays, combined with use of myotube cultures to explore the effects of the antibodies, enable us to begin to identify new antigenic targets and test antibody pathogenicity in vitro.

Congenital myasthenic syndromes (CMS) are disorders caused by mutations in genes encoding proteins that are essential for maintaining the integrity of neuromuscular transmission. In recent years we have identified an unexpected relationship between glycosylation defects and CMS by using next generation sequencing. Patients with mutations affecting the early stages of glycosylation constitute a clinically distinctive group of disorders. Unlike other CMS subtypes, the presentation is delayed beyond infancy with prominent limb-girdle weakness and minimal or absent craniobulbar manifestations. In fact, we find that they occur in the absence of classic myasthenic manifestations such as ptosis, ophthalmoplegia or facial weakness, and therefore are likely to be under-diagnosed. In addition, the presence of additional features such as mild cognitive delay, cataracts, raised CK levels, myopathic abnormalities on muscle biopsy and muscle MRI, and selective abnormalities of neuromuscular transmission on electrophysiological testing, make them easily misdiagnosed as myopathies or muscular dystrophies. The recognition of these disorders is important since they can be treated symptomatically with cholinesterase inhibitors.

Asymmetric mRNA localisation to specific compartments of the cell is a fundamental mechanism of -spatial and temporal regulation of gene expression. It is used by a variety of organisms and cell types to achieve different cellular functions. However, the mechanisms of mRNA localisation are not well understood. An important advance in this field has been the development of techniques that allow the visualisation of mRNA movements in living cells in real time. In this paper, we describe one approach to visualising mRNA localisation in vivo, in which RNAs containing MS2 binding sites are labelled by the MS2 coat protein fused to fluorescent reporters. We discuss the use of this mRNA-MS2/MS2CP-FP system to study mRNA localisation during Drosophila oogenesis, and provide a detailed explanation of the steps required for this approach, including the design of the mRNA-MS2 and MS2CP-FP constructs, the preparation of fly oocytes for imaging, the optimal microscope configurations for live cell imaging, and strategies for image processing and analysis.

Asymmetric mRNA localisation to specific compartments of the cell is a fundamental mechanism of -spatial and temporal regulation of gene expression. It is used by a variety of organisms and cell types to achieve different cellular functions. However, the mechanisms of mRNA localisation are not well understood. An important advance in this field has been the development of techniques that allow the visualisation of mRNA movements in living cells in real time. In this paper, we describe one approach to visualising mRNA localisation in vivo, in which RNAs containing MS2 binding sites are labelled by the MS2 coat protein fused to fluorescent reporters. We discuss the use of this mRNA-MS2/MS2CP-FP system to study mRNA localisation during Drosophila oogenesis, and provide a detailed explanation of the steps required for this approach, including the design of the mRNA-MS2 and MS2CP-FP constructs, the preparation of fly oocytes for imaging, the optimal microscope configurations for live cell imaging, and strategies for image processing and analysis.

Summary oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.

Nucleo-cytoplasmic shuttling is an important feature of proteins involved in nuclear export/import of RNAs, proteins, and also large ribonucleoprotein complexes such as ribosomes. The vast amount of proteomic data available shows that many of these processes are highly dynamic. Therefore, methods are needed to reliably assess whether a protein shuttles between nucleus and cytoplasm, and the kinetics with which it exchanges. Here we describe a combination of the classical heterokaryon assay with fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques, which allows an assessment of the kinetics of protein shuttling in the yeast Saccharomyces cerevisiae.