Prof David Beeson

Research Area: Ion Channels and Disease
Scientific Themes: Molecular, Cell & Systems Biology and Genes, Genetics, Epigenetics & Genomics
Keywords: Ion channels, AChR, Congenital myasthenic syndromes, neuromuscular junction, synaptic disorders
Web Links:
Fluorescence images of the neuromuscular junction stained with antibodies to the acetylcholine receptor (red) and terminal region of the motor nerve (green)

Fluorescence images of the neuromuscular junction stained with antibodies to the acetylcholine ...

Fluorescence images of the neuromuscular junction stained with antibodies to the acetylcholine receptor (red) and terminal region of the motor nerve (green)

Fluorescence images of the neuromuscular junction stained with antibodies to the acetylcholine ...

Molecular Neurosciences forms an integral part of the Neurosciences Group, and collaborates with Professor Vincent, Professor Willcox and Dr Lang.

We are studying inherited diseases that affect neuromuscular transmission, focusing both on mutations that directly affect the muscle acetylcholine receptors (AChR) and on mutations in proteins that control synaptic structure and formation. The neuromuscular junction is both well understood and accessible for study. Functional analysis of mutations at the molecular level can be directly correlated with measurements of defective synaptic transmission in vivo and with the clinical features of the patients. Thus a detailed knowledge of inherited dysfunction of neuromuscular transmission forms a paradigm for investigation of other neurological syndromes that may result from defective synaptic transmission in the CNS.

At the clinical level the congenital myasthenic syndromes provide a fascinating series of subtly different phenotypes that often require different treatments. Although a series of different syndromes have been characterized in detail, including the slow channel syndrome, fast channel syndrome, AChR deficiency syndrome acetylcholinesterase deficiency, and CMS due to DOK7 mutations, in about 40% of cases the underlying genetics is still unknown.

Our present work aims to identify candidate genes, define underlying mutations, investigate the disease mechanisms at the molecular level, and then explore new therapeutic strategies.

The projects involve close collaboration with clinicians in the Department of Clinical Neurology in Oxford, and with neurologists from the UK and overseas. The success of the work has led to the group being commissioned to provide a National Service for mutation detection and treatment of congenital myasthenic syndromes, as part of a consortium chosen by the National Specialist Advisory Commissioning Service for the diagnosis of rare muscle diseases. The service provides a means for the rapid translation of discoveries in the laboratory into clinical practice.

Our studies also include a series of laboratory-based collaborative projects. Studies on proteins involved in neuromuscular junction synaptogenesis are performed with Professor Yamanashi (Tokyo Medical and Dental School, Japan), and gene therapies in disease models using catalytic nucleic acids is being studied with Dr Matthew Wood, Department of Human Anatomy, Oxford. In addition, we make up part of the Wellcome Trust-funded OXION consortium that aims to understand the function of ion channels and their role in disease.

Name Department Institution Country
Prof Yuji Yamanashi Tokyo Dental Hospital Japan
Rodríguez Cruz PM, Palace J, Ramjattan H, Jayawant S, Robb SA, Beeson D. 2015. Salbutamol and ephedrine in the treatment of severe AChR deficiency syndromes. Neurology, 85 (12), pp. 1043-1047. | Show Abstract | Read more

OBJECTIVE: To evaluate the response to salbutamol and ephedrine in the treatment of congenital myasthenic syndromes due to CHRNE mutations causing severe acetylcholine receptor (AChR)deficiency. METHODS: A cohort study of 6 patients with severe AChR deficiency, symptomatic despite optimal therapy with anticholinesterase and 3,4-diaminopyridine, were analyzed for their response to the addition of salbutamol or ephedrine to their medication. Baseline quantitative myasthenia gravis (QMG) (severity) scores were worse than 15 of 39. Patients were assessed in clinic with QMG and mobility scores. Pretreatment and 6- to 8-month follow-up scores were evaluated. RESULTS: All 6 patients tolerated treatment well and reported no side effects. There was a strong positive response to treatment over the 6- to 8-month assessment period with significant improvement in QMG (p = 0.027) and mobility scores. The analysis of subcomponents of the QMG score revealed marked improvement in upper (p = 0.028) and lower (p = 0.028) limb raise times. All patients reported enhanced activities of daily living at 6 to 8 months. CONCLUSIONS: Oral salbutamol and ephedrine appear to be effective treatments in severe cases ofAChR deficiency on pyridostigmine. They are well tolerated and improvement in strength can be dramatic. Classification of evidence: This study provides Class IV evidence that salbutamol or ephedrine improves muscle strength in patients with congenital myasthenia from severe AChR deficiency.

Belaya K, Rodríguez Cruz PM, Liu WW, Maxwell S, McGowan S, Farrugia ME, Petty R, Walls TJ et al. 2015. Mutations in GMPPB cause congenital myasthenic syndrome and bridge myasthenic disorders with dystroglycanopathies. Brain, 138 (Pt 9), pp. 2493-2504. | Show Abstract | Read more

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.

Taylor JC, Martin HC, Lise S, Broxholme J, Cazier JB, Rimmer A, Kanapin A, Lunter G et al. 2015. Factors influencing success of clinical genome sequencing across a broad spectrum of disorders. Nat Genet, 47 (7), pp. 717-726. | Show Abstract | Read more

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.

Rodríguez Cruz PM, Al-Hajjar M, Huda S, Jacobson L, Woodhall M, Jayawant S, Buckley C, Hilton-Jones D et al. 2015. Clinical Features and Diagnostic Usefulness of Antibodies to Clustered Acetylcholine Receptors in the Diagnosis of Seronegative Myasthenia Gravis. JAMA Neurol, 72 (6), pp. 642-649. | Show Abstract | Read more

IMPORTANCE: Cell-based assays (CBAs) were shown to improve detection of acetylcholine receptor (AChR) antibodies in patients with myasthenia gravis (MG). Herein, we asked whether these assays were able to help determine the diagnosis in patients studied in routine clinical practice. OBJECTIVES: To determine the diagnostic usefulness of CBAs in the diagnosis of MG and to compare the clinical features of patients with antibodies only to clustered AChRs with those of patients with seronegative MG (SNMG). DESIGN, SETTING, AND PARTICIPANTS: All patients with clinical suspicion of MG who were seen within the Division of Clinical Neurology at the John Radcliffe Hospital in Oxford, England, between November 1, 2009, and November 30, 2013. Their serum antibodies and clinical features were studied. EXPOSURES: Radioimmunoprecipitation assay (RIPA) and CBA were used to test for standard AChR antibodies and antibodies to clustered AChRs in 138 patients. All available samples from patients with SNMG were retrospectively tested for lipoprotein receptor-related protein 4 (LRP4) antibodies. MAIN OUTCOMES AND MEASURES: Demographic, clinical, neurophysiological, and laboratory data. RESULTS: In total, 138 patients were tested for antibodies to clustered AChRs, and 42 had a final diagnosis of MG. The clustered AChR CBA detected antibodies in 38.1% (16 of 42) of RIPA-negative patients with MG with 100% specificity. All patients with SNMG who were tested for LRP4 antibodies (21 of 26) were negative by CBA. Compared with patients with SNMG, patients with antibodies only to clustered AChRs had frequent prepubertal onset (62.5% [median age, 6 years; age range, 1-52 years] vs 11.5% [median age, 38 years; age range, 2-72 years], P ≤ .05), high prevalence of ocular MG (62.5% vs 42.3%), milder disease severity with less bulbar involvement (25.0% vs 46.2%), and absence of respiratory symptoms (0% vs 23.1%). Response to treatment and prognosis was good, with a reduced need for thymectomy (6.3% vs 19.2%) and a high proportion of patients going into remission (50.0% vs 8.3%, P ≤ .05). These observations also apply to the classic AChR MG phenotype seen in large series. CONCLUSIONS AND RELEVANCE: Cell-based assay is a useful procedure in the routine diagnosis of RIPA-negative MG, particularly in children. Patients with antibodies only to clustered AChRs appear to be younger and have milder disease than other patients with MG. These observations will have implications in planning treatment.

Pettingill P, Kramer HB, Coebergh JA, Pettingill R, Maxwell S, Nibber A, Malaspina A, Jacob A et al. 2015. Antibodies to GABAA receptor α1 and γ2 subunits: clinical and serologic characterization. Neurology, 84 (12), pp. 1233-1241. | Show Abstract | Read more

OBJECTIVE: To search for antibodies against neuronal cell surface proteins. METHODS: Using immunoprecipitation from neuronal cultures and tandem mass spectrometry, we identified antibodies against the α1 subunit of the γ-aminobutyric acid A receptor (GABAAR) in a patient whose immunoglobulin G (IgG) antibodies bound to hippocampal neurons. We searched 2,548 sera for antibodies binding to GABAAR α, β, and γ subunits on live HEK293 cells and identified the class, subclass, and GABAAR subunit specificities of the positive samples. RESULTS: GABAAR-Abs were identified in 40 of 2,046 (2%) referred sera previously found negative for neuronal antibodies, in 5/502 (1%) previously positive for other neuronal surface antibodies, but not in 92 healthy individuals. The antibodies in 40% bound to either the α1 (9/45, 20%) or the γ2 subunits (9/45, 20%) and were of IgG1 (94%) or IgG3 (6%) subclass. The remaining 60% had lower antibody titers (p = 0.0005), which were mainly immunoglobulin M (IgM) (p = 0.0025), and showed no defined subunit specificity. Incubation of primary hippocampal neurons with GABAAR IgG1 sera reduced surface GABAAR membrane expression. The clinical features of 15 patients (GABAAR α1 n = 6, γ2 n = 5, undefined n = 4) included seizures (47%), memory impairment (47%), hallucinations (33%), or anxiety (20%). Most patients had not been given immunotherapies, but one with new-onset treatment-resistant catatonia made substantial improvement after plasma exchange. CONCLUSIONS: The GABAAR α1 and γ2 are new targets for antibodies in autoimmune neurologic disease. The full spectrum of clinical features, treatment responses, correlation with antibody specificity, and in particular the role of the IgM antibodies will need to be assessed in future studies.

Zoltowska Katarzyna M, Belaya K, Leite M, Patrick W, Vincent A, Beeson D. 2015. Collagen Q - A potential target for autoantibodies in myasthenia gravis Journal of the Neurological Sciences, 348 (1-2), pp. 241-244. | Show Abstract | Read more

© 2014 Elsevier B.V. All rights reserved.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.

Zoltowska Katarzyna M, Belaya K, Leite M, Patrick W, Vincent A, Beeson D. 2015. Collagen Q--a potential target for autoantibodies in myasthenia gravis. J Neurol Sci, 348 (1-2), pp. 241-244. | Show Abstract | Read more

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.

Rodríguez Cruz PM, Sewry C, Beeson D, Jayawant S, Squier W, McWilliam R, Palace J. 2014. Congenital myopathies with secondary neuromuscular transmission defects; A case report and review of the literature Neuromuscular Disorders, 24 (12), pp. 1103-1110. | Show Abstract | Read more

© 2014 Elsevier B.V.Congenital myopathies are a clinically and genetically heterogeneous group of disorders characterized by early onset hypotonia, weakness and characteristic, but not pathognomonic, structural abnormalities in muscle fibres. The clinical features overlap with muscular dystrophies, myofibrillar myopathies, neurogenic conditions and congenital myasthenic syndromes. We describe a case of cap myopathy with myasthenic features due to a mutation in the TPM2 gene that responded to anticholinesterase therapy. We also review other published cases of congenital myopathies with neuromuscular transmission abnormalities. This report expands the spectrum of congenital myopathies with secondary neuromuscular transmission defects. The recognition of these cases is important since these conditions can benefit from treatment with drugs enhancing neuromuscular transmission.

Klein A, Robb S, Rushing E, Liu WW, Belaya K, Beeson D. 2015. Congenital myasthenic syndrome caused by mutations in DPAGT. Neuromuscul Disord, 25 (3), pp. 253-256. | Show Abstract | Read more

Congenital myasthenic syndromes with prominent limb girdle involvement are an important differential diagnosis for congenital myopathies because of the therapeutic considerations. We present a case where accurate diagnosis was delayed for many years. Fluctuations of weakness were misinterpreted as effects of alternative treatments. Weakness was generalised, most prominently in the arms. Fatigability was more prominent in less affected muscles revealed by a positive Simpson test. Stimulation single fibre electromyography confirmed the suspected neuromuscular transmission defect. The marked response to pyridostigmine and cognitive impairment pointed to a myasthenic syndrome due to impaired glycosylation. Two mutations in trans were found in DPAGT1, the gene coding for dolichyl-phosphate N-acetylglucosaminephosphotransferase, one novel, the other previously reported in a rare form of congenital disorder of glycosylation. Gene expression studies revealed that both mutations reduce DPAGT1 expression. Phenotypic features not previously described for DPAGT1 CMS included restricted ocular abduction and long finger flexor contractures.

Rodríguez Cruz PM, Palace J, Beeson D. 2014. Inherited disorders of the neuromuscular junction: an update. J Neurol, 261 (11), pp. 2234-2243. | Show Abstract | Read more

Congenital myasthenic syndromes (CMSs) are a group of heterogeneous inherited disorders caused by mutations in genes affecting the function and structure of the neuromuscular junction. This review updates the reader on established and novel subtypes of congenital myasthenia, and the treatment strategies for these increasingly heterogeneous disorders. The discovery of mutations associated with the N-glycosylation pathway and in the family of serine peptidases has shown that causative genes encoding ubiquitously expressed molecules can produce defects at the human neuromuscular junction. By contrast, mutations in lipoprotein-like receptor 4 (LRP4), a long-time candidate gene for congenital myasthenia, and a novel phenotype of myasthenia with distal weakness and atrophy due to mutations in AGRN have now been described. In addition, a pathogenic splicing mutation in a nonfunctional exon of CHRNA1 has been reported emphasizing the importance of analysing nonfunctional exons in genetic analysis. The benefit of salbutamol and ephedrine alone or combined with pyridostigmine or 3,4-DAP is increasingly being reported for particular subtypes of CMS.

Arimura S, Okada T, Tezuka T, Chiyo T, Kasahara Y, Yoshimura T, Motomura M, Yoshida N, Beeson D, Takeda S, Yamanashi Y. 2014. Neuromuscular disease. DOK7 gene therapy benefits mouse models of diseases characterized by defects in the neuromuscular junction. Science, 345 (6203), pp. 1505-1508. | Show Abstract | Read more

The neuromuscular junction (NMJ) is the synapse between a motor neuron and skeletal muscle. Defects in NMJ transmission cause muscle weakness, termed myasthenia. The muscle protein Dok-7 is essential for activation of the receptor kinase MuSK, which governs NMJ formation, and DOK7 mutations underlie familial limb-girdle myasthenia (DOK7 myasthenia), a neuromuscular disease characterized by small NMJs. Here, we show in a mouse model of DOK7 myasthenia that therapeutic administration of an adeno-associated virus (AAV) vector encoding the human DOK7 gene resulted in an enlargement of NMJs and substantial increases in muscle strength and life span. When applied to model mice of another neuromuscular disorder, autosomal dominant Emery-Dreifuss muscular dystrophy, DOK7 gene therapy likewise resulted in enlargement of NMJs as well as positive effects on motor activity and life span. These results suggest that therapies aimed at enlarging the NMJ may be useful for a range of neuromuscular disorders.

Rodríguez Cruz PM, Palace J, Beeson D. 2014. Congenital myasthenic syndromes and the neuromuscular junction. Curr Opin Neurol, 27 (5), pp. 566-575. | Show Abstract | Read more

PURPOSE OF REVIEW: Congenital myasthenic syndromes (CMS) are a group of heterogeneous inherited disorders caused by mutations in genes encoding proteins essential for the integrity of neuromuscular transmission. This review updates the reader on the new mutations of known CMS genes, new causative genes and the treatment strategies for these increasingly heterogeneous disorders. It also provides a brief summary of the congenital myopathies with myasthenic features. RECENT FINDINGS: The discovery of causative genes encoding for ubiquitously expressed and extrajunctional molecules has changed our previous view of congenital myasthenia. Mutations associated with the N-glycosylation pathway and in the family of serine peptidases have shown that abnormalities in the post-translational modification of proteins can produce defects at the human neuromuscular junction. However, mutations in lipoprotein-like receptor 4, a long-time candidate gene for congenital myasthenia, have now been described and a new pathogenic splicing mutation in the nonfunctional exon of CHRNA1 has been reported. The use of salbutamol and ephedrine alone or combined with pyridostigmine or 3,4-DAP is increasingly being reported in different CMS subtypes with significant benefit. SUMMARY: Recent studies of the CMS illustrate the increasing complexity of the genetics, pathophysiological mechanisms and therapy of impaired synaptic transmission at the neuromuscular junction.

Parr JR, Andrew MJ, Finnis M, Beeson D, Vincent A, Jayawant S. 2014. How common is childhood myasthenia? The UK incidence and prevalence of autoimmune and congenital myasthenia. Arch Dis Child, 99 (6), pp. 539-542. | Show Abstract | Read more

OBJECTIVE: To ascertain the frequency of childhood myasthenia in the UK. Specifically, we aimed to identify the detected incidence of autoimmune myasthenia and the detected prevalence of genetically confirmed congenital myasthenic syndrome (CMS) in children. METHODS: All children under 18 years of age on 31 December 2009 with a confirmed CMS genetic mutation were identified by the only UK laboratory undertaking CMS genetic testing. All cases with positive acetylcholine receptor (AChR) and muscle specific kinase (MuSK) receptor antibodies in the 5 years between 2003 and 2007 inclusive were identified by the testing laboratories. UK census data from 2001 were used as the denominator for analyses. RESULTS: The UK detected prevalence of genetically confirmed CMS was 9.2 per million children under 18 years of age. CMS was equally prevalent in girls and boys. CHRNE, RAPSN and DOK7 were the most commonly identified mutations. Prevalence varied across geographical regions in England (between 2.8 and 14.8 per million children). The mean incidence of antibody-positive autoimmune myasthenia was 1.5 per million children per year over the period of the study. Girls were affected more frequently than boys; this difference persisted across the age range. Antibodies were identified during the neonatal period in 17 children. CONCLUSIONS: This laboratory based study shows that childhood myasthenia is very rare. This condition is treatable, and these definitive detected incidence and prevalence data can be used to help plan diagnostic and supporting services for affected children and their families, and maximise research opportunities.

Illingworth MA, Main M, Pitt M, Feng L, Sewry CA, Gunny R, Vorstman E, Beeson D, Manzur A, Muntoni F, Robb SA. 2014. RYR1-related congenital myopathy with fatigable weakness, responding to pyridostigimine. Neuromuscul Disord, 24 (8), pp. 707-712. | Show Abstract | Read more

The spectrum of RYR1 mutation associated disease encompasses congenital myopathies, exercise induced rhabdomyolysis, malignant hyperthermia susceptibility and King-Denborough syndrome. We report the clinical phenotype of two siblings who presented in infancy with hypotonia and striking fatigable ptosis. Their response to pyridostigimine was striking, but genetic screening for congenital myasthenic syndromes was negative, prompting further evaluation. Muscle MRI was abnormal with a selective pattern of involvement evocative of RYR1-related myopathy. This directed sequencing of the RYR1 gene, which revealed two heterozygous c.6721C>T (p.Arg2241X) nonsense mutations and novel c.8888T>C (p.Leu2963Pro) mutations in both siblings. These cases broaden the RYR1-related disease spectrum to include a myasthenic-like phenotype, including partial response to pyridostigimine. RYR1-related myopathy should be considered in the presence of fatigable weakness especially if muscle imaging demonstrates structural abnormalities. Single fibre electromyography can also be helpful in cases like this.

Webster R, Liu WW, Chaouch A, Lochmüller H, Beeson D. 2014. Fast-channel congenital myasthenic syndrome with a novel acetylcholine receptor mutation at the α-ε subunit interface Neuromuscular Disorders, 24 (2), pp. 143-147. | Show Abstract | Read more

Congenital myasthenic syndromes (CMS) result from the failure to achieve muscle depolarisation due to disorders in the structure and/or function of the neuromuscular synapse. Mutations of the nicotinic acetylcholine receptor (nAChR) form a major subset of CMS. We describe a patient who presented with recurrent apnoeic crises in the neonatal period requiring ventilator support. Electromyography revealed compound muscle action potential decrement upon repetitive stimulation. Sequencing of nAChR subunit genes revealed two missense mutations. One previously reported null mutation p.εTyr15His, and a second novel missense mutation, p.εThr38Lys, that is well expressed in mammalian cell culture and thus likely to exert its effect via alteration of ion channel kinetics. Functional analysis revealed abbreviated ion channel bursts characteristic of a fast channel CMS. The mutation p.εThr38Lys occurs at the interface between the α and ε subunits of the nAChR pentamer and leads to instability of the open channel. The effects of this mutation on channel function were investigated in relation to other fast channel mutants at an analogous subunit interface within the nAChR pentamer. Fast channel syndromes are frequently characterised by severe myasthenic weakness with apnoeic crises; knowledge of the underlying mutation and its functional consequences can be vital for appropriate therapy and patient management. © 2013 Elsevier B.V.

Cruz PMR, Palace J, Beeson D. 2014. Congenital myasthenic syndromes and the neuromuscular junction Current Opinion in Neurology, 27 (5), pp. 566-575. | Show Abstract | Read more

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.Purpose of review: Congenital myasthenic syndromes (CMS) are a group of heterogeneous inherited disorders caused by mutations in genes encoding proteins essential for the integrity of neuromuscular transmission. This review updates the reader on the new mutations of known CMS genes, new causative genes and the treatment strategies for these increasingly heterogeneous disorders. It also provides a brief summary of the congenital myopathies with myasthenic features. Recent findings: The discovery of causative genes encoding for ubiquitously expressed and extrajunctional molecules has changed our previous view of congenital myasthenia. Mutations associated with the N-glycosylation pathway and in the family of serine peptidases have shown that abnormalities in the post-translational modification of proteins can produce defects at the human neuromuscular junction. However, mutations in lipoprotein-like receptor 4, a long-time candidate gene for congenital myasthenia, have now been described and a new pathogenic splicing mutation in the nonfunctional exon of CHRNA1 has been reported. The use of salbutamol and ephedrine alone or combined with pyridostigmine or 3,4-DAP is increasingly being reported in different CMS subtypes with significant benefit. Summary: Recent studies of the CMS illustrate the increasing complexity of the genetics, pathophysiological mechanisms and therapy of impaired synaptic transmission at the neuromuscular junction.

Rodríguez Cruz PM, Sewry C, Beeson D, Jayawant S, Squier W, McWilliam R, Palace J. 2014. Congenital myopathies with secondary neuromuscular transmission defects; a case report and review of the literature. Neuromuscul Disord, 24 (12), pp. 1103-1110. | Show Abstract | Read more

Congenital myopathies are a clinically and genetically heterogeneous group of disorders characterized by early onset hypotonia, weakness and characteristic, but not pathognomonic, structural abnormalities in muscle fibres. The clinical features overlap with muscular dystrophies, myofibrillar myopathies, neurogenic conditions and congenital myasthenic syndromes. We describe a case of cap myopathy with myasthenic features due to a mutation in the TPM2 gene that responded to anticholinesterase therapy. We also review other published cases of congenital myopathies with neuromuscular transmission abnormalities. This report expands the spectrum of congenital myopathies with secondary neuromuscular transmission defects. The recognition of these cases is important since these conditions can benefit from treatment with drugs enhancing neuromuscular transmission.

Lewis AJM, Finlayson S, Mahmod M, Karamitsos TD, Dass S, Ashrafian H, Francis JM, Watkins H, Beeson D, Palace J, Neubauer S. 2014. A novel cardiac phenotype in patients with GFPT1 or DPAGT1 mutations Experimental and Clinical Cardiology, 20 (8), pp. 3139-3145. | Show Abstract

Mutations in the GFPT1 and DPAGT1 genes, which encode enzymes associated with roles in protein N-linked glycosylation, have been recently identified in a rare subgroup of patients with congenital myasthenic syndromes (CMSs). Aberrant glycosylation is implicated in the development of cardiomyopathies in the congenital disorders of glycosylation. We investigated whether patients with CMS and GFPT1 or DPAGT1 mutations also had evidence of a cardiac phenotype. Cardiovascular magnetic resonance and echocardiography were used to evaluate cardiac structure and function in patients with GFPT1 (n=2) and DPAGT1 (n=2) mutations. Electrocardiography was abnormal in all, with abnormal repolarization and deep S waves (n=3) or left ventricular hypertrophy by voltage criteria (n=1). Despite normal biventricular size and systolic function, GFPT1/DPAGT1 patients demonstrated late gadolinium enhancement suggestive of myocardial fibrosis (n=4), diastolic dysfunction (n=3) and impaired phosphocreatine to adenosine triphosphate ratio (an indicator of myocardial energetic state), assessed using 31P magnetic resonance spectroscopy (n=2). These findings may reflect incipient cardiomyopathy due to aberrant cardiac glycoprotein function and reinforce the need for cardiac surveillance in patients with disorders due to glycosylation pathway defects. © 2013 et al.; licensee Cardiology Academic Press.

Webster R, Liu WW, Chaouch A, Lochmüller H, Beeson D. 2014. Fast-channel congenital myasthenic syndrome with a novel acetylcholine receptor mutation at the α-ε subunit interface. Neuromuscul Disord, 24 (2), pp. 143-147. | Show Abstract | Read more

Congenital myasthenic syndromes (CMS) result from the failure to achieve muscle depolarisation due to disorders in the structure and/or function of the neuromuscular synapse. Mutations of the nicotinic acetylcholine receptor (nAChR) form a major subset of CMS. We describe a patient who presented with recurrent apnoeic crises in the neonatal period requiring ventilator support. Electromyography revealed compound muscle action potential decrement upon repetitive stimulation. Sequencing of nAChR subunit genes revealed two missense mutations. One previously reported null mutation p.εTyr15His, and a second novel missense mutation, p.εThr38Lys, that is well expressed in mammalian cell culture and thus likely to exert its effect via alteration of ion channel kinetics. Functional analysis revealed abbreviated ion channel bursts characteristic of a fast channel CMS. The mutation p.εThr38Lys occurs at the interface between the α and ε subunits of the nAChR pentamer and leads to instability of the open channel. The effects of this mutation on channel function were investigated in relation to other fast channel mutants at an analogous subunit interface within the nAChR pentamer. Fast channel syndromes are frequently characterised by severe myasthenic weakness with apnoeic crises; knowledge of the underlying mutation and its functional consequences can be vital for appropriate therapy and patient management.

Klein A, Pitt MC, McHugh JC, Niks EH, Sewry CA, Phadke R, Feng L, Manzur AY et al. 2013. DOK7 congenital myasthenic syndrome in childhood: Early diagnostic clues in 23 children Neuromuscular Disorders, 23 (11), pp. 883-891. | Show Abstract | Read more

Mutations in DOK7 are a common cause of congenital myasthenia. Treatment with ephedrine or salbutamol is effective, but diagnosis is often delayed. The aim of our study was to find early clues to the diagnosis of DOK7 congenital myasthenic syndrome. We included 23 children of 20 families. Onset of symptoms ranged from birth to age 3. years. 13 presented at birth with feeding difficulties, 11 with stridor (documented vocal cord palsy in 7), 3/11 with hypotonia/poor head control. Weakness was more pronounced proximally in all, axial in early presenting infants. Muscle biopsy showed non-specific features in 15/16, type 1 fibre predominance in 14/16, areas devoid of oxidative enzyme activity in 7/16. Muscle imaging was normal in 8/10, 2/10 showed mild non-specific changes. A diagnostic clue suggesting CMS rather than myopathy was the discrepancy between muscle imaging or histology findings compared with the degree of weakness. Repetitive nerve stimulation and stimulation single fibre electromyography were pathological in 9/17 and 13/14, respectively. In conclusion, stridor and feeding difficulties at birth or progressive weakness despite normal milestones in infancy point to the diagnosis and should lead to neurophysiological and genetic investigation. Fatigability can be absent or easily missed in the first years of life. © 2013 Elsevier B.V.

Klein A, Pitt MC, McHugh JC, Niks EH, Sewry CA, Phadke R, Feng L, Manzur AY et al. 2013. DOK7 congenital myasthenic syndrome in childhood: early diagnostic clues in 23 children. Neuromuscul Disord, 23 (11), pp. 883-891. | Show Abstract | Read more

Mutations in DOK7 are a common cause of congenital myasthenia. Treatment with ephedrine or salbutamol is effective, but diagnosis is often delayed. The aim of our study was to find early clues to the diagnosis of DOK7 congenital myasthenic syndrome. We included 23 children of 20 families. Onset of symptoms ranged from birth to age 3 years. 13 presented at birth with feeding difficulties, 11 with stridor (documented vocal cord palsy in 7), 3/11 with hypotonia/poor head control. Weakness was more pronounced proximally in all, axial in early presenting infants. Muscle biopsy showed non-specific features in 15/16, type 1 fibre predominance in 14/16, areas devoid of oxidative enzyme activity in 7/16. Muscle imaging was normal in 8/10, 2/10 showed mild non-specific changes. A diagnostic clue suggesting CMS rather than myopathy was the discrepancy between muscle imaging or histology findings compared with the degree of weakness. Repetitive nerve stimulation and stimulation single fibre electromyography were pathological in 9/17 and 13/14, respectively. In conclusion, stridor and feeding difficulties at birth or progressive weakness despite normal milestones in infancy point to the diagnosis and should lead to neurophysiological and genetic investigation. Fatigability can be absent or easily missed in the first years of life.

Webster RG, Cossins J, Lashley D, Maxwell S, Liu WW, Wickens JR, Martinez-Martinez P, de Baets M, Beeson D. 2013. A mouse model of the slow channel myasthenic syndrome: Neuromuscular physiology and effects of ephedrine treatment. Exp Neurol, 248 pp. 286-298. | Show Abstract | Read more

In the slow channel congenital myasthenic syndrome mutations in genes encoding the muscle acetylcholine receptor give rise to prolonged ion channel activations. The resulting cation overload in the postsynaptic region leads to damage of synaptic structures, impaired neuromuscular transmission and fatigable muscle weakness. Previously we identified and characterised in detail the properties of the slow channel syndrome mutation εL221F. Here, using this mutation, we generate a transgenic mouse model for the slow channel syndrome that expresses mutant human ε-subunits harbouring an EGFP tag within the M3-M4 cytoplasmic region, driven by a ~1500 bp region of the CHRNB promoter. Fluorescent mutant acetylcholine receptors are assembled, cluster at the motor endplates and give rise to a disease model that mirrors the human condition. Mice demonstrate mild fatigable muscle weakness, prolonged endplate and miniature endplate potentials, and variable degeneration of the postsynaptic membrane. We use our model to investigate ephedrine as a potential treatment. Mice were assessed before and after six weeks on oral ephedrine (serum ephedrine concentration 89 ± 3 ng/ml) using an inverted screen test and in vivo electromyography. Treated mice demonstrated modest benefit for screen hang time, and in measures of compound muscle action potentials and mean jitter that did not reach statistical significance. Ephedrine and salbutamol show clear benefit when used in the treatment of DOK7 or COLQ congenital myasthenic syndromes. Our results highlight only a modest potential benefit of these β2-adrenergic receptor agonists for the treatment of the slow channel syndrome.

Zoltowska K, Webster R, Finlayson S, Maxwell S, Cossins J, Müller J, Lochmüller H, Beeson D. 2013. Mutations in GFPT1 that underlie limb-girdle congenital myasthenic syndrome result in reduced cell-surface expression of muscle AChR. Hum Mol Genet, 22 (14), pp. 2905-2913. | Show Abstract | Read more

Mutations in GFPT1 underlie a congenital myasthenic syndrome (CMS) characterized by a limb-girdle pattern of muscle weakness. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is a key rate-limiting enzyme in the hexosamine biosynthetic pathway providing building blocks for the glycosylation of proteins and lipids. It is expressed ubiquitously and it is not readily apparent why mutations in this gene should cause a syndrome with symptoms restricted to muscle and, in particular, to the neuromuscular junction. Data from a muscle biopsy obtained from a patient with GFPT1 mutations indicated that there were reduced endplate acetylcholine receptors. We, therefore, further investigated the relationship between identified mutations in GFPT1 and expression of the muscle acetylcholine receptor. Cultured myotubes derived from two patients with GFPT1 mutations showed a significant reduction in cell-surface AChR expression (Pt1 P < 0.0001; Pt2 P = 0.0097). Inhibition of GFPT1 enzymatic activity or siRNA silencing of GFPT1 expression both resulted in reduced AChR cell-surface expression. Western blot and gene-silencing experiments indicate this is due to reduced steady-state levels of AChR α, δ, ε, but not β subunits rather than altered transcription of AChR-subunit RNA. Uridine diphospho-N-acetylglucosamine, a product of the hexosamine synthetic pathway, acts as a substrate at an early stage in the N-linked glycosylation pathway. Similarity between CMS due to GFPT1 mutations and CMS due to DPAGT1 mutations would suggest that reduced endplate AChR due to defective N-linked glycosylation is a primary disease mechanism in this disorder.

Finlayson S, Beeson D, Palace J. 2013. Congenital myasthenic syndromes: an update. Pract Neurol, 13 (2), pp. 80-91. | Read more

Finlayson S, Palace J, Belaya K, Walls TJ, Norwood F, Burke G, Holton JL, Pascual-Pascual SI, Cossins J, Beeson D. 2013. Clinical features of congenital myasthenic syndrome due to mutations in DPAGT1. J Neurol Neurosurg Psychiatry, 84 (10), pp. 1119-1125. | Show Abstract | Read more

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.

Cossins J, Belaya K, Hicks D, Salih MA, Finlayson S, Carboni N, Liu WW, Maxwell S et al. 2013. Congenital myasthenic syndromes due to mutations in ALG2 and ALG14. Brain, 136 (Pt 3), pp. 944-956. | Show Abstract | Read more

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 linkage analysis, whole-exome and whole-genome sequencing to determine the underlying defect in patients with an inherited limb-girdle pattern of myasthenic weakness. We identify ALG14 and ALG2 as novel genes in which mutations cause a congenital myasthenic syndrome. Through analogy with yeast, ALG14 is thought to form a multiglycosyltransferase complex with ALG13 and DPAGT1 that catalyses the first two committed steps of asparagine-linked protein glycosylation. We show that ALG14 is concentrated at the muscle motor endplates and small interfering RNA silencing of ALG14 results in reduced cell-surface expression of muscle acetylcholine receptor expressed in human embryonic kidney 293 cells. ALG2 is an alpha-1,3-mannosyltransferase that also catalyses early steps in the asparagine-linked glycosylation pathway. Mutations were identified in two kinships, with mutation ALG2p.Val68Gly found to severely reduce ALG2 expression both in patient muscle, and in cell cultures. Identification of DPAGT1, ALG14 and ALG2 mutations as a cause of congenital myasthenic syndrome underscores the importance of asparagine-linked protein glycosylation for proper functioning of the neuromuscular junction. These syndromes form part of the wider spectrum of congenital disorders of glycosylation caused by impaired asparagine-linked glycosylation. It is likely that further genes encoding components of this pathway will be associated with congenital myasthenic syndromes or impaired neuromuscular transmission as part of a more severe multisystem disorder. Our findings suggest that treatment with cholinesterase inhibitors may improve muscle function in many of the congenital disorders of glycosylation.

Koneczny I, Cossins J, Waters P, Beeson D, Vincent A. 2013. MuSK myasthenia gravis IgG4 disrupts the interaction of LRP4 with MuSK but both IgG4 and IgG1-3 can disperse preformed agrin-independent AChR clusters. PLoS One, 8 (11), pp. e80695. | Show Abstract | Read more

A variable proportion of patients with generalized myasthenia gravis (MG) have autoantibodies to muscle specific tyrosine kinase (MuSK). During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4), which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs) on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a) agrin, reflecting neuromuscular development, and b) by Dok7- overexpression, producing AChR clusters that more closely resemble the adult neuromuscular synapse. Total IgG, IgG4 or IgG1-3 MuSK antibodies were not endocytosed unless cross-linked by divalent anti-human IgG. MuSK IgG, Fab fragments and IgG4 inhibited the binding of LRP4 to MuSK and reduced agrin-induced AChR clustering in C2C12 cells. By contrast, IgG1-3 antibodies did not inhibit LRP4-MuSK binding but, surprisingly, did inhibit agrin-induced clustering. Moreover, both IgG4 and IgG1-3 preparations dispersed agrin-independent AChR clusters in Dok7-overexpressing C2C12 cells. Thus interference by IgG4 antibodies of the LRP4-MuSK interaction will be one pathogenic mechanism of MuSK antibodies, but IgG1-3 MuSK antibodies will also contribute to the reduced AChR density and neuromuscular dysfunction in myasthenia patients with MuSK antibodies.

Beeson D. 2013. Hereditary and Autoimmune Myasthenias pp. 1-17. | Read more

Basiri K, Belaya K, Liu WW, Maxwell S, Sedghi M, Beeson D. 2013. Clinical features in a large Iranian family with a limb-girdle congenital myasthenic syndrome due to a mutation in DPAGT1. Neuromuscul Disord, 23 (6), pp. 469-472. | Show Abstract | Read more

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.

Cited:

36

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Cossins J, Belaya K, Hicks D, Salih MA, Finlayson S, Carboni N, Liu WW, Maxwell S et al. 2013. Congenital myasthenic syndromes due to mutations in ALG2 and ALG14 Brain, 136 (3), pp. 944-956. | Show Abstract | Read more

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 linkage analysis, whole-exome and whole-genome sequencing to determine the underlying defect in patients with an inherited limb-girdle pattern of myasthenic weakness. We identify ALG14 and ALG2 as novel genes in which mutations cause a congenital myasthenic syndrome. Through analogy with yeast, ALG14 is thought to form a multiglycosyltransferase complex with ALG13 and DPAGT1 that catalyses the first two committed steps of asparagine-linked protein glycosylation. We show that ALG14 is concentrated at the muscle motor endplates and small interfering RNA silencing of ALG14 results in reduced cell-surface expression of muscle acetylcholine receptor expressed in human embryonic kidney 293 cells. ALG2 is an alpha-1,3-mannosyltransferase that also catalyses early steps in the asparagine-linked glycosylation pathway. Mutations were identified in two kinships, with mutation ALG2p.Val68Gly found to severely reduce ALG2 expression both in patient muscle, and in cell cultures. Identification of DPAGT1, ALG14 and ALG2 mutations as a cause of congenital myasthenic syndrome underscores the importance of asparagine-linked protein glycosylation for proper functioning of the neuromuscular junction. These syndromes form part of the wider spectrum of congenital disorders of glycosylation caused by impaired asparagine-linked glycosylation. It is likely that further genes encoding components of this pathway will be associated with congenital myasthenic syndromes or impaired neuromuscular transmission as part of a more severe multisystem disorder. Our findings suggest that treatment with cholinesterase inhibitors may improve muscle function in many of the congenital disorders of glycosylation. © 2013 The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain.

Belaya K, Finlayson S, Cossins J, Liu WW, Maxwell S, Palace J, Beeson D. 2012. Identification of DPAGT1 as a new gene in which mutations cause a congenital myasthenic syndrome. Ann N Y Acad Sci, 1275 (1), pp. 29-35. | Show Abstract | Read more

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.

Cossins J, Belaya K, Zoltowska K, Koneczny I, Maxwell S, Jacobson L, Leite MI, Waters P, Vincent A, Beeson D. 2012. The search for new antigenic targets in myasthenia gravis. Ann N Y Acad Sci, 1275 (1), pp. 123-128. | Show Abstract | Read more

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.

Beeson D. 2012. Synaptic dysfunction in congenital myasthenic syndromes. Ann N Y Acad Sci, 1275 (1), pp. 63-69. | Show Abstract | Read more

Congenital myasthenic syndromes (CMS) are hereditary disorders of neuromuscular transmission characterized by fatigable muscle weakness. The number of cases recognized is increasing with improved diagnosis. To date we have identified over 300 different mutations present in over 350 unrelated kinships. The underlying genetic defects are diverse, involving a series of different genes with a variety of different phenotypes. The type of treatment and its effectiveness will depend on the underlying pathogenic mechanism. We aim to define the molecular mechanism for each mutation identified and feed this information back to the clinic as a basis to tailor patient treatment. Here, we describe some of the methods that can be used to define if a DNA sequence variant is pathogenic with reference to variants in DOK7. We highlight a new mechanism for disruption of AChR function, where a mutation in the AChR ɛ-subunit gene causes reduced ion channel conductance and discuss new methods for identifying gene mutations. The study of these disorders is proving highly informative for understanding the diverse molecular mechanisms that can underlie synaptic dysfunction.

Vincent A, Waters P, Leite MI, Jacobson L, Koneczny I, Cossins J, Beeson D. 2012. Antibodies identified by cell-based assays in myasthenia gravis and associated diseases. Ann N Y Acad Sci, 1274 (1), pp. 92-98. | Show Abstract | Read more

We have established cell-based assays for the improved detection of acetylcholine receptor (AChR) and muscle-specific kinase (MuSK) antibodies in myasthenia gravis. This approach has enabled us to demonstrate antibodies to "clustered" AChRs in patients who were previously AChR antibody negative and can also be used to distinguish between adult and fetal AChR antibodies in mothers of babies with arthrogryposis multiplex congenita. We summarize our recent evidence for the pathogenicity of MuSK and clustered AChR antibodies using in vivo models. Cell-based assays are now also being used for the detection of other antibodies, such as those directed to components of the VGKC/CASPR2/LGI1 complex in Morvan's syndrome, and to AQP4 antibodies in neuromyelitis optica; both of these diseases can be associated with MG and sometimes thymoma. The cell-based method is time consuming but has many advantages and may provide a gold standard for the future in the detection of pathogenic autoantibodies in patients with immune-mediated diseases.

Finlayson S, Spillane J, Kullmann DM, Howard R, Webster R, Palace J, Beeson D. 2013. Slow channel congenital myasthenic syndrome responsive to a combination of fluoxetine and salbutamol. Muscle Nerve, 47 (2), pp. 279-282. | Show Abstract | Read more

INTRODUCTION: Slow channel congenital myasthenic syndrome is a dominant disorder characterized by prolonged acetylcholine receptor ion-channel activation. METHODS: Molecular genetic techniques, electrophysiology, and binding studies in human embryonic kidney (HEK) 293 cells determined mutant function and expression levels. Patient response to treatment was measured by quantitative myasthenic gravis and Medical Research Council grade strength scores. RESULTS: We report an unusual case due to heteroallelic mutations in CHRNE. The slow channel mutation, p.εS278del, is accompanied by a severe low-expression mutation, p.εR217L, on the second allele. Expression studies and cosegregation of p.εS278del with the disorder in the patient's offspring demonstrate robust expression of the p.εS278del mutation. The patient showed modest benefits from standard treatment with fluoxetine, but there was dramatic improvement when salbutamol was combined with fluoxetine. CONCLUSIONS: This case suggests that salbutamol, which is beneficial in some other congenital myasthenic syndromes, might also be considered in addition to fluoxetine in slow channel syndrome.

Belaya K, Finlayson S, Slater CR, Cossins J, Liu WW, Maxwell S, McGowan SJ, Maslau S et al. 2012. Mutations in DPAGT1 cause a limb-girdle congenital myasthenic syndrome with tubular aggregates. Am J Hum Genet, 91 (1), pp. 193-201. | Show Abstract | Read more

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.

Cossins J, Liu WW, Belaya K, Maxwell S, Oldridge M, Lester T, Robb S, Beeson D. 2012. The spectrum of mutations that underlie the neuromuscular junction synaptopathy in DOK7 congenital myasthenic syndrome. Hum Mol Genet, 21 (17), pp. 3765-3775. | Show Abstract | Read more

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.

Webster R, Maxwell S, Spearman H, Tai K, Beckstein O, Sansom M, Beeson D. 2012. A novel congenital myasthenic syndrome due to decreased acetylcholine receptor ion-channel conductance. Brain, 135 (Pt 4), pp. 1070-1080. | Show Abstract | Read more

Muscle acetylcholine receptor ion channels mediate neurotransmission by depolarizing the postsynaptic membrane at the neuromuscular junction. Inherited disorders of neuromuscular transmission, termed congenital myasthenic syndromes, are commonly caused by mutations in genes encoding the five subunits of the acetylcholine receptor that severely reduce endplate acetylcholine receptor numbers and/or cause kinetic abnormalities of acetylcholine receptor function. We tracked the cause of the myasthenic disorder in a female with onset of first symptoms at birth, who displayed mildly progressive bulbar, respiratory and generalized limb weakness with ptosis and ophthalmoplegia. Direct DNA sequencing revealed heteroallelic mutations in exon 8 of the acetylcholine receptor ε-subunit gene. Two alleles were identified: one with the missense substitution p.εP282R, and the second with a deletion, c.798_800delCTT, which result in the loss of a single amino acid, residue F266, within the M2 transmembrane domain. When these acetylcholine receptor mutations were expressed in HEK 293 cells, the p.εP282R mutation caused severely reduced expression on the cell surface, whereas p.εΔF266 gave robust surface expression. Single-channel analysis for p.εΔF266 acetylcholine receptor channels showed the longest burst duration population was not different from wild-type acetylcholine receptor (4.39 ± 0.6 ms versus 4.68 ± 0.7 ms, n = 5 each) but that the amplitude of channel openings was reduced. Channel amplitudes at different holding potentials showed that single-channel conductance was significantly reduced in p.εΔF266 acetylcholine receptor channels (42.7 ± 1.4 pS, n = 8, compared with 70.9 ± 1.6 pS for wild-type, n = 6). Although a phenylalanine residue at this position within M2 is conserved throughout ligand-gated excitatory cys-loop channel subunits, deletion of equivalent residues in the other subunits of muscle acetylcholine receptor did not have equivalent effects. Modelling the impact of p.εΔF266 revealed only a minor alteration to channel structure. In this study we uncover the novel mechanism of reduced acetylcholine receptor channel conductance as an underlying cause of congenital myasthenic syndrome, with the 'low conductance' phenotype that results from the p.εΔF266 deletion mutation revealed by the coinheritance of the low-expressor mutation p.εP282R.

Chaouch A, Beeson D, Hantaï D, Lochmüller H. 2012. 186th ENMC International Workshop: Congenital myasthenic syndromes 24-26 June 2011, Naarden, The Netherlands Neuromuscular Disorders,

Burke G, Hiscock A, Klein A, Niks EH, Main M, Manzur AY, Ng J, de Vile C, Muntoni F, Beeson D, Robb S. 2013. Salbutamol benefits children with congenital myasthenic syndrome due to DOK7 mutations. Neuromuscul Disord, 23 (2), pp. 170-175. | Show Abstract | Read more

Congenital myasthenic syndromes due to DOK7 mutations cause fatigable limb girdle weakness. Treatment with ephedrine improves muscle strength. Salbutamol, a β(2)-adrenergic receptor agonist with fewer side effects and more readily available, has been effective in adult and anecdotal childhood cases. This study reports the effects of salbutamol on motor function in childhood DOK7 congenital myasthenic syndrome. Nine children (age range 5.9-15.1years) were treated with oral salbutamol, 2-4mg TDS. The effect on timed tests of motor function, pre- and up to 28months post-treatment, was audited retrospectively. All 9 reported functional benefit within 1month, with progressive improvement to a plateau at 12-18months. Within the first month, all 3 non-ambulant children resumed walking with assistance. Although improvements were seen in some timed tests (timed 10m, arm raise time, 6min walk time) this did not fully reflect the observed functional benefits in daily living activities. No major side effects were reported. We conclude that oral salbutamol treatment significantly improves strength in children with DOK7 congenital myasthenic syndrome and is well tolerated. Outcome measures need to be refined further, both to accurately reflect functional abilities in children and to document progress and treatment response.

Cited:

59

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Belaya K, Finlayson S, Slater CR, Cossins J, Liu WW, Maxwell S, McGowan SJ, Maslau S et al. 2012. Mutations in DPAGT1 cause a limb-girdle congenital myasthenic syndrome with tubular aggregates American Journal of Human Genetics, 91 (1), pp. 193-201. | Show Abstract | Read more

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.

Maselli RA, Fernandez JM, Arredondo J, Navarro C, Ngo M, Beeson D, Cagney O, Williams DC, Wollmann RL, Yarov-Yarovoy V, Ferns MJ. 2012. LG2 agrin mutation causing severe congenital myasthenic syndrome mimics functional characteristics of non-neural (z-) agrin. Hum Genet, 131 (7), pp. 1123-1135. | Show Abstract | Read more

We describe a severe form of congenital myasthenic syndrome (CMS) caused by two heteroallelic mutations: a nonsense and a missense mutation in the gene encoding agrin (AGRN). The identified mutations, Q353X and V1727F, are located at the N-terminal and at the second laminin G-like (LG2) domain of agrin, respectively. A motor-point muscle biopsy demonstrated severe disruption of the architecture of the neuromuscular junction (NMJ), including: dispersion and fragmentation of endplate areas with normal expression of acetylcholinesterase; simplification of postsynaptic membranes; pronounced reduction of the axon terminal size; widening of the primary synaptic cleft; and, collection of membranous debris material in the primary synaptic cleft and in the subsynaptic cytoplasm. Expression studies in heterologous cells revealed that the Q353X mutation abolished expression of full-length agrin. Moreover, the V1727F mutation decreased agrin-induced clustering of the acetylcholine receptor (AChR) in cultured C2 muscle cells by >100-fold, and phosphorylation of the MuSK receptor and AChR beta subunit by ~tenfold. Surprisingly, the V1727F mutant also displayed increased binding to α-dystroglycan but decreased binding to a neural (z+) agrin-specific antibody. Our findings demonstrate that agrin mutations can associate with a severe form of CMS and cause profound distortion of the architecture and function of the NMJ. The impaired ability of V1727F agrin to activate MuSK and cluster AChRs, together with its increased affinity to α-dystroglycan, mimics non-neural (z-) agrin and are important determinants of the pathogenesis of the disease.

Chaouch A, Beeson D, Hantaï D, Lochmüller H. 2012. 186th ENMC international workshop: congenital myasthenic syndromes 24-26 June 2011, Naarden, The Netherlands. Neuromuscul Disord, 22 (6), pp. 566-576. | Read more

Webster R, Maxwell S, Spearman H, Tai K, Beckstein O, Sansom M, Beeson D. 2012. A novel congenital myasthenic syndrome due to decreased acetylcholine receptor ion-channel conductance Brain, 135 (4), pp. 1070-1080. | Show Abstract | Read more

Muscle acetylcholine receptor ion channels mediate neurotransmission by depolarizing the postsynaptic membrane at the neuromuscular junction. Inherited disorders of neuromuscular transmission, termed congenital myasthenic syndromes, are commonly caused by mutations in genes encoding the five subunits of the acetylcholine receptor that severely reduce endplate acetylcholine receptor numbers and/or cause kinetic abnormalities of acetylcholine receptor function. We tracked the cause of the myasthenic disorder in a female with onset of first symptoms at birth, who displayed mildly progressive bulbar, respiratory and generalized limb weakness with ptosis and ophthalmoplegia. Direct DNA sequencing revealed heteroallelic mutations in exon 8 of the acetylcholine receptor ε-subunit gene. Two alleles were identified: one with the missense substitution p.εP282R, and the second with a deletion, c.798-800delCTT, which result in the loss of a single amino acid, residue F266, within the M2 transmembrane domain. When these acetylcholine receptor mutations were expressed in HEK 293 cells, the p.εP282R mutation caused severely reduced expression on the cell surface, whereas p.εΔF266 gave robust surface expression. Single-channel analysis for p.εΔF266 acetylcholine receptor channels showed the longest burst duration population was not different from wild-type acetylcholine receptor (4.39±0.6ms versus 4.68±0.7ms, n=5 each) but that the amplitude of channel openings was reduced. Channel amplitudes at different holding potentials showed that single-channel conductance was significantly reduced in p.εΔF266 acetylcholine receptor channels (42.7±1.4 pS, n=8, compared with 70.9±1.6 pS for wild-type, n=6). Although a phenylalanine residue at this position within M2 is conserved throughout ligand-gated excitatory cys-loop channel subunits, deletion of equivalent residues in the other subunits of muscle acetylcholine receptor did not have equivalent effects. Modelling the impact of p.εΔF266 revealed only a minor alteration to channel structure. In this study we uncover the novel mechanism of reduced acetylcholine receptor channel conductance as an underlying cause of congenital myasthenic syndrome, with the 'low conductance' phenotype that results from the p.εΔF266 deletion mutation revealed by the coinheritance of the low-expressor mutation p.εP282R. © 2012 The Author.

Guergueltcheva V, Müller JS, Dusl M, Senderek J, Oldfors A, Lindbergh C, Maxwell S, Colomer J et al. 2012. Congenital myasthenic syndrome with tubular aggregates caused by GFPT1 mutations. J Neurol, 259 (5), pp. 838-850. | Show Abstract | Read more

Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous group of inherited disorders of the neuromuscular junction. A difficult to diagnose subgroup of CMS is characterised by proximal muscle weakness and fatigue while ocular and facial involvement is only minimal. DOK7 mutations have been identified as causing the disorder in about half of the cases. More recently, using classical positional cloning, we have identified mutations in a previously unrecognised CMS gene, GFPT1, in a series of DOK7-negative cases. However, detailed description of clinical features of GFPT1 patients has not been reported yet. Here we describe the clinical picture of 24 limb-girdle CMS (LG-CMS) patients and pathological findings of 18 of them, all carrying GFPT1 mutations. Additional patients with CMS, but without tubular aggregates, and patients with non-fatigable weakness with tubular aggregates were also screened. In most patients with GFPT1 mutations, onset of the disease occurs in the first decade of life with characteristic limb-girdle weakness and fatigue. A common feature was beneficial and sustained response to acetylcholinesterase inhibitor treatment. Most of the patients who had a muscle biopsy showed tubular aggregates in myofibers. Analysis of endplate morphology in one of the patients revealed unspecific abnormalities. Our study delineates the phenotype of CMS associated with GFPT1 mutations and expands the understanding of neuromuscular junction disorders. As tubular aggregates in context of a neuromuscular transmission defect appear to be highly indicative, we suggest calling this condition congenital myasthenic syndrome with tubular aggregates (CMS-TA).

Palace J, Lashley D, Bailey S, Jayawant S, Carr A, McConville J, Robb S, Beeson D. 2012. Clinical features in a series of fast channel congenital myasthenia syndrome. Neuromuscul Disord, 22 (2), pp. 112-117. | Show Abstract | Read more

Fast channel congenital myasthenic syndromes are rare, but frequently result in severe weakness. We report a case of 12 fast channel patients to highlight clinical features and management difficulties. Patients were diagnosed through genetic screening and identification of mutations shown to cause fast channel syndrome. Data was obtained from clinical notes, history, examination and follow up. Patterns of muscle weakness involved limb, trunk, bulbar, respiratory, facial and extraocular muscles. Patients responded to treatment with anticholinesterase medication and 3,4-diaminopyridine. Fast channel syndrome contrasted with AChR deficiency in the occurrence of severe respiratory crises in infancy and childhood. The death of two children even when on treatment and the family histories of sibling deaths re-inforces the need for accurate genetic diagnosis, optimised pharmacological treatment and additional supportive measures to manage acute respiratory crises. Referral to a specialist paediatric respiratory centre and regular resuscitation training for parents are recommended.

Salih MA, Oystreck DT, Al-Faky YH, Kabiraj M, Omer MI, Subahi EM, Beeson D, Abu-Amero KK, Bosley TM. 2011. Congenital myasthenic syndrome due to homozygous CHRNE mutations: report of patients in Arabia. J Neuroophthalmol, 31 (1), pp. 42-47. | Show Abstract | Read more

We describe the clinical characteristics of 3 siblings from 1 family with congenital myasthenic syndrome due to homozygous mutations of the gene coding for the epsilon subunit of the acetylcholine receptor (CHRNE). Onset of symptoms occurred in the first few months of life with ptosis, restricted ocular motility, mild proximal weakness, and difficulty swallowing. Multiple hospital admissions were required due to recurrent pulmonary infections. There was no decremental conduction on repetitive nerve stimulation, but jitter was increased on single fiber electromyographic. Since early childhood, our patients have done well without pulmonary or bulbar symptoms and with partial improvement on pyridostigmine therapy. Response of ptosis to diagnostic ice pack test was striking. Although these siblings have a clinical history and examination findings typical of homozygous CHRNE mutations, the clinical presentation of congenital myasthenia subtypes is variable, and accurate genotyping is essential in choosing the appropriate treatment.

Yang L, Maxwell S, Leite MI, Waters P, Clover L, Fan X, Zhang D, Yang C, Beeson D, Vincent A. 2011. Non-radioactive serological diagnosis of myasthenia gravis and clinical features of patients from Tianjin, China. J Neurol Sci, 301 (1-2), pp. 71-76. | Show Abstract | Read more

PURPOSE: To establish non-radioactive assays for detection of antibodies (Abs) to the acetylcholine receptor (AChR) and to muscle specific kinase (MuSK). To show that the assays can be used in Tianjin for testing patients with MG. METHOD: AChR and MuSK Abs in 51 Chinese MG patients' sera were tested in Oxford, UK, using the conventional radioimmunoprecipitation assay (RIPA), a recently described cell-based assay (CBA), and a new non-radioactive fluorescence immunoprecipitation assay (FIPA) which can measure both AChR and MuSK antibodies in one step. 102 MG sera were subsequently tested by CBA and FIPA in Tianjin, China. Based on the different serological subgroups, the clinical features of these 153 MG patients were analyzed. RESULTS: We first confirmed the sensitivity and specificity of the assays in Oxford. There was good agreement between the FIPA and the RIPA for AChR Abs (r² = 0.6; p < 0.0001) with 80% positivity in the RIPA and 76% in the FIPA. Two patients were positive for MuSK Abs (4% of total) by both RIPA and FIPA assays. CBA was more sensitive for MuSK Abs, identifying an additional 3 patients. In Tianjin, using the FIPA and CBA, 84/102 (82%) AChR Ab positive patients and four MuSK Ab positive patients were identified. In the whole group of 153 MG patients, there were no differences in clinical features between different antibody subgroups. However, there were 30 patients with thymomas (20%), of whom one had MuSK antibodies. Moreover, two patients with purely ocular MG also had MuSK antibodies. CONCLUSIONS: Both the FIPA and CBA were established in China and should prove useful for diagnostic testing. Including the CBA for MuSK antibodies increased the number of MuSK-MG patients to 6% of the total, and 33% of the patients without AChR antibodies. The clinical features were mainly relatively mild but thymic tumors were common.

Senderek J, Müller JS, Dusl M, Strom TM, Guergueltcheva V, Diepolder I, Laval SH, Maxwell S et al. 2011. Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect. Am J Hum Genet, 88 (2), pp. 162-172. | Show Abstract | Read more

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.

Palace J, Lashley D, Bailey S, Jayawant S, Carr A, McConville J, Robb S, Beeson D. 2012. Clinical features in a series of fast channel congenital myasthenia syndrome NEUROMUSCULAR DISORDERS, 22 (2), pp. 112-117. | Read more

Guergueltcheva V, Müller JS, Dusl M, Senderek J, Oldfors A, Lindbergh C, Maxwell S, Colomer J et al. 2011. Congenital myasthenic syndrome with tubular aggregates caused by GFPT1 mutations Journal of Neurology, pp. 1-13.

Robb SA, Sewry CA, Dowling JJ, Feng L, Cullup T, Lillis S, Abbs S, Lees MM et al. 2011. Impaired neuromuscular transmission and response to acetylcholinesterase inhibitors in centronuclear myopathies. Neuromuscul Disord, 21 (6), pp. 379-386. | Show Abstract | Read more

Many clinical features of autosomal centronuclear myopathies (CNM) and X-linked myotubular myopathy (XLMTM) are common to congenital myasthenic syndromes (CMS). We describe three children whose clinical and electrophysiological findings originally suggested CMS, in whom CNM was diagnosed pathologically, though not yet genetically characterised. A fourth case, with XLMTM, also showed electrophysiological features of a neuromuscular transmission defect. Three (including the XLMTM case) showed improved strength with acetylcholinesterase inhibitor treatment. We also studied neuromuscular junction structure and function in the MTM1 knockdown zebrafish model of XLMTM, demonstrating abnormal neuromuscular junction organization; anticholinesterase therapy resulted in marked clinical response. These observations suggest that a neuromuscular transmission defect may accompany CNM and contribute to muscle weakness. Muscle biopsy should be considered in infants suspected to have CMS, especially if treatment response is incomplete, or no CMS gene mutation is identified. Treatment with acetylcholinesterase inhibitors may benefit some CNM patients. This warrants further confirmation.

Maselli RA, Fernandez JM, Arredondo J, Navarro C, Ngo M, Beeson D, Cagney O, Williams DC, Wollmann RL, Yarov-Yarovoy V, Ferns MJ. 2011. LG2 agrin mutation causing severe congenital myasthenic syndrome mimics functional characteristics of non-neural (z-) agrin Human Genetics, pp. 1-13.

Munot P, Lashley D, Jungbluth H, Feng L, Pitt M, Robb SA, Palace J, Jayawant S et al. 2010. Congenital fibre type disproportion associated with mutations in the tropomyosin 3 (TPM3) gene mimicking congenital myasthenia. Neuromuscul Disord, 20 (12), pp. 796-800. | Show Abstract | Read more

Congenital myopathy with fibre type disproportion (CFTD) has been associated with mutations in ACTA1, SEPN1, RYR1 and TPM3 genes. We report the clinico-pathological and electrophysiological features of 2 unrelated cases with heterozygous TPM3 mutation. Case 1 is a 19-year-old lady who presented with motor delay in infancy, respiratory failure in early teens requiring non-invasive ventilation despite being ambulant, ptosis, axial more than proximal weakness and scoliosis. Case 2 is a 7-year-old boy with hypotonia, feeding difficulties, motor delay and scoliosis, also requiring non-invasive ventilation while ambulant. Muscle biopsies in both cases showed fibre type disproportion. Muscle MRI (Case 1) showed mild uniformly increased interstitial tissue in and around the muscles. Sequencing of TPM3 in case 1 revealed a previously described heterozygous c.503G > A(pArg168His) missense variant in exon 5 and a novel heterozygous missense mutation c.521A > C(pGlu174Ala), also in exon 5, in case 2. A mild abnormality in the single fibre EMG was documented on electrophysiology in both cases. These cases highlight the neuromuscular transmission defect in CFTD secondary to TPM3 mutations.

Nicholl DJ, Hilton-Jones D, Palace J, Richmond S, Finlayson S, Winer J, Weir A, Maddison P et al. 2010. Orphan Drugs Open letter to prime minister David Cameron and health secretary Andrew Lansley BRITISH MEDICAL JOURNAL, 341 (nov16 3), pp. c6466-c6466. | Read more

Clark RH, McTaggart JS, Webster R, Mannikko R, Iberl M, Sim XL, Rorsman P, Glitsch M, Beeson D, Ashcroft FM. 2010. Muscle dysfunction caused by a KATP channel mutation in neonatal diabetes is neuronal in origin. Science, 329 (5990), pp. 458-461. | Show Abstract | Read more

Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel, cause neonatal diabetes. Many patients also suffer from hypotonia (weak and flaccid muscles) and balance problems. The diabetes arises from suppressed insulin secretion by overactive KATP channels in pancreatic beta-cells, but the source of the motor phenotype is unknown. By using mice carrying a human Kir6.2 mutation (Val59-->Met59) targeted to either muscle or nerve, we show that analogous motor impairments originate in the central nervous system rather than in muscle or peripheral nerves. We also identify locomotor hyperactivity as a feature of KATP channel overactivity. These findings suggest that drugs targeted against neuronal, rather than muscle, KATP channels are needed to treat the motor deficits and that such drugs require high blood-brain barrier permeability.

Irani SR, Bera K, Waters P, Zuliani L, Maxwell S, Zandi MS, Friese MA, Galea I et al. 2010. N-methyl-D-aspartate antibody encephalitis: temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes. Brain, 133 (Pt 6), pp. 1655-1667. | Show Abstract | Read more

Antibodies to the N-methyl-d-aspartate subtype of glutamate receptor have been associated with a newly-described encephalopathy that has been mainly identified in young females with ovarian tumours. However, the full clinical spectrum and treatment responses are not yet clear. We established a sensitive cell-based assay for detection of N-methyl-d-aspartate receptor antibodies in serum or cerebrospinal fluid, and a quantitative fluorescent immunoprecipitation assay for serial studies. Although there was marked intrathecal synthesis of N-methyl-d-aspartate receptor antibodies, the absolute levels of N-methyl-d-aspartate receptor antibodies were higher in serum than in cerebrospinal fluid. N-methyl-d-aspartate receptor antibodies were of the immunoglobulin G1 subclass and were able to activate complement on N-methyl d-aspartate receptor-expressing human embryonic kidney cells. From questionnaires returned on 44 N-methyl-d-aspartate receptor antibody-positive patients, we identified a high proportion without a detected tumour (35/44, 80%: follow-up 3.6-121 months, median 16 months). Among the latter were 15 adult females (43%), 10 adult males (29%) and 10 children (29%), with four in the first decade of life. Overall, there was a high proportion (29%) of non-Caucasians. Good clinical outcomes, as defined by reductions in modified Rankin scores, correlated with decreased N-methyl-d-aspartate receptor antibody levels and were associated with early (<40 days) administration of immunotherapies in non-paraneoplastic patients (P < 0.0001) and earlier tumour removal in paraneoplastic patients (P = 0.02). Ten patients (23%) who were first diagnosed during relapses had no evidence of tumours but had received minimal or no immunotherapy during earlier episodes. Temporal analysis of the onset of the neurological features suggested progression through two main stages. The time of onset of the early features, characterized by neuropsychiatric symptoms and seizures preceded by a median of 10-20 days, the onset of movement disorders, reduction in consciousness and dysautonomia. This temporal dichotomy was also seen in the timing of cerebrospinal fluid, electroencephalographic and in the rather infrequent cerebral imaging changes. Overall, our data support a model in which the early features are associated with cerebrospinal fluid lymphocytosis, and the later features with appearance of oligoclonal bands. The immunological events and neuronal mechanisms underlying these observations need to be explored further, but one possibility is that the early stage represents diffusion of serum antibodies into the cortical grey matter, whereas the later stage results from secondary expansion of the immunological repertoire within the intrathecal compartment acting on subcortical neurons. Four patients, who only had temporal lobe epilepsy without oligoclonal bands, may represent restriction to the first stage.

Lashley D, Palace J, Jayawant S, Robb S, Beeson D. 2010. Ephedrine treatment in congenital myasthenic syndrome due to mutations in DOK7. Neurology, 74 (19), pp. 1517-1523. | Show Abstract | Read more

BACKGROUND: Mutations in the postsynaptic adaptor protein Dok-7 underlie congenital myasthenic syndrome (CMS) with a characteristic limb girdle pattern of muscle weakness. Patients usually do not respond to or worsen with the standard CMS treatments: cholinesterase inhibitors and 3,4-diaminopyridine. However, anecdotal reports suggest they may improve with ephedrine. METHODS: This was an open prospective follow-up study to determine muscle strength in response to ephedrine in Dok-7 CMS. Patients were first evaluated as inpatients for suitability for a trial of treatment with ephedrine. The response was assessed at 2 and 6 to 8 months follow-up clinic visits using a quantitative myasthenia gravis (severity) score (QMG) and mobility measures. RESULTS: Ten out of 12 of the cohort with DOK7 mutations tolerated ephedrine. We noted a progressive response to treatment over the 6 to 8 months assessment period with a significant improvement at the final QMG score (p = 0.009). Mobility scores also improved (p = 0.0006). Improvements in the subcomponents of the QMG score that measured proximal muscle function (those muscle groups most severely affected) were most marked, and in some cases were dramatic. All patients reported enhanced activities of daily living at 6-8 months. CONCLUSION: Ephedrine appears to be an effective treatment for Dok-7 CMS. It is well-tolerated by most patients and improvement in strength can be profound. Determining the long-term response and the most effective dosing regimen will require further research. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that ephedrine given at doses between 15 and 90 mg/day improves muscle strength in patients with documented mutations in DOK7.

Gattenloehner S, Joerissen H, Huhn M, Vincent A, Beeson D, Tzartos S, Mamalaki A, Etschmann B et al. 2010. A Human Recombinant Autoantibody-Based Immunotoxin Specific for the Fetal Acetylcholine Receptor Inhibits Rhabdomyosarcoma Growth In Vitro and in a Murine Transplantation Model JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY, 2010 pp. 1-11. | Show Abstract | Read more

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) γ-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA).While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms. Copyright © 2010 S. Gattenlöhner et al.

Jephson CG, Mills NA, Pitt MC, Beeson D, Aloysius A, Muntoni F, Robb SA, Bailey CM. 2010. Congenital stridor with feeding difficulty as a presenting symptom of Dok7 congenital myasthenic syndrome. Int J Pediatr Otorhinolaryngol, 74 (9), pp. 991-994. | Show Abstract | Read more

OBJECTIVE: The congenital myasthenic syndromes (CMS) are a group of genetic disorders of neuromuscular transmission causing fatigable weakness. Symptoms may be present from birth, but diagnosis is often delayed for several years, notably in post-synaptic CMS due to mutations in the DOK7 gene. Recently, we noted a subgroup of children with CMS in whom congenital stridor and bilateral vocal cord palsy predated other symptoms. All had mutations in the DOK7 gene. The purpose of this study was to review our population of DOK7 CMS patients with congenital stridor and assess whether there were other phenotypic features which might raise suspicion of a diagnosis of CMS in the neonatal period, in the absence of limb weakness and ptosis and prompt earlier referral for neurophysiological investigation, genetic diagnosis and appropriate treatment. METHODS: A retrospective case review of 11 DOK7 CMS patients at a tertiary referral centre. RESULTS: Six patients were identified with DOK7 mutations and congenital stridor, four requiring intubation soon after birth. Four patients had a diagnosis of bilateral vocal cord palsy and three required tracheostomy, successfully decannulated in one after 3 years. All six patients had difficulty with feeding, with weak suck and swallow necessitating nasogastric feeding in five, two of whom required gastrostomy. Despite all six children having had neonatal symptoms, the mean age at CMS diagnosis was 5 years and 9 months. CONCLUSION: CMS, particularly caused by mutations in the DOK7 gene, is a rare but treatable cause of congenital stridor in the neonate. A combination of congenital stridor, especially with an apparently idiopathic bilateral vocal cord palsy and weak suck and swallow should alert the clinician to the possibility of CMS and prompt early referral for neurophysiology and genetic investigations. Confirmation of a CMS diagnosis enables treatment to be initiated, informed management of the VCP and anticipation of myasthenic symptoms, particularly life-threatening respiratory decompensation. Treatment may allow early decannulation or possible avoidance of tracheostomy. At least 12 genes are known to cause CMS; the presence of congenital stridor may help target genetic diagnosis.

Spillane J, Beeson DJ, Kullmann DM. 2010. Myasthenia and related disorders of the neuromuscular junction. J Neurol Neurosurg Psychiatry, 81 (8), pp. 850-857. | Show Abstract | Read more

Our understanding of transmission at the neuromuscular junction has increased greatly in recent years. We now recognise a wide variety of autoimmune and genetic diseases that affect this specialised synapse, causing muscle weakness and fatigue. These disorders greatly affect quality of life and rarely can be fatal. Myasthenia gravis is the most common disorder and is most commonly caused by autoantibodies targeting postsynaptic acetylcholine receptors. Antibodies to muscle-specific kinase (MuSK) are detected in a variable proportion of the remainder. Treatment is symptomatic and immunomodulatory. Lambert-Eaton myasthenic syndrome is caused by antibodies to presynaptic calcium channels, and approximately 50% of cases are paraneoplastic, most often related to small cell carcinoma of the lung. Botulism is an acquired disorder caused by neurotoxins produced by Clostridium botulinum, impairing acetylcholine release into the synaptic cleft. In addition, several rare congenital myasthenic syndromes have been identified, caused by inherited defects in presynaptic, synaptic basal lamina and postsynaptic proteins necessary for neuromuscular transmission. This review focuses on recent advances in the diagnosis and treatment of these disorders.

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Irani SR, Bera K, Waters P, Zuliani L, Maxwell S, Zandi MS, Friese MA, Galea I et al. 2010. N-methyl-d-aspartate antibody encephalitis: Temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes Brain, 133 (6), pp. 1655-1667. | Show Abstract | Read more

Antibodies to the N-methyl-d-aspartate subtype of glutamate receptor have been associated with a newly-described encephalopathy that has been mainly identified in young females with ovarian tumours. However, the full clinical spectrum and treatment responses are not yet clear. We established a sensitive cell-based assay for detection of N-methyl-d-aspartate receptor antibodies in serum or cerebrospinal fluid, and a quantitative fluorescent immunoprecipitation assay for serial studies. Although there was marked intrathecal synthesis of N-methyl-d-aspartate receptor antibodies, the absolute levels of N-methyl-d-aspartate receptor antibodies were higher in serum than in cerebrospinal fluid. N-methyl-d-aspartate receptor antibodies were of the immunoglobulin G1 subclass and were able to activate complement on N-methyl d-aspartate receptor-expressing human embryonic kidney cells. From questionnaires returned on 44 N-methyl-d-aspartate receptor antibody-positive patients, we identified a high proportion without a detected tumour (35/44, 80: follow-up 3.6-121 months, median 16 months). Among the latter were 15 adult females (43), 10 adult males (29) and 10 children (29), with four in the first decade of life. Overall, there was a high proportion (29) of non-Caucasians. Good clinical outcomes, as defined by reductions in modified Rankin scores, correlated with decreased N-methyl-d-aspartate receptor antibody levels and were associated with early (<40 days) administration of immunotherapies in non-paraneoplastic patients (P < 0.0001) and earlier tumour removal in paraneoplastic patients (P = 0.02). Ten patients (23) who were first diagnosed during relapses had no evidence of tumours but had received minimal or no immunotherapy during earlier episodes. Temporal analysis of the onset of the neurological features suggested progression through two main stages. The time of onset of the early features, characterized by neuropsychiatric symptoms and seizures preceded by a median of 10-20 days, the onset of movement disorders, reduction in consciousness and dysautonomia. This temporal dichotomy was also seen in the timing of cerebrospinal fluid, electroencephalographic and in the rather infrequent cerebral imaging changes. Overall, our data support a model in which the early features are associated with cerebrospinal fluid lymphocytosis, and the later features with appearance of oligoclonal bands. The immunological events and neuronal mechanisms underlying these observations need to be explored further, but one possibility is that the early stage represents diffusion of serum antibodies into the cortical grey matter, whereas the later stage results from secondary expansion of the immunological repertoire within the intrathecal compartment acting on subcortical neurons. Four patients, who only had temporal lobe epilepsy without oligoclonal bands, may represent restriction to the first stage. © The Author(s) 2010.

Faber CG, Molenaar PC, Vles JS, Bonifati DM, Verschuuren JJ, van Doorn PA, Kuks JB, Wokke JH, Beeson D, De Baets M. 2009. AChR deficiency due to epsilon-subunit mutations: two common mutations in the Netherlands. J Neurol, 256 (10), pp. 1719-1723. | Show Abstract | Read more

Congenital myasthenic syndromes are a clinically and genetically heterogeneous group of hereditary disorders affecting neuromuscular transmission. We have identified mutations within the acetylcholine receptor (AChR) epsilon-subunit gene underlying congenital myasthenic syndromes in nine patients (seven kinships) of Dutch origin. Previously reported mutations epsilon1369delG and epsilonR311Q were found to be common; epsilon1369delG was present on at least one allele in seven of the nine patients, and epsilonR311Q in six. Phenotypes ranged from relatively mild ptosis and external ophthalmoplegia to generalized myasthenia. The common occurrence of epsilonR311Q and epsilon1369delG suggests a possible founder for each of these mutations originating in North Western Europe, possibly in Holland. Knowledge of the ethnic or geographic origin within Europe of AChR deficiency patients can help in targeting genetic screening and it may be possible to provide a rapid genetic diagnosis for patients of Dutch origin by screening first for epsilonR311Q and epsilon1369delG.

Vogt J, Morgan NV, Marton T, Maxwell S, Harrison BJ, Beeson D, Maher ER. 2009. Germline mutation in DOK7 associated with fetal akinesia deformation sequence. J Med Genet, 46 (5), pp. 338-340. | Show Abstract | Read more

BACKGROUND: Fetal akinesia deformation sequence syndrome (FADS) is a heterogeneous disorder characterised by fetal akinesia and developmental defects including, in some case, pterygia. Multiple pterygium syndromes (MPS) are traditionally divided into prenatally lethal and non-lethal (such as Escobar) types. Previously, we and others reported that homozygous mutations in the fetal acetylcholine receptor gamma subunit (CHRNG) can cause both lethal and non-lethal MPS, demonstrating that pterygia resulted from fetal akinesia, and that mutations in the acetylcholine receptor subunits CHRNA1, CHRND, and Rapsyn (RAPSN) can also result in a MPS/FADS phenotype. METHODS: We hypothesised that mutations in other acetylcholine receptor related genes may interfere with neurotransmission at the neuromuscular junction and so we analysed 14 cases of lethal MPS/FADS without CHRNG, CHRNA1, CHRNB1, CHRND, or RAPSN mutations for mutations in DOK7. RESULTS: A homozygous DOK7 splice site mutation, c.331+1G>T, was identified in a family with three children affected with lethal FADS. Previously DOK7 mutations have been reported to underlie a congenital myaesthenic syndrome with a characteristic "limb girdle" pattern of muscle weakness. CONCLUSION: This finding is consistent with the hypothesis that whereas incomplete loss of DOK7 function may cause congenital myasthenia, more severe loss of function can result in a lethal fetal akinesia phenotype.

Irani SR, Maxwell S, Zandi M, Cossins J, Beeson D, Vincent A. 2009. CLINICAL FEATURES AND FOLLOW-UP OF 13 PATIENTS WITH ANTIBODIES AGAINST NMDA RECEPTORS JOURNAL OF NEUROLOGY NEUROSURGERY AND PSYCHIATRY, 80 (4), pp. 452-452.

Burke G, Allen D, Arunachalam R, Beeson D, Hammans S. 2009. A treatable muscle disease. Pract Neurol, 9 (4), pp. 233-236. | Read more

Irani SR, Waters P, Beeson D, Lang B, Vincent A. 2008. A novel antigenic target in Morvan's syndrome JOURNAL OF NEUROIMMUNOLOGY, 203 (2), pp. 181-181.

Waters P, Jacobson L, Leite MI, Maxwell S, Beeson D, Vincent A. 2008. A novel fluorescent immunoprecipitation (FIPA) method to detect antibodies to new targets in antibody-mediated diseases JOURNAL OF NEUROIMMUNOLOGY, 203 (2), pp. 184-184.

Jacob S, Leite MI, Waters P, Viegas S, Cossins J, Beeson D, Morgan BP, Vincent A. 2008. IgG subclasses of disease-specific antibodies and complement activation in-vitro in seronegative myasthenia gravis and neuromyelitis optica JOURNAL OF NEUROIMMUNOLOGY, 203 (2), pp. 180-181.

Kinali M, Beeson D, Pitt MC, Jungbluth H, Simonds AK, Aloysius A, Cockerill H, Davis T et al. 2008. Congenital myasthenic syndromes in childhood: diagnostic and management challenges. J Neuroimmunol, 201-202 (C), pp. 6-12. | Show Abstract | Read more

The Congenital Myasthenic Syndromes (CMS), a group of heterogeneous genetic disorders of neuromuscular transmission, are often misdiagnosed as congenital muscular dystrophy (CMD) or myopathies and present particular management problems. We present our experience of 46 children with CMS, referred to us between 1992-2007 with provisional diagnoses of congenital myopathy (22/46), CMS or limb-girdle myasthenia (9/46), central hypotonia or neurometabolic disease (5/46), myasthenia gravis (4/46), limb-girdle or congenital muscular dystrophy (4/46) and SMA (2/46). Diagnosis was often considerably delayed (up to 18y4 m), despite the early symptoms in most cases. Diagnostic clues in the neonates were feeding difficulties (29/46), hypotonia with or without limb weakness (21/46), ptosis (19/46), respiratory insufficiency (12/46), contractures (4/46) and stridor (6/46). Twenty-five children had delayed motor milestones. Fatigability developed in 43 and a variable degree of ptosis was eventually present in 40. Over the period of the study, the mainstay of EMG diagnosis evolved from repetitive nerve stimulation to stimulation single fibre EMG. The patients were studied by several different operators. 66 EMGs were performed in 40 children, 29 showed a neuromuscular junction abnormality, 7 were myopathic, 2 had possible neurogenic changes and 28 were normal or inconclusive. A repetitive CMAP was detected in only one of seven children with a COLQ mutation and neither of the two children with Slow Channel Syndrome mutations. Mutations have been identified so far in 32/46 children: 10 RAPSN, 7 COLQ, 6 CHRNE, 7 DOK7, 1 CHRNA1 and 1 CHAT. 24 of 25 muscle biopsies showed myopathic changes with fibre size variation; 14 had type-1 fibre predominance. Three cases showed small type-1 fibres resembling fibre type disproportion, and four showed core-like lesions. No specific myopathic features were associated with any of the genes. Twenty children responded to Pyridostigmine treatment alone, 11 to Pyridostigmine with either 3, 4 DAP or Ephedrine and five to Ephedrine alone. Twenty one children required acute or chronic respiratory support, with tracheostomy in 4 and nocturnal or emergency non-invasive ventilation in 9. Eight children had gastrostomy. Another 11 were underweight for height indicative of failure to thrive and required dietetic input. A high index of clinical suspicion, repeat EMG by an experienced electromyographer and, if necessary, a therapeutic trial of Pyridostigmine facilitates the diagnosis of CMS with subsequent molecular genetic confirmation. This guides rational therapy and multidisciplinary management, which may be crucial for survival, particularly in pedigrees where previous deaths have occurred in infancy.

Maclennan CA, Vincent A, Marx A, Willcox N, Gilhus NE, Newsom-Davis J, Beeson D. 2008. Preferential expression of AChR epsilon-subunit in thymomas from patients with myasthenia gravis. J Neuroimmunol, 201-202 (C), pp. 28-32. | Show Abstract | Read more

The role of antigen expression by thymomas in myasthenia gravis (MG) is not clear. Previous reports of acetylcholine receptor (AChR) mRNA expression by the highly sensitive reverse transcription-polymerase chain reactions (RT-PCR) produced varying results. To try to clarify this issue, we first used RT-PCR but then turned to the more accurate and quantitative RNase protection assays (RPA) to assess AChR subunit mRNA expression in thymomas from 25 patients (22 with MG). By RT-PCR, all five AChR subunits could be detected in many thymomas. However, by RPA, the mRNA for the adult-specific AChR epsilon-subunit was found in 13/25 (52%) thymomas, but not mRNA for the other subunits. AChR epsilon-subunit was more frequently detected in thymomas of A or AB histology (WHO classification) than those with B1-B3 histology. Overall, 6/6 with thymomas of A or AB histology were positive compared with only 8/19 with B histology (p=0.02). Autoantibodies in the two patients with the highest levels of epsilon-subunit mRNA bound better to adult (alpha(2)betadeltaepsilon) AChR than to fetal (alpha(2)betadeltagamma) AChR, whereas the other sera bound better to fetal AChR. The greater abundance of mRNA for AChR epsilon-subunit than for other subunits suggests that the AChR epsilon-subunit may play a distinctive role in autosensitization in MG-associated thymomas, particularly those of type A or AB.

Palace J, Beeson D. 2008. The congenital myasthenic syndromes. J Neuroimmunol, 201-202 (C), pp. 2-5. | Show Abstract | Read more

The congenital myasthenic syndromes (CMS) are rare inherited disorders of neuromuscular transmission characterised by fatigable muscle weakness. Thus far, genetic analysis has identified mutations in eleven different genes but it is clear that additional phenotypic subgroups exist where the underlying genetics has not yet been defined. Although each syndrome results from defective synaptic transmission at the neuromuscular junction, the patients show a variable set of phenotypes. Here, we provide a brief clinical review.

Waters P, Jarius S, Littleton E, Leite MI, Jacob S, Gray B, Geraldes R, Vale T et al. 2008. Aquaporin-4 antibodies in neuromyelitis optica and longitudinally extensive transverse myelitis. Arch Neurol, 65 (7), pp. 913-919. | Show Abstract | Read more

BACKGROUND: There is increasing recognition of antibody-mediated immunotherapy-responsive neurologic diseases and a need for appropriate immunoassays. OBJECTIVES: To develop a clinically applicable quantitative assay to detect the presence of aquaporin-4 (AQP4) antibodies in patients with neuromyelitis optica and to characterize the anti-AQP4 antibodies. DESIGN: We compared a simple new quantitative fluorescence immunoprecipitation assay (FIPA) with both indirect immunofluorescence and an AQP4-transfected cell-based assay, both previously described. We used the cell-based assay to characterize the antibodies for their immunoglobulin class, IgG subclass, and ability to induce complement C3b deposition in vitro. SETTING: United Kingdom and Germany. PARTICIPANTS: Serum samples from patients with neuromyelitis optica (n = 25) or longitudinally extensive transverse myelitis (n = 11) and from relevant controls (n = 78) were studied. MAIN OUTCOME MEASURES: Comparison of different assays for AQP4 antibodies and characterization of anti-AQP4 antibodies in patients with neuromyelitis optica. RESULTS: We found antibodies to AQP4 in 19 of 25 patients with neuromyelitis optica (76%) using FIPA, in 20 of 25 patients with neuromyelitis optica (80%) using the cell-based assay, and in 6 of 11 patients with longitudinally extensive transverse myelitis (55%) with both assays; these assays were more sensitive than indirect immunofluorescence and 100% specific. The antibodies bound to extracellular epitope(s) of AQP4, were predominantly IgG1, and strongly induced C3b deposition. CONCLUSIONS: Aquaporin-4 is a major antigen in neuromyelitis optica, and antibodies can be detected in more than 75% of patients. Further studies on larger samples will show whether this novel FIPA is suitable for clinical use. The IgG1 antibodies bind to AQP4 on the cell surface and can initiate complement deposition. These approaches will be useful for investigation of other antibody-mediated diseases.

Yamanashi Y, Higuch O, Beeson D. 2008. Dok-7/MuSK signaling and a congenital myasthenic syndrome. Acta Myol, 27 (JULY), pp. 25-29. | Show Abstract

Skeletal muscle contraction is controlled by motor neurons, which contact the muscle at the neuromuscular junction (NMJ). The formation and maintenance of the NMJ, which includes the aggregation of densely packed clusters of acetylcholine receptor (AChR) opposite the motor nerve terminal, is orchestrated by muscle-specific receptor tyrosine kinase, MuSK. Recently, a MuSK-interacting cytoplasmic adaptor-like protein Dok-7 was identified and its localization at the postsynaptic region of the NMJ was revealed. Mice lacking Dok-7 have a phenotype indistinguishable from MuSK-deficient mice, and fail to form both AChR clusters and NMJs. In cultured myotubes, Dok-7 is required for MuSK activation and AChR clustering. Thus, Dok-7 is essential for neuromuscular synaptogenesis and it appears that the regulatory interaction of Dok-7 with MuSK is integrally involved in this process. In humans there are both autoimmune and genetic causes of defective neuromuscular transmission that gives rise to the fatigable muscle weakness known as myasthenia. DOK7 has been found to be a major locus for mutations that underlie a genetic form of myasthenia with a characteristic 'limb girdle' pattern of muscle weakness (DOK7 CMS). Patients with DOK7 CMS have small, simplified NMJs but normal AChR function. The most common mutation causes a COOH-terminal truncation, which greatly impairs Dok-7's ability to activate MuSK. Recently, a series of differing DOK7 mutations have been identified, which affect not only the COOH-terminal region but also the NH2-terminal moiety. The study of these mutations may help understand the underlying pathogenic mechanism of DOK7 CMS.

Leite MI, Jacob S, Viegas S, Cossins J, Clover L, Morgan BP, Beeson D, Willcox N, Vincent A. 2008. IgG1 antibodies to acetylcholine receptors in 'seronegative' myasthenia gravis. Brain, 131 (Pt 7), pp. 1940-1952. | Show Abstract | Read more

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.

Vogt J, Harrison BJ, Spearman H, Cossins J, Vermeer S, ten Cate LN, Morgan NV, Beeson D, Maher ER. 2008. Mutation analysis of CHRNA1, CHRNB1, CHRND, and RAPSN genes in multiple pterygium syndrome/fetal akinesia patients. Am J Hum Genet, 82 (1), pp. 222-227. | Show Abstract | Read more

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.

Hamuro J, Higuchi O, Okada K, Ueno M, Iemura S, Natsume T, Spearman H, Beeson D, Yamanashi Y. 2008. Mutations causing DOK7 congenital myasthenia ablate functional motifs in Dok-7. J Biol Chem, 283 (9), pp. 5518-5524. | Show Abstract | Read more

Dok-7 is a cytoplasmic activator of muscle-specific receptor-tyrosine kinase (MuSK). Both Dok-7 and MuSK are required for neuromuscular synaptogenesis. Mutations in DOK7 underlie a congenital myasthenic syndrome (CMS) associated with small and simplified neuromuscular synapses likely due to impaired Dok-7/MuSK signaling. The overwhelming majority of patients with DOK7 CMS have at least one allele with a frameshift mutation that causes a truncation in the COOH-terminal region of Dok-7 and affects MuSK activation. Dok-7 has pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains in the NH2-terminal moiety, both of which are indispensable for MuSK activation in myotubes, but little is known about additional functional elements. Here, we identify a chromosome region maintenance 1-dependent nuclear export signal (NES) in the COOH-terminal moiety and demonstrate that the NES-mediated cytoplasmic location of Dok-7 is essential for regulating the interaction with MuSK in myotubes. The NH2-terminal PH domain is responsible for the nuclear import of Dok-7. We also show that the Src homology 2 target motifs in the COOH-terminal moiety of Dok-7 are active and crucial for MuSK activation in myotubes. In addition, CMS-associated missense mutations found in the PH or PTB domain inactivate Dok-7. Together, these findings demonstrate that, in addition to the NH2-terminal PH and PTB domains, the COOH-terminal NES and Src homology 2 target motifs play key roles in Dok-7/MuSK signaling for neuromuscular synaptogenesis. Ablation or disruption of these functional elements in Dok-7 probably underlies the neuromuscular junction synaptopathy observed in DOK7 CMS.

Vincent A, Leite MI, Farrugia ME, Jacob S, Viegas S, Shiraishi H, Benveniste O, Morgan BP et al. 2008. Myasthenia gravis seronegative for acetylcholine receptor antibodies. Ann N Y Acad Sci, 1132 (1), pp. 84-92. | Show Abstract | Read more

Antibodies to muscle-specific kinase (MuSK) are found in a variable proportion of patients with myasthenia without typical acetylcholine receptor (AChR) antibodies, but their characteristics and pathogenic mechanisms are not fully understood. We discuss the incidence and pathogenicity of MuSK antibodies and how clinical studies, animal models, and cultured cell lines can be used to elucidate their pathogenic mechanisms. Patients without either AChR or MuSK antibodies (seronegative myasthenia) appear to present another disease subtype that is highly similar to that of typical myasthenia gravis. We demonstrate a new method that detects AChR antibodies in these patients and show that these low-affinity AChR antibodies are predominantly IgG1 and can activate complement C3b deposition. Similarly MuSK antibodies, although mainly IgG4, are partially IgG1 and can activate C3b deposition. Overall, these results suggest that complement-activation may be an important pathogenic mechanism even in patients without conventional AChR antibodies.

Abdelgany A, Wood M, Beeson D. 2007. Hairpin DNAzymes: a new tool for efficient cellular gene silencing. J Gene Med, 9 (8), pp. 727-738. | Show Abstract | Read more

BACKGROUND: RNA-based gene silencing is potentially a powerful therapeutic strategy. Catalytic 10-23 DNAzymes bind to target RNA by complimentary sequence arms on a Watson-Crick basis and thus can be targeted to effectively cleave specific mRNA species. However, for in vivo applications it is necessary to stabilise DNAzymes against nucleolytic attack. Chemical modifications can be introduced into the binding arms to increase stability but these may alter catalytic activity and in some cases increase cell toxicity. METHODS: We designed novel 10-23 DNAzyme structures that incorporate stem-loop hairpins at either end on the DNAzyme binding arms. The catalytic activity of hairpin DNAzymes (hpDNAzyme) were tested in vitro against 32P-labelled cRNA encoding the muscle acetylcholine receptor (AChR) alpha-subunit. Resistance of hpDNAzymes to nucleolytic degradation was tested by incubation of the hpDNAzymes with Bal-31, DNase1 or HeLa cell extract. Gene silencing by hpDNAzymes was assessed by measuring reduced fluorescence from DsRed2 and EGFP reporters in cell culture systems, and reduced 125I-alpha-bungarotoxin binding in cells transfected with cDNA encoding the AChR. RESULTS: We show that hpDNAzymes show remarkable resistance to nucleolytic degradation, and demonstrate that in cell culture systems the hpDNAzymes are far more effective than standard 10-23 DNAzymes in down-regulating protein expression from target mRNA species. CONCLUSION: hpDNAzymes provide new molecular tools that, without chemical modification, give highly efficient gene silencing in cells, and may have potential therapeutic applications.

Palace J, Lashley D, Newsom-Davis J, Cossins J, Maxwell S, Kennett R, Jayawant S, Yamanashi Y, Beeson D. 2007. Clinical features of the DOK7 neuromuscular junction synaptopathy. Brain, 130 (Pt 6), pp. 1507-1515. | Show Abstract | Read more

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.

Farrugia ME, Bonifati DM, Clover L, Cossins J, Beeson D, Vincent A. 2007. Effect of sera from AChR-antibody negative myasthenia gravis patients on AChR and MuSK in cell cultures. J Neuroimmunol, 185 (1-2), pp. 136-144. | Show Abstract | Read more

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.

Giraud M, Taubert R, Vandiedonck C, Ke X, Lévi-Strauss M, Pagani F, Baralle FE, Eymard B et al. 2007. An IRF8-binding promoter variant and AIRE control CHRNA1 promiscuous expression in thymus. Nature, 448 (7156), pp. 934-937. | Show Abstract | Read more

Promiscuous expression of tissue-restricted auto-antigens in the thymus imposes T-cell tolerance and provides protection from autoimmune diseases. Promiscuous expression of a set of self-antigens occurs in medullary thymic epithelial cells and is partly controlled by the autoimmune regulator (AIRE), a nuclear protein for which loss-of-function mutations cause the type 1 autoimmune polyendocrine syndrome. However, additional factors must be involved in the regulation of this promiscuous expression. Here we describe a mechanism controlling thymic transcription of a prototypic tissue-restricted human auto-antigen gene, CHRNA1. This gene encodes the alpha-subunit of the muscle acetylcholine receptor, which is the main target of pathogenic auto-antibodies in autoimmune myasthenia gravis. On re-sequencing the CHRNA1 gene, we identified a functional bi-allelic variant in the promoter that is associated with early onset of disease in two independent human populations (France and United Kingdom). We show that this variant prevents binding of interferon regulatory factor 8 (IRF8) and abrogates CHRNA1 promoter activity in thymic epithelial cells in vitro. Notably, both the CHRNA1 promoter variant and AIRE modulate CHRNA1 messenger RNA levels in human medullary thymic epithelial cells ex vivo and also in a transactivation assay. These findings reveal a critical function of AIRE and the interferon signalling pathway in regulating quantitative expression of this auto-antigen in the thymus, suggesting that together they set the threshold for self-tolerance versus autoimmunity.

Müller JS, Herczegfalvi A, Vilchez JJ, Colomer J, Bachinski LL, Mihaylova V, Santos M, Schara U et al. 2007. Phenotypical spectrum of DOK7 mutations in congenital myasthenic syndromes. Brain, 130 (Pt 6), pp. 1497-1506. | Show Abstract | Read more

Dok ('downstream-of-kinase') family of cytoplasmic proteins play a role in signalling downstream of receptor and non-receptor phosphotyrosine kinases. Recently, a skeletal muscle receptor tyrosine kinase (MuSK)-interacting cytoplasmic protein termed Dok-7 has been identified. Subsequently, we and others identified mutations in DOK7 as a cause of congenital myasthenic syndromes (CMS), providing evidence for a crucial role of Dok-7 in maintaining synaptic structure. Here we present clinical and molecular genetic data of 14 patients from 12 independent kinships with 13 different mutations in the DOK7 gene. The clinical picture of CMS with DOK7 mutations is highly variable. The age of onset may vary between birth and the third decade. However, most of the patients display a characteristic 'limb-girdle' pattern of weakness with a waddling gait and ptosis, but without ophthalmoparesis. Respiratory problems were frequent. Patients did not benefit from long-term therapy with esterase inhibitors; some of the patients even worsened. DOK7 mutations have emerged as one of the major genetic defects in CMS. The clinical picture differs significantly from CMS caused by mutations in other genes, such as the acetylcholine receptor (AChR) subunit genes. None of the patients with DOK7 mutations had tubular aggregates in the muscle biopsy, implying that 'limb-girdle myasthenia (LGM) with tubular aggregates' previously described in literature may be a pathogenic entity distinct from CMS caused by DOK7 mutations.

Cossins J, Burke G, Maxwell S, Spearman H, Man S, Kuks J, Vincent A, Palace J, Fuhrer C, Beeson D. 2006. Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations. Brain, 129 (Pt 10), pp. 2773-2783. | Show Abstract | Read more

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.

Beeson D, Higuchi O, Palace J, Cossins J, Spearman H, Maxwell S, Newsom-Davis J, Burke G et al. 2006. Dok-7 mutations underlie a neuromuscular junction synaptopathy. Science, 313 (5795), pp. 1975-1978. | Show Abstract | Read more

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.

Slater CR, Fawcett PR, Walls TJ, Lyons PR, Bailey SJ, Beeson D, Young C, Gardner-Medwin D. 2006. Pre- and post-synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with 'limb-girdle myasthenia'. Brain, 129 (Pt 8), pp. 2061-2076. | Show Abstract | Read more

The properties of neuromuscular junctions (NMJs) were studied in motor-point biopsy samples from eight patients with congenital myasthenic syndromes affecting primarily proximal limb muscles ['limb-girdle myasthenia' (LGM)]. All had moderate to severe weakness of the proximal muscles, without short-term clinical fatigability but with marked variation in strength over periods of weeks or months, with little or no facial weakness or ptosis and no ophthalmoplegia. Most had a characteristic gait and stance. All patients showed decrement of the compound muscle action potential (CMAP) on repetitive stimulation at 3 Hz, and increased jitter and blocking was detected by SFEMG, confirming the presence of impaired neuromuscular transmission. None of the patients had serum antibodies against acetylcholine receptors (AChRs). Two of the patients had similarly affected siblings. Intracellular recording from isolated nerve-muscle preparations revealed that the quantal content (the number of ACh quanta released per nerve impulse) was only approximately 50% of that in controls. However, the quantal size (amplitude of miniature end-plate currents) and the kinetic properties of synaptic potentials and currents were similar to control values. The area of synaptic contact and extent of post-synaptic folding were approximately 50% of control values. Thus, the quantal content per unit area of synaptic contact was normal. The number of AChRs per NMJ was also reduced to approximately 50% of normal, so the local AChR density was normal. Immunolabelling studies revealed qualitatively normal distributions and abundance of each of 14 proteins normally concentrated at the NMJ, including components of the basal lamina, post-synaptic membrane and post-synaptic cytoskeleton. DNA analysis failed to detect mutations in the genes encoding any of the following proteins: AChR subunits, rapsyn, ColQ, ChAT or muscle-specific kinase. Response of these patients to treatment was varied: few showed long-term improvement with pyridostigmine and some even deteriorated with treatments, while others had intolerable side-effects. Several patients showed improvement with 3,4-diaminopyridine, but this was generally only transient. Ephedrine was helpful in half of the patients. We conclude that impaired neuromuscular transmission in these LGM patients results from structural abnormalities of the NMJ, including reduced size and post-synaptic folding, rather from any abnormality in the immediate events of neuromuscular transmission.

Burke G, Jayawant S, Palace J, Newsom-Davis J, Beeson D. 2006. A nationwide service for the diagnosis and treatment of congenital myasthenic syndromes DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY, 48 pp. 5-5.

Müller JS, Baumeister SK, Schara U, Cossins J, Krause S, von der Hagen M, Huebner A, Webster R, Beeson D, Lochmüller H, Abicht A. 2006. CHRND mutation causes a congenital myasthenic syndrome by impairing co-clustering of the acetylcholine receptor with rapsyn. Brain, 129 (Pt 10), pp. 2784-2793. | Show Abstract | Read more

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.

Mewasingh LD, Hussein N, Jayawant S, Newsom-Davis J, Beeson D. 2006. Chronic stridor as an early presentation of congenital myasthenic syndrome due to RAPSN mutation DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY, 48 pp. 30-30.

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Cossins J, Burke G, Maxwell S, Spearman H, Man S, Kuks J, Vincent A, Palace J, Fuhrer C, Beeson D. 2006. Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations Brain, 129 (10), pp. 2773-2783. | Show Abstract | Read more

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. © The Author (2006). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.

Gattenlöhner S, Marx A, Markfort B, Pscherer S, Landmeier S, Juergens H, Müller-Hermelink HK, Matthews I, Beeson D, Vincent A, Rossig C. 2006. Rhabdomyosarcoma lysis by T cells expressing a human autoantibody-based chimeric receptor targeting the fetal acetylcholine receptor. Cancer Res, 66 (1), pp. 24-28. | Show Abstract | Read more

Rhabdomyosarcomas are the most frequent malignant soft tissue tumors of childhood; however, because current multimodality treatments fail to improve the poor survival rate of children with metastatic rhabdomyosarcoma, new treatments are required. We previously identified the gamma-subunit of the fetal acetylcholine receptor (fAChR) as a specific cell surface target in rhabdomyosarcoma. Here, we engineered human T lymphocytes to express chimeric receptors composed of the antigen-binding domain of a human anti-fAChR antibody joined to the signaling domain of the human T-cell receptor zeta-chain. The interaction of fAChRzeta-transduced T cells with fAChR-positive rhabdomyosarcoma cell lines, but not with fAChR-negative control cells, induced T-cell activation characterized by strong secretion of IFN-gamma and delayed lysis of tumor cells. Importantly, we found that in six of six rhabdomyosarcoma patients, chemotherapy increased fAChR expression on residual tumor cells in vivo. Our observations suggest that these fully human chimeric fAChRzeta-transduced T cells, which should be well tolerated by the patient, have potential use in vivo both as a primary treatment for rhabdomyosarcoma and as a complementary approach to eradicate residual tumor cells after chemotherapy.

Watson R, Jepson JE, Bermudez I, Alexander S, Hart Y, McKnight K, Roubertie A, Fecto F et al. 2005. Alpha7-acetylcholine receptor antibodies in two patients with Rasmussen encephalitis. Neurology, 65 (11), pp. 1802-1804. | Show Abstract | Read more

Rasmussen encephalitis (RE) sera were screened for antibodies to human alpha7 nicotinic acetylcholine receptors (nAChRs) using electrophysiology, calcium imaging, and ligand binding assays. Sera from two of nine patients with RE blocked ACh-induced currents through alpha7 nAChRs and the ACh-induced rise in intracellular free calcium ([Ca2+]i) and inhibited (125)I-alpha-bungarotoxin binding in cells expressing alpha7 nAChRs. Thus, the alpha7 nAChR is a potential target for pathogenic antibodies in patients with RE.

Beeson D, Hantaï D, Lochmüller H, Engel AG. 2005. 126th International Workshop: congenital myasthenic syndromes, 24-26 September 2004, Naarden, the Netherlands. Neuromuscul Disord, 15 (7), pp. 498-512. | Read more

Spreadbury I, Kishore U, Beeson D, Vincent A. 2005. Inhibition of acetylcholine receptor function by seronegative myasthenia gravis non-IgG factor correlates with desensitisation. J Neuroimmunol, 162 (1-2), pp. 149-156. | Show Abstract | Read more

15% of myasthenia gravis (MG) patients do not have antibodies against the acetylcholine receptor (AChR). Some of these "seronegative" MG patients have antibodies against muscle specific kinase (MuSK), and many have a non-IgG factor that acutely inhibits AChR function in a muscle-like cell line, CN21. Here we show, using mainly one plasma negative for both AChR and MuSK antibodies, that the inhibitory effect of the non-IgG fraction correlates well with the desensitisation caused by 100 microM nicotine, and is found also when AChRs are expressed in a non-muscle cell line (HEK). Moreover, a similar effect was seen with M3C7-a monoclonal antibody against human AChR. The results suggest that, rather than acting indirectly as previously proposed, the SNMG factor may bind directly to an allosteric site that induces or enhances AChR desensitisation.

Fostieri E, Tzartos SJ, Berrih-Aknin S, Beeson D, Mamalaki A. 2005. Isolation of potent human Fab fragments against a novel highly immunogenic region on human muscle acetylcholine receptor which protect the receptor from myasthenic autoantibodies. Eur J Immunol, 35 (2), pp. 632-643. | Show Abstract | Read more

In the autoimmune disease myasthenia gravis (MG), antibodies against the muscle nicotinic acetylcholine receptor (AChR) cause loss of functional AChR in the neuromuscular junction. To isolate AChR-specific human antibody fragments (Fab), a phage-display library was constructed from an MG patient's thymic B lymphocytes. The first Fab isolated had a low affinity for human AChR, but two sequential antibody chain shufflings using the MG donor heavy and light chain gene repertoires resulted in isolating two new Fab with an approximately 30-fold higher binding ability. The selected Fab contained extensively mutated heavy and light chains and probably represent intraclonal variants of a common progenitor having diverged in vivo by somatic hypermutation. Interestingly, the isolated Fab bound to an extracellular highly immunogenic region located either on an alpha-subunit site affected by the gamma/epsilon-subunits or on the interface between alpha- and gamma/epsilon-subunits. This region is not the previously described "main immunogenic region" (MIR), although it seems to be close to it, as one improved Fab and an anti-MIR mAb competed for AChR binding with distinctly different subpopulations of MG sera. Furthermore, this Fab protected surface AChR in cell cultures against MG autoantibody-induced antigenic modulation, suggesting a potential therapeutic use in MG, especially in combination with a human anti-MIR Fab.

Abdelgany A, Ealing J, Wood M, Beeson D. 2005. Selective cleavage of AChR cRNAs harbouring mutations underlying the slow channel myasthenic syndrome by hammerhead ribozymes. J RNAi Gene Silencing, 1 (1), pp. 26-31. | Show Abstract

Slow channel congenital myasthenic syndrome (SCCMS) is a dominant disorder caused by missense mutations in muscle acetylcholine receptors (AChR). Expression from mutant alleles causes prolonged AChR ion-channel activations. This 'gain of function' results in excitotoxic damage due to excess entry of calcium ions that manifests as an endplate myopathy. The biology of SCCMS provides a model system to investigate the potential of catalytic nucleic acids for therapy in dominantly inherited disorders involving single missense mutations. Hammerhead ribozymes can catalytically cleave RNA transcripts in a sequence-specific manner. We designed hammerhead ribozymes to target transcripts from four SCCMS mutations, alphaT254I, alphaS226F, alphaS269I and epsilonL221F. Ribozymes were incubated with cRNA transcripts encoding wild type and mutant AChR subunits. The ribozymes efficiently cleaved the mutant allele cRNA transcripts but left the wild type cRNA intact. Cleavage efficiency was optimised for alphaS226F. We were able to demonstrate robust catalytic activity under simulated physiological conditions and at high Ca(2+) concentrations, which is likely to be accumulated at the endplate region of the SCCMS patient muscles. These results demonstrate the potential for gene therapy applications of ribozymes to specifically down-regulate expression of mutant alleles in dominantly inherited disorders.

Abdelgany A, Ealing J, Wood M, Beeson D. 2005. Selective DNAzyme-mediated cleavage of AChR mutant transcripts by targeting the mutation site or through mismatches in the binding arm. J RNAi Gene Silencing, 1 (1), pp. 32-37. | Show Abstract

Many dominantly inherited disorders are caused by missense amino acid substitutions resulting from a single nucleotide exchange in the encoding gene. For these disorders, where proteins expressed from the mutant alleles are often pathogenic and present throughout life, gene silencing, through intervention at the mRNA level, holds promise as a therapeutic approach. We have used mutations that underlie the slow channel congenital myasthenic syndrome (SCCMS) as a model system to study allele-specific gene silencing of RNA transcripts by DNAzymes. We tested the ability of DNAzymes to give allele-specific cleavage for i) mutations that create cleavage sites, and ii) mutations located close to a DNAzyme cleavage site that create a potential mismatch in the binding arms. For both we demonstrate selective cleavage of mutant transcripts under simulated physiological conditions. For DNAzymes with binding arm mismatches the degree of selectivity for mutant over wild type may be enhanced by optimising the mismatch position as well as the binding arm length. The optimal sites for mismatches are 1.1 and 1.2 in arm I, and 16.2 in arm II. Asymmetric binding arm DNAzymes with a shorter arm I are more discriminative. Our results show it should be possible to apply DNAzyme-mediated cleavage of mutant alleles even when the mutant does not itself create a putative cleavage site. This therapeutic approach may be well suited to dominantly inherited disorders such as SCCMS, where loss of some wild type transcripts is unlikely to have pathogenic consequences.

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Scopus

Watson R, Jepson JEC, Bermudez I, Alexander S, Hart Y, McKnight K, Roubertie A, Fecto F et al. 2005. α7-Acetylcholine receptor antibodies in two patients with Rasmussen encephalitis Neurology, 65 (11), pp. 1802-1804. | Show Abstract | Read more

Rasmussen encephalitis (RE) sera were screened for antibodies to human α7 nicotinic acetylcholine receptors (nAChRs) using electrophysiology, calcium imaging, and ligand binding assays. Sera from two of nine patients with RE blocked ACh-induced currents through α7 nAChRs and the ACh-induced rise in intracellular free calcium ([Ca2+]i) and inhibited 125I-α-bungarotoxin binding in cells expressing α7 nAChRs. Thus, the α7 nAChR is a potential target for pathogenic antibodies in patients with RE. Copyright © 2005 by AAN Enterprises, Inc.

Cossins J, Webster R, Maxwell S, Burke G, Vincent A, Beeson D. 2004. A mouse model of AChR deficiency syndrome with a phenotype reflecting the human condition. Hum Mol Genet, 13 (23), pp. 2947-2957. | Show Abstract | Read more

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.

Morar B, Gresham D, Angelicheva D, Tournev I, Gooding R, Guergueltcheva V, Schmidt C, Abicht A et al. 2004. Mutation history of the roma/gypsies. Am J Hum Genet, 75 (4), pp. 596-609. | Show Abstract | Read more

The 8-10 million European Roma/Gypsies are a founder population of common origins that has subsequently split into multiple socially divergent and geographically dispersed Gypsy groups. Unlike other founder populations, whose genealogy has been extensively documented, the demographic history of the Gypsies is not fully understood and, given the lack of written records, has to be inferred from current genetic data. In this study, we have used five disease loci harboring private Gypsy mutations to examine some missing historical parameters and current structure. We analyzed the frequency distribution of the five mutations in 832-1,363 unrelated controls, representing 14 Gypsy populations, and the diversification of chromosomal haplotypes in 501 members of affected families. Sharing of mutations and high carrier rates supported a strong founder effect, and the identity of the congenital myasthenia 1267delG mutation in Gypsy and Indian/Pakistani chromosomes provided the best evidence yet of the Indian origins of the Gypsies. However, dramatic differences in mutation frequencies and haplotype divergence and very limited haplotype sharing pointed to strong internal differentiation and characterized the Gypsies as a founder population comprising multiple subisolates. Using disease haplotype coalescence times at the different loci, we estimated that the entire Gypsy population was founded approximately 32-40 generations ago, with secondary and tertiary founder events occurring approximately 16-25 generations ago. The existence of multiple subisolates, with endogamy maintained to the present day, suggests a general approach to complex disorders in which initial gene mapping could be performed in large families from a single Gypsy group, whereas fine mapping would rely on the informed sampling of the divergent subisolates and searching for the shared genomic region that displays the strongest linkage disequilibrium with the disease.

Bonifati DM, Webster R, Maxwell S, Brydson M, Polizzi A, Vincent A, Beeson D. 2004. Congenital myasthenic syndrome patients due to AChR epsilon subunit mutations EUROPEAN JOURNAL OF NEUROLOGY, 11 pp. 25-26.

Bonifati DM, Webster R, Maxwell S, Brydson M, Polizzi A, Vincent A, Beeson D. 2004. Correlation between polymorphisms in the AChR gamma and delta subunit promoters and clinical phenotype in AChR deficiency patients EUROPEAN JOURNAL OF NEUROLOGY, 11 pp. 271-271.

Müller JS, Abicht A, Burke G, Cossins J, Richard P, Baumeister SK, Stucka R, Eymard B, Hantaï D, Beeson D, Lochmüller H. 2004. The congenital myasthenic syndrome mutation RAPSN N88K derives from an ancient Indo-European founder. J Med Genet, 41 (8), pp. e104. | Read more

Watson R, Jiang Y, Bermudez I, Houlihan L, Clover L, McKnight K, Cross JH, Hart IK et al. 2004. Absence of antibodies to glutamate receptor type 3 (GluR3) in Rasmussen encephalitis. Neurology, 63 (1), pp. 43-50. | Show Abstract | Read more

OBJECTIVE: To determine the prevalence of serum antibodies to the ionotropic glutamate receptor 3 (GluR3) in patients with Rasmussen encephalitis (RE), a severe epileptic disorder, and to compare with serum from control subjects and patients with intractable epilepsy (IE). METHODS: The authors looked for serum immunoglobulin (Ig) G antibodies to GluR3 in 30 patients with RE, including two patients who had plasma exchange and 12 who had been treated with IV Igs with varying results, and 49 patients with IE and 23 healthy individuals, using ELISA with GluR3B peptide, Western blot analysis of recombinant full-length GluR3, immunoprecipitation of [35S]- and [125I]-labeled GluR3 extracellular domains, immunohistochemistry on rat brain sections, and electrophysiology of GluR3 expressed in Xenopus oocytes. RESULTS: Low levels of antibodies to the GluR3B peptide were detected using ELISA in only 4 of the 79 patients with epilepsy (2 with RE and 2 with IE); binding to GluR3B in other sera was shown to be nonspecific. One other patient with IE had antibodies to recombinant GluR3 on Western blot analysis. However, none of the sera tested precipitated either the [35S]- or the [125I]-labeled GluR3 domains; none bound to rat brain sections in a manner similar to rabbit antibodies to GluR3; and none of the nine sera tested affected the electrophysiologic function of GluR3. CONCLUSIONS: GluR3 antibodies were only infrequently found in Rasmussen encephalitis or intractable epilepsy.

Burke G, Cossins J, Maxwell S, Robb S, Nicolle M, Vincent A, Newsom-Davis J, Palace J, Beeson D. 2004. Distinct phenotypes of congenital acetylcholine receptor deficiency. Neuromuscul Disord, 14 (6), pp. 356-364. | Show Abstract | Read more

We contrast the phenotypes associated with hereditary acetylcholine receptor deficiency arising from mutations in either the acetylcholine receptor epsilon subunit or the endplate acetylcholine receptor clustering protein rapsyn. Mutational screening was performed by amplification of promoter and coding regions by PCR and direct DNA sequencing. We identified mutations in 37 acetylcholine receptor deficiency patients; 18 had acetylcholine receptor-epsilon mutations, 19 had rapsyn mutations. Mutated acetylcholine receptor-epsilon associated with bulbar symptoms, ptosis and ophthalmoplegia at birth, and generalized weakness. Mutated rapsyn caused either an early onset (rapsyn-EO) or late onset (rapsyn-LO) phenotype. Rapsyn-EO associated with arthrogryposis and life-threatening exacerbations during early childhood. Rapsyn-LO presented with limb weakness in adolescence or adulthood resembling seronegative myasthenia gravis. Awareness of distinct phenotypic features of acetylcholine receptor deficiency resulting from acetylcholine receptor-epsilon or rapsyn mutations should facilitate targeted genetic diagnosis, avoid inappropriate immunological therapy and, in some infants, prompt the rapid introduction of treatment that could be life saving.

Webster R, Brydson M, Croxen R, Newsom-Davis J, Vincent A, Beeson D. 2004. Mutation in the AChR ion channel gate underlies a fast channel congenital myasthenic syndrome. Neurology, 62 (7), pp. 1090-1096. | Show Abstract | Read more

BACKGROUND: Most congenital myasthenic syndromes (CMS) have postsynaptic defects from mutations within the muscle acetylcholine receptor (AChR). Mutations underlying the slow channel syndrome cause a "gain of function" and usually show dominant inheritance, whereas mutations underlying AChR deficiency or the fast channel syndrome cause a "loss of function" and show recessive inheritance. OBJECTIVE: To characterize the disease mechanism underlying an apparently dominantly inherited CMS that responds to IV edrophonium. METHODS: DNA from CMS patients was analyzed for mutations by single-strand conformation polymorphism analysis, DNA sequence analysis, and restriction endonuclease digestion. Functional analysis of mutations was by alpha-bungarotoxin binding studies and by patch clamp analysis of mutant AChR expressed in human embryonic kidney cells. RESULTS: Analysis of muscle biopsies from father and son in an affected kinship showed normal endplate morphology and AChR number but severely reduced miniature endplate potentials. DNA analysis revealed that each harbors a single missense mutation in the AChR alpha-subunit gene, alphaF256L. Expression studies demonstrate this mutation underlies a fast channel phenotype with fewer and shorter ion channel activations. The major effect of alphaF256L, located within the M2 transmembrane domain, is on channel gating, both reducing the opening and increasing the closure rate. CONCLUSIONS: Mutation alphaF256L results in fast channel kinetics. Expression studies suggest a dominant-negative effect within the AChR pentamer, severely compromising receptor function.

McConville J, Farrugia ME, Beeson D, Kishore U, Metcalfe R, Newsom-Davis J, Vincent A. 2004. Detection and characterization of MuSK antibodies in seronegative myasthenia gravis. Ann Neurol, 55 (4), pp. 580-584. | Show Abstract | Read more

Antibodies to rat muscle specific kinase, MuSK, have recently been identified in some generalized "seronegative" myasthenia gravis (SNMG) patients, who are often females with marked bulbar symptoms. Using immunoprecipitation of (125)I-labelled-human MuSK, 27 of 66 (41%) seronegative patients were positive, but 18 ocular SNMG patients, 105 AChR antibody positive MG patients, and 108 controls were negative. The antibodies are of high affinity (Kds around 100 pM) with titers between 1 and 200 nM. They bind to the extracellular Ig-like domains of soluble or native MuSK. Surprisingly they are predominantly in the IgG4 subclass. MuSK-antibody associated MG may be different in etiological and pathological mechanisms.

Bonifati DM, Willcox N, Vincent A, Beeson D. 2004. Lack of association between acetylcholine receptor epsilon polymorphisms and early-onset myasthenia gravis. Muscle Nerve, 29 (3), pp. 436-439. | Show Abstract | Read more

A patient with mutations in the acetylcholine receptor (AChR) epsilon subunit, who subsequently developed autoimmune myasthenia gravis (MG), led us to search for epsilon AChR mutations and polymorphisms in 167 patients with early-onset MG. No epsilon-subunit mutations or increased incidence of exonic epsilon-subunit polymorphisms were found. The allelic frequency of the intron polymorphism IVS11+ 20del20 was more prevalent in non-United Kingdom subjects, both patients and healthy individuals, than in United Kingdom subjects (15.8 vs. 6.2%, P = 0.0008) but not between MG patients and healthy individuals. These data provide no evidence that heteroallelic mutations or polymorphisms in the AChR epsilon subunit are involved in the development of autoimmune early-onset MG but raise issues for future studies.

Beeson D. 2004. Autoantibodies in the CNS: encephalopathy and potassium channelopathy. Neurology, 62 (7), pp. 1040-1041. | Read more

Abdelgany A, Wood M, Beeson D. 2003. Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference. Hum Mol Genet, 12 (20), pp. 2637-2644. | Show Abstract | Read more

Slow channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular synapse caused by dominantly inherited missense mutations in genes that encode the muscle acetylcholine receptor (AChR) subunits. Here we investigate the potential of post-transcriptional gene silencing using RNA interference (RNAi) for the selective down-regulation of pathogenic mutant AChR. By transfection of both siRNA and shRNA into mammalian cells expressing wild-type or mutant AChR subunits, we show, using 125I-alpha-bungarotoxin binding and immunofluorescence to measure cell surface AChR expression, efficient discrimination between the silencing of alphaS226F AChR mutant RNA transcripts and the wild-type. In this model we find that selectivity between mutant and wild-type transcripts is optimized with the nucleotide mismatch at position 9 in the shRNA complementary sequence. We also find that allele-specific silencing using shRNA has comparable efficiency to that using siRNA, underlining the general potential of stable expression of shRNA molecules as a long term therapeutic approach for allele-specific silencing of mutant transcripts in dominant genetic disorders.

Wood MJ, Trülzsch B, Abdelgany A, Beeson D. 2003. Therapeutic gene silencing in the nervous system. Hum Mol Genet, 12 Spec No 2 (suppl 2), pp. R279-R284. | Show Abstract | Read more

Progress in the understanding of RNA biology has brought into focus the prospect of using RNA-based therapeutics as a novel approach to treat human disease. In particular, following the discovery of the RNA interference (RNAi) pathway, the emergence of technology based on small interfering RNA (siRNA) now offers a powerful and highly specific tool for therapeutic gene silencing. Many neurological diseases, including neurodegenerative disorders, tumours and retinal disease are likely candidates to benefit from such advances. The challenges ahead will be to identify appropriate disease gene targets and, crucially, to understand the biological parameters that determine safe, precise and effective delivery and function of RNA-based therapeutic molecules within the unique environment of the nervous system.

Burke G, Cossins J, Maxwell S, Owens G, Vincent A, Robb S, Nicolle M, Hilton-Jones D, Newsom-Davis J, Palace J, Beeson D. 2003. Rapsyn mutations in hereditary myasthenia: distinct early- and late-onset phenotypes. Neurology, 61 (6), pp. 826-828. | Show Abstract | Read more

Rapsyn mutations in 16 unrelated patients with a congenital/hereditary myasthenic syndrome were identified, and a mutation (N88K) common to each of them was found. Two distinct phenotypes were noted: early and late onset. The former is frequently associated with arthrogryposis multiplex congenita and life-threatening crises. The late-onset phenotype developed in adolescence or adulthood and was initially mistaken for seronegative myasthenia gravis. Recognition of this late-onset phenotype should prevent inappropriate immunotherapy.

Vincent A, McConville J, Farrugia ME, Bowen J, Plested P, Tang T, Evoli A, Matthews I et al. 2003. Antibodies in myasthenia gravis and related disorders. Ann N Y Acad Sci, 998 (1), pp. 324-335. | Show Abstract | Read more

Acetylcholine receptor (AChR) antibodies are present in around 85% of patients with myasthenia gravis (MG) as measured by the conventional radioimmunoprecipitation assay. Antibodies that block the fetal form of the AChR are occasionally present in mothers who develop MG after pregnancy, especially in those whose babies are born with arthrogryposis multiplex congenita. The antibodies cross the placenta and block neuromuscular transmission, leading to joint deformities and often stillbirth. In these mothers, antibodies made in the thymus are mainly specific for fetal AChR and show restricted germline origins, suggesting a highly mutated clonal response; subsequent spread to involve adult AChR could explain development of maternal MG in those cases who first present after pregnancy. In the 15% of "seronegative" MG patients without AChR antibodies (SNMG), there are serum factors that increase AChR phosphorylation and reduce AChR function, probably acting via a different membrane receptor. These factors are not IgG and could be IgM or even non-Ig serum proteins. In a proportion of SNMG patients, however, IgG antibodies to the muscle-specific kinase, MuSK, are present. These antibodies are not found in AChR antibody-positive MG and are predominantly IgG4. MuSK antibody positivity appears to be associated with more severe bulbar disease that can be difficult to treat effectively.

Beeson D, Webster R, Ealing J, Croxen R, Brownlow S, Brydson M, Newsom-Davis J, Slater C et al. 2003. Structural abnormalities of the AChR caused by mutations underlying congenital myasthenic syndromes. Ann N Y Acad Sci, 998 (1), pp. 114-124. | Show Abstract | Read more

The objective was to define the molecular mechanisms underlying congenital myasthenic syndromes (CMS) by studying mutations within genes encoding the acetylcholine receptor (AChR) and related proteins at the neuromuscular junction. It was found that mutations within muscle AChRs are the most common cause of CMS. The majority are located within the epsilon-subunit gene and result in AChR deficiency.

Wood MJ, Trülzsch B, Abdelgany A, Beeson D. 2003. Ribozymes and siRNA for the treatment of diseases of the nervous system. Curr Opin Mol Ther, 5 (4), pp. 383-388. | Show Abstract

Recent advances in our understanding of RNA biology have focused attention on the potential of developing RNA-based strategies to treat human disease. Naturally occurring catalytic RNA molecules (ribozymes), their synthetic DNA counterparts (deoxyribozymes or DNAzymes), as well as the exciting, emerging technology of small interfering RNA which utilizes the highly conserved cellular RNA interference pathway, are being developed for therapeutic gene silencing purposes. The challenges for the application of this technology to neurological disease will be to identify appropriate disease targets, and to optimize the function, and particularly delivery of these RNA-based therapeutic molecules within the complex environment of the nervous system. This review will assess the potential of these RNA-based therapeutic strategies and the challenges ahead in their application to the treatment of neurological disease.

Hatton CJ, Shelley C, Brydson M, Beeson D, Colquhoun D. 2003. Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant epsilonL221F, inferred from maximum likelihood fits. J Physiol, 547 (Pt 3), pp. 729-760. | Show Abstract | Read more

The mechanisms that underlie activation of nicotinic receptors are investigated using human recombinant receptors, both wild type and receptors that contain the slow channel myasthenic syndrome mutation, epsilonL221F. The method uses the program HJCFIT, which fits the rate constants in a specified mechanism directly to a sequence of observed open and shut times by maximising the likelihood of the sequence with exact correction for missed events. A mechanism with two different binding sites was used. The rate constants that apply to the diliganded receptor (opening, shutting and total dissociation rates) were estimated robustly, being insensitive to the exact assumptions made during fitting, as expected from simulation studies. They are sufficient to predict the main physiological properties of the receptors. The epsilonL221F mutation causes an approximately 4-fold reduction in dissociation rate from diliganded receptors, and a smaller increase in opening rate and mean open time. These are sufficient to explain the approximately 6-fold slowing of decay of miniature synaptic currents seen in patients. The distinction between the two binding sites was less robust, the estimates of rate constants being dependent to some extent on assumptions, e.g. whether an extra short-lived shut state was included or whether the EC50 was constrained. The results suggest that the two binding sites differ by roughly 10-fold in the affinity of the shut receptor for ACh in the wild type, and that in the epsilonL221F mutation the lower affinity is increased so the sites become more similar.

Shiono H, Roxanis I, Zhang W, Sims GP, Meager A, Jacobson LW, Liu JL, Matthews I et al. 2003. Scenarios for autoimmunization of T and B cells in myasthenia gravis. Ann N Y Acad Sci, 998 (1), pp. 237-256. | Show Abstract | Read more

We have studied responses in thymoma patients to interferon-alpha and to the acetylcholine receptor (AChR) in early-onset myasthenia gravis (EOMG), seeking clues to autoimmunizing mechanisms. Our new evidence implicates a two-step process: (step 1) professional antigen-presenting cells and thymic epithelial cells prime AChR-specific T cells; then (step 2) thymic myoid cells subsequently provoke germinal center formation in EOMG. Our unifying hypothesis proposes that AChR epitopes expressed by neoplastic or hyperplastic thymic epithelial cells aberrantly prime helper T cells, whether generated locally or infiltrating from the circulation. These helper T cells then induce antibody responses against linear epitopes that cross-react with whole AChR and attack myoid cells in the EOMG thymus. The resulting antigen-antibody complexes and the recruitment of professional antigen-presenting cells increase the exposure of thymic cells to the infiltrates and provoke local germinal center formation and determinant spreading. Both these and the consequently enhanced heterogeneity and pathogenicity of the autoantibodies should be minimized by early thymectomy.

Shiono H, Wong YL, Matthews I, Liu JL, Zhang W, Sims G, Meager A, Beeson D, Vincent A, Willcox N. 2003. Spontaneous production of anti-IFN-alpha and anti-IL-12 autoantibodies by thymoma cells from myasthenia gravis patients suggests autoimmunization in the tumor. Int Immunol, 15 (8), pp. 903-913. | Show Abstract | Read more

Myasthenia gravis (MG) is mediated by autoantibodies to the acetylcholine receptor (AChR), expressed in muscle and rare thymic myoid cells. Most early-onset cases show thymic lymph node-type infiltrates, including pre-activated plasma cells spontaneously producing anti-AChR antibodies. Since these are not evident in the associated thymomas found in another 10% of MG patients, AChR-specific B cells must be autosensitized elsewhere. Unexpectedly, at diagnosis, >70% of MG/thymoma patients also have high-titer neutralizing autoantibodies to IFN-alpha, and >50% to IL-12; moreover, titers increase strikingly if the thymomas recur, indicating a closer tumor relationship than for anti-AChR. To investigate this, we have measured autoantibody production by cells cultured from thymomas, any available thymic remnants and blood, with or without the B cell stimulant pokeweed mitogen (PWM). To check autoantibody specificity and clonal origins, we isolated Fabs from two combinatorial libraries from producer thymus/thymoma cells. Surprisingly, thymoma cells spontaneously produced antibodies to IFN-alpha and/or IL-12 in >40% of seropositive cases, showing typical plasma cell behavior, whereas they produced anti-AChR only after PWM stimulation. We isolated 15 combinatorial Fabs to IFN-alpha (versus only one to AChR). Their strong binding in radio-immunoprecipitation and Western blots implies high affinities. The four Fabs tested neutralized anti-viral actions of IFN-alpha. The diverse V genes clearly showed ongoing antigen-driven selection. These results imply pre-activation in situ by native IFN-alpha/IL-12 expressed within a 'dangerous' tumor microenvironment. With these molecules, it should be easier to identify provoking cell type(s) that may give novel additional clues to autoimmunization against T-cell epitopes from the more complex AChR.

Ealing J, Webster R, Brownlow S, Abdelgany A, Oosterhuis H, Muntoni F, Vaux DJ, Vincent A, Beeson D. 2002. Mutations in congenital myasthenic syndromes reveal an epsilon subunit C-terminal cysteine, C470, crucial for maturation and surface expression of adult AChR. Hum Mol Genet, 11 (24), pp. 3087-3096. | Show Abstract | Read more

Many congenital myasthenic syndromes (CMS) are associated with mutations in the genes encoding the acetylcholine receptor (AChR), an oligomeric protein with the structure alpha(2)betadelta epsilon. AChR deficiency is frequently due to homozygous or heteroallelic mutations in the AChR epsilon subunit, most of which cause truncation of the polypeptide chain and loss of surface expression of AChR. Here we identified mutations epsilon 1369delG and epsilon Y458X, located in the 18 amino acid epsilon subunit C-terminus that lies extracellular to the M4 transmembrane domain. We then incorporated green fluorescent protein (GFP) into the intracellular loop between M3 and M4 of mutant or wild-type epsilon subunits and expressed the AChRs in RD or HEK 293 cells. AChR containing wild-type GFP-tagged epsilon subunits were incorporated into the surface membrane, whereas the GFP-tagged AChR mutant epsilon subunits co-localized with an endoplasmic reticulum (ER) marker and were not expressed on the cell surface. In addition, mutant AChRs did not reach the cell surface, as measured by labelling of intact cells with (125)I-alpha-bungarotoxin and precipitation with an epsilon-subunit-specific antiserum. Mutagenesis studies showed that cysteine 470, located four amino acids from the C-terminus, is essential for alpha/epsilon assembly and surface expression of adult AChR. Replacement of cysteine 470 by serine does not restore alpha/epsilon assembly or surface expression. Our results provide the first use of GFP-tagged AChR as a tool for investigation of CMS and demonstrate a previously undetermined role for a disulphide-bonded cystine in the epsilon subunit C-terminus, which plays a crucial role in expression of the adult AChR.

Matthews I, Sims G, Ledwidge S, Stott D, Beeson D, Willcox N, Vincent A. 2002. Antibodies to acetylcholine receptor in parous women with myasthenia: evidence for immunization by fetal antigen. Lab Invest, 82 (10), pp. 1407-1417. | Show Abstract | Read more

The weakness in myasthenia gravis (MG) is mediated by autoantibodies against adult muscle acetylcholine receptors (AChR) at the neuromuscular junction; most of these antibodies also bind to fetal AChR, which is present in the thymus. In rare cases, babies of mothers with MG, or even of asymptomatic mothers, develop a severe developmental condition, arthrogryposis multiplex congenita, caused by antibodies that inhibit the ion channel function of the fetal AChR while not affecting the adult AChR. Here we show that these fetal AChR inhibitory antibodies are significantly more common in females sampled after pregnancy than in those who present before pregnancy, suggesting that they may be induced by the fetus. Moreover, we were able to clone high-affinity combinatorial Fab antibodies from thymic cells of two mothers with MG who had babies with arthrogryposis multiplex congenita. These Fabs were highly specific for fetal AChR and did not bind the main immunogenic region that is common to fetal and adult AChR. The Fabs show strong biases to VH3 heavy chains and to a single Vkappa1 light chain in one mother. Nevertheless, they each show extensive intraclonal diversification from a highly mutated consensus sequence, consistent with antigen-driven selection in successive steps. Collectively, our results suggest that, in some cases of MG, initial immunization against fetal AChR is followed by diversification and expansion of B cells in the thymus; maternal autoimmunity will result if the immune response spreads to the main immunogenic region and other epitopes common to fetal and adult AChR.

Dalton P, Clover L, Beeson D, Vincent A. 2002. Antibodies to fetal antigens in mothers of babies with arthrogryposis multiplex congenita (AMC) or other neurodevelopmental disorders JOURNAL OF NEUROLOGY NEUROSURGERY AND PSYCHIATRY, 73 (2), pp. 229-229.

McConville J, Hoch W, Beeson D, Newsom-Davis J, Vincent A. 2002. Detection of musk antibodies in seronegative myasthenia gravis using human musk as antigen JOURNAL OF NEUROLOGY NEUROSURGERY AND PSYCHIATRY, 73 (2), pp. 222-222.

Webster R, Vincent A, Newsom-Davis J, Beeson D. 2002. Novel mutation in the muscle acetylcholine receptor a-subunit underlies a fast channel congenital myasthenic syndrome JOURNAL OF NEUROLOGY NEUROSURGERY AND PSYCHIATRY, 73 (2), pp. 230-230.

Watson R, Lang B, Beeson D, Bermudez I. 2002. Presence of anti-glur3 antibodies in Rasmussen's encephalitis JOURNAL OF NEUROLOGY NEUROSURGERY AND PSYCHIATRY, 73 (2), pp. 224-224.

Croxen R, Hatton C, Shelley C, Brydson M, Chauplannaz G, Oosterhuis H, Vincent A, Newsom-Davis J, Colquhoun D, Beeson D. 2002. Recessive inheritance and variable penetrance of slow-channel congenital myasthenic syndromes. Neurology, 59 (2), pp. 162-168. | Show Abstract | Read more

BACKGROUND: Slow-channel congenital myasthenic syndromes (SCCMS) typically show dominant inheritance. They are caused by missense mutations within the subunits of muscle nicotinic acetylcholine receptors (AChR) that result in prolonged ion channel activations. SCCMS mutations within the AChR subunit are located in various functional domains, whereas fully described mutations in AChR non- subunits have, thus far, been located only in the M2 channel-lining domain. The authors identified and characterized two -subunit mutations, located outside M2, that underlie SCCMS in three kinships. In two of the three kinships, the syndrome showed an atypical inheritance pattern. METHODS: These methods included clinical diagnosis, mutation detection, haplotype analysis, and functional expression studies using single-channel recordings of mutant AChR transiently transfected into HEK293 cells. RESULTS: The authors identified two SCCMS mutations in the AChR subunit, L78P and L221F. Both mutations prolonged ACh-induced ion channel activations. L78P is present in a consanguineous family and appears to be pathogenic only when present on both alleles, and L221F shows variable penetrance in one of the two families that were identified harboring this mutation. CONCLUSION: SCCMS mutations may show a recessive inheritance pattern and variable penetrance. A diagnosis of SCCMS should not be ruled out in cases of CMS with an apparent recessive inheritance pattern.

Cited:

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Croxen R, Hatton C, Shelley C, Brydson M, Chauplannaz G, Oosterhuis H, Vincent A, Newsom-Davis J, Colquhoun D, Beeson D. 2002. Recessive inheritance and variable penetrance of slow-channel congenital myasthenic syndromes (Retracted Article. See vol 72, pg 294, 2009) NEUROLOGY, 59 (2), pp. 162-168.

Croxen R, Vincent A, Newsom-Davis J, Beeson D. 2002. Myasthenia gravis in a woman with congenital AChR deficiency due to epsilon-subunit mutations. Neurology, 58 (10), pp. 1563-1565. | Show Abstract | Read more

A reduction in the number of acetylcholine receptors (AChR) on the postsynaptic membrane is characteristic of MG. This may be inherited (AChR deficiency syndrome) or acquired (MG). The authors report two sisters with AChR deficiency caused by heteroallelic mutations in the AChR epsilon-subunit gene. The younger sister developed MG at 34 years. This unusual case raises the possibility that genetic defects of the AChR might be a factor in the etiology of autoimmune MG.

Ealing J, Brownlow S, Al-Murani M, Brydson M, Vincent A, Vaux D, Beeson D. 2002. A cysteine residue close to the C-terminus of the E subunit is essential for surface acetylcholine receptor (AChR) expression: Implications for AChR deficiency syndrome JOURNAL OF NEUROLOGY NEUROSURGERY AND PSYCHIATRY, 72 (1), pp. 137-137.

Liyanage Y, Hoch W, Beeson D, Vincent A. 2002. The agrin/muscle-specific kinase pathway: new targets for autoimmune and genetic disorders at the neuromuscular junction. Muscle Nerve, 25 (1), pp. 4-16. | Show Abstract | Read more

The increasing understanding of the structural complexity of the neuromuscular junction (NMJ), and the processes that are important in its development, suggests many possible new disease targets. Here, we summarize briefly the genetic and autoimmune disorders that affect neuromuscular transmission, and the identified targets, including new evidence that antibodies to muscle-specific receptor tyrosine kinase (MuSK) are involved in the pathogenesis of acetylcholine receptor (AChR) antibody-negative myasthenia gravis. We then review the development of the NMJ, focusing on the important roles of nerve-derived agrin and MuSK in clustering of AChRs and other essential components of the NMJ.

Palace J, Vincent A, Beeson D. 2001. Myasthenia gravis: diagnostic and management dilemmas. Curr Opin Neurol, 14 (5), pp. 583-589. | Show Abstract | Read more

This review focuses on the diagnostic tests that may help to confirm myasthenia in patients without acetylcholine receptor antibodies, including the newly discovered anti-muscle-specific receptor tyrosine kinase antibody and other investigations. We discuss management dilemmas, particularly those that revolve around treatments whose efficacy is questionable or unproved, such as thymectomy. We review the important issue of how treatments are assessed, and examine future treatment trial designs.

Brownlow S, Webster R, Croxen R, Brydson M, Neville B, Lin JP, Vincent A, Newsom-Davis J, Beeson D. 2001. Acetylcholine receptor delta subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita. J Clin Invest, 108 (1), pp. 125-130. | Show Abstract | Read more

Limitation of movement during fetal development may lead to multiple joint contractures in the neonate, termed arthrogryposis multiplex congenita. Neuromuscular disorders are among the many different causes of reduced fetal movement. Many congenital myasthenic syndromes (CMSs) are due to mutations of the adult-specific epsilon subunit of the acetylcholine receptor (AChR), and, thus, functional deficits do not arise until late in gestation. However, an earlier effect on the fetus might be predicted with some defects of other AChR subunits. We studied a child who presented at birth with joint contractures and was subsequently found to have a CMS. Mutational screening revealed heteroallelic mutation within the AChR delta subunit gene, delta 756ins2 and delta E59K. Expression studies demonstrate that delta 756ins2 is a null mutation. By contrast, both fetal and adult AChR containing delta E59K have shorter than normal channel activations that predict fast decay of endplate currents. Thus, delta E59K causes dysfunction of fetal as well as the adult AChR and would explain the presence of joint contractures on the basis of reduced fetal movement. This is the first report of the association of AChR gene mutations with arthrogryposis multiplex congenita. It is probable that mutations that severely disrupt function of fetal AChR will underlie additional cases.

Croxen R, Young C, Slater C, Haslam S, Brydson M, Vincent A, Beeson D. 2001. End-plate gamma- and epsilon-subunit mRNA levels in AChR deficiency syndrome due to epsilon-subunit null mutations. Brain, 124 (Pt 7), pp. 1362-1372. | Show Abstract | Read more

Acetylcholine receptor (AChR) deficiency is the most common of the congenital myasthenic syndromes (CMS). Typically, the number of AChRs, measured by alpha-bungarotoxin binding, is reduced to 10-30% of normal levels, the miniature end-plate potentials are correspondingly reduced, and there are morphological changes at the motor end-plates. The majority of these syndromes are due to either missense or frameshift mutations within the gene encoding the adult-specific epsilon-subunit. These are often null mutations, but some mutant epsilon-subunits can be incorporated, at low levels, into functional AChRs in transfected cell lines. It is not clear, therefore, whether upregulation of the mutant epsilon-subunit mRNA could generate sufficient AChR to support neuromuscular transmission, albeit at a reduced level. Conversely, it might be that the mutant epsilon-subunit transcripts are subject to mRNA surveillance and 'nonsense-mediated' loss, leading to reduced epsilon-subunit mRNA expression. In either case, it is thought that neuromuscular transmission may be provided partly or entirely by incorporation of the foetal-specific gamma-subunit into end-plate AChR. gamma-Subunit mRNA is expressed at low levels in normal human muscle, but might be upregulated in CMS. The study of mRNA levels for AChR subunits should improve our understanding of genotype-phenotype relationships in CMS. Here we have defined homozygous epsilon-subunit mutations in four unrelated families with AChR deficiency and studied the steady-state levels of mRNA for AChR subunits at the motor end-plates by in situ hybridization. Although we demonstrated that each mutation would lead to almost complete absence of surface adult AChR expression, we detected similar robust expression of alpha- and epsilon-subunit mRNAs at end-plates of patient and control muscles, suggesting that mRNA transcripts for the epsilon-subunit are neither upregulated nor degraded preferentially. Interestingly, we were unable to detect any increase in gamma-subunit mRNA expression at CMS end-plates. Transgenic mice lacking the epsilon-subunit die 2-3 months after birth, suggesting that alpha(2)betadelta(2) pentamers cannot sustain neuromuscular transmission. Therefore, we tentatively conclude that the persistent low level expression of the gamma-subunit, which is present in normal human muscles as well as in AChR deficiency syndromes, is sufficient to enable patients with epsilon-subunit null alleles to survive.

Li F, Szobor A, Croxen R, Anselmo V, Yuan QP, Lindblad K, Schalling M, Komoly S, Beeson D, Larsson C. 2001. Dominantly inherited familial myasthenia gravis as a separate genetic entity without involvement of defined candidate gene loci. Int J Mol Med, 7 (3), pp. 289-294. | Show Abstract

Myasthenia gravis (MG) is a sporadic autoimmune disorder affecting neuromuscular transmission. Very rarely autoimmune myasthenia gravis may be inherited within a family. We present here the genetic analysis of a Hungarian family where nine members from two generations are affected by myasthenia gravis. Genetic characterisation of this unique Hungarian family using linkage analysis and mutation screening excludes the involvement of defined candidate gene loci. These findings point to familial MG as a separate genetic entity. Identification of the underlying genetic defect in this family may greatly enhance our understanding of the pathogenesis of myasthenia gravis.

Benatar M, Blaes F, Johnston I, Wilson K, Vincent A, Beeson D, Lang B. 2001. Presynaptic neuronal antigens expressed by a small cell lung carcinoma cell line. J Neuroimmunol, 113 (1), pp. 153-162. | Show Abstract | Read more

Small cell lung carcinoma (SCLC) is a tumour of neuroendocrine origin often found in association with autoimmune paraneoplastic neurological disorders. We established a SCLC cell line from a woman with Lambert-Eaton myasthenic syndrome (LEMS) who developed antibodies to both the P/Q-type voltage-gated calcium channels (VGCC) and the muscle acetycholine receptor (AChR). We used a range of techniques to establish which neuronal antigens were expressed in her tumour cell line. The results show that many proteins involved in exocytosis are present in the SCLC cells, and that depolarisation-dependent release of [(3)H]-serotonin is linked to calcium influx through P/Q-type VGCCs. In addition, some of the subunits encoding the AChR and both agrin and ARIA, molecules released from the motor nerve during development, were expressed. These results suggest that many potential antigenic targets are present in SCLC, and indicate a surprising 'motor nerve terminal'-like characteristic of this line.

Spelbrink JN, Li FY, Tiranti V, Nikali K, Yuan QP, Tariq M, Wanrooij S, Garrido N et al. 2001. Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat Genet, 28 (3), pp. 223-231. | Show Abstract | Read more

The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

Vincent A, Beeson D, Lang B. 2000. Molecular targets for autoimmune and genetic disorders of neuromuscular transmission. Eur J Biochem, 267 (23), pp. 6717-6728. | Show Abstract | Read more

The neuromuscular junction is the target of a variety of autoimmune, neurotoxic and genetic disorders, most of which result in muscle weakness. Most of the diseases, and many neurotoxins, target the ion channels that are essential for neuromuscular transmission. Myasthenia gravis is an acquired autoimmune disease caused in the majority of patients by antibodies to the acetylcholine receptor, a ligand-gated ion channel. The antibodies lead to loss of acetylcholine receptor, reduced efficiency of neuromuscular transmission and muscle weakness and fatigue. Placental transfer of these antibodies in women with myasthenia can cause fetal or neonatal weakness and occasionally severe deformities. Lambert Eaton myasthenic syndrome and acquired neuromyotonia are caused by antibodies to voltage-gated calcium or potassium channels, respectively. In the rare acquired neuromyotonia, reduced repolarization of the nerve terminal leads to spontaneous and repetitive muscle activity. In each of these disorders, the antibodies are detected by immunoprecipitation of the relevant ion channel labelled with radioactive neurotoxins. Genetic disorders of neuromuscular transmission are due mainly to mutations in the genes for the acetylcholine receptor. These conditions show recessive or dominant inheritance and result in either loss of receptors or altered kinetics of acetylcholine receptor channel properties. Study of these conditions has greatly increased our understanding of synaptic function and of disease aetiology.

Fostieri E, Beeson D, Tzartos SJ. 2000. The conformation of the main immunogenic region on the alpha-subunit of muscle acetylcholine receptor is affected by neighboring receptor subunits. FEBS Lett, 481 (2), pp. 127-130. | Show Abstract | Read more

Myasthenia gravis (MG) is caused by autoantibodies to the acetylcholine receptor (AChR). Experiments with fetal (alpha(2)betagammadelta) and adult (alpha(2)betaepsilondelta) AChR and with recombinant subunit dimers showed that some monoclonal antibodies (mAbs) against the main immunogenic region (MIR), located on the alpha-subunit of the AChR, bind better to fetal AChR and to alphagamma subunit dimer than to adult AChR and alphaepsilon dimer and equally to both alphabeta and alphadelta. However, other anti-MIR mAbs prefer adult AChR and alphaepsilon dimer, bind well to alphabeta but weakly to alphadelta. These results suggest that the MIR conformation is affected by the neighboring gamma/epsilon- and delta-subunits and may contribute to understanding the antibody specificities in MG.

Hatton C, Chen J, Shelley C, Croxen R, Beeson D, Colquhoun D. 2000. The defect in a slow channel myasthenic syndrome mutant, epsilon L221F JOURNAL OF PHYSIOLOGY-LONDON, 527 pp. 110P-111P.

Blaes F, Beeson D, Plested P, Lang B, Vincent A. 2000. IgG from "seronegative" myasthenia gravis patients binds to a muscle cell line, TE671, but not to human acetylcholine receptor. Ann Neurol, 47 (4), pp. 504-510. | Show Abstract | Read more

Antibodies to acetylcholine receptor (AChR) are found in 85% of patients with myasthenia gravis (seropositive MG [SPMG]) and are thought to be pathogenic; but in 15% of MG patients, the standard immunoprecipitation test for anti-AChR is negative (seronegative MG [SNMG]). Here, we used a novel approach, fluorescence-activated cell sorting analysis, to measure binding of SPMG and SNMG IgG antibodies to rhabdomyosarcoma cell lines that express human adult (TE671-epsilon) or fetal (TE671-gamma) AChR, and to human embryonic kidney (HEK) fibroblasts that express adult human AChR (HEK-AChR). We found that whereas most SPMG antibodies bind to all three cell lines, IgG from 8 of 15 SNMG sera/plasmas bind to the surface of both TE671 cell lines but not to HEK-AChR cells. These results indicate that SNMG antibodies bind to a muscle surface antigen that is not the AChR, which strongly supports previous studies that suggest that SNMG should be considered a distinct subtype of MG.

Brownlow S, Croxen R, Vincent A, Neville B, Lin JP, Newsom-Davis J, Beeson D. 2000. Arthrogryposis multiplex congenita associated with congenital myasthenic syndrome due to mutations in the acetylcholine receptor delta subunit EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 392-392.

Sheridan RD, Beeson D, Tattersall JEH. 2000. Non-competitive block of the human muscle adult nicotinic acetylcholine receptor ion channel by the bispyridinium compounds, SAD-128 (SAD), toxogonin (TOX) and HI-6 EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 36-36.

Croxen R, Vincent A, Newsom-Davis J, Beeson D. 2000. Splice-site mutations in the achr epsilon subunit gene associated with congenital myasthenic syndrome EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 36-36.

Beeson DMW, Newland C, Croxen R, Vincent A. 2000. The fourth transmembrane domain of the muscle nicotinic acetylcholine receptor epsilon subunit is not required for channel function EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 392-392.

Zoltowska K, Webster R, Finlayson S, Maxwell S, Cossins J, Müller J, Lochmüller H, Beeson D. 2013. Mutations in GFPT1 that underlie limb-girdle congenital myasthenic syndrome result in reduced cell-surface expression of muscle AChR. Hum Mol Genet, 22 (14), pp. 2905-2913. | Show Abstract | Read more

Mutations in GFPT1 underlie a congenital myasthenic syndrome (CMS) characterized by a limb-girdle pattern of muscle weakness. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is a key rate-limiting enzyme in the hexosamine biosynthetic pathway providing building blocks for the glycosylation of proteins and lipids. It is expressed ubiquitously and it is not readily apparent why mutations in this gene should cause a syndrome with symptoms restricted to muscle and, in particular, to the neuromuscular junction. Data from a muscle biopsy obtained from a patient with GFPT1 mutations indicated that there were reduced endplate acetylcholine receptors. We, therefore, further investigated the relationship between identified mutations in GFPT1 and expression of the muscle acetylcholine receptor. Cultured myotubes derived from two patients with GFPT1 mutations showed a significant reduction in cell-surface AChR expression (Pt1 P < 0.0001; Pt2 P = 0.0097). Inhibition of GFPT1 enzymatic activity or siRNA silencing of GFPT1 expression both resulted in reduced AChR cell-surface expression. Western blot and gene-silencing experiments indicate this is due to reduced steady-state levels of AChR α, δ, ε, but not β subunits rather than altered transcription of AChR-subunit RNA. Uridine diphospho-N-acetylglucosamine, a product of the hexosamine synthetic pathway, acts as a substrate at an early stage in the N-linked glycosylation pathway. Similarity between CMS due to GFPT1 mutations and CMS due to DPAGT1 mutations would suggest that reduced endplate AChR due to defective N-linked glycosylation is a primary disease mechanism in this disorder.

Finlayson S, Beeson D, Palace J. 2013. Congenital myasthenic syndromes: an update. Pract Neurol, 13 (2), pp. 80-91. | Read more

Cossins J, Belaya K, Hicks D, Salih MA, Finlayson S, Carboni N, Liu WW, Maxwell S et al. 2013. Congenital myasthenic syndromes due to mutations in ALG2 and ALG14. Brain, 136 (Pt 3), pp. 944-956. | Show Abstract | Read more

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 linkage analysis, whole-exome and whole-genome sequencing to determine the underlying defect in patients with an inherited limb-girdle pattern of myasthenic weakness. We identify ALG14 and ALG2 as novel genes in which mutations cause a congenital myasthenic syndrome. Through analogy with yeast, ALG14 is thought to form a multiglycosyltransferase complex with ALG13 and DPAGT1 that catalyses the first two committed steps of asparagine-linked protein glycosylation. We show that ALG14 is concentrated at the muscle motor endplates and small interfering RNA silencing of ALG14 results in reduced cell-surface expression of muscle acetylcholine receptor expressed in human embryonic kidney 293 cells. ALG2 is an alpha-1,3-mannosyltransferase that also catalyses early steps in the asparagine-linked glycosylation pathway. Mutations were identified in two kinships, with mutation ALG2p.Val68Gly found to severely reduce ALG2 expression both in patient muscle, and in cell cultures. Identification of DPAGT1, ALG14 and ALG2 mutations as a cause of congenital myasthenic syndrome underscores the importance of asparagine-linked protein glycosylation for proper functioning of the neuromuscular junction. These syndromes form part of the wider spectrum of congenital disorders of glycosylation caused by impaired asparagine-linked glycosylation. It is likely that further genes encoding components of this pathway will be associated with congenital myasthenic syndromes or impaired neuromuscular transmission as part of a more severe multisystem disorder. Our findings suggest that treatment with cholinesterase inhibitors may improve muscle function in many of the congenital disorders of glycosylation.

Belaya K, Finlayson S, Slater CR, Cossins J, Liu WW, Maxwell S, McGowan SJ, Maslau S et al. 2012. Mutations in DPAGT1 cause a limb-girdle congenital myasthenic syndrome with tubular aggregates. Am J Hum Genet, 91 (1), pp. 193-201. | Show Abstract | Read more

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.

Cossins J, Liu WW, Belaya K, Maxwell S, Oldridge M, Lester T, Robb S, Beeson D. 2012. The spectrum of mutations that underlie the neuromuscular junction synaptopathy in DOK7 congenital myasthenic syndrome. Hum Mol Genet, 21 (17), pp. 3765-3775. | Show Abstract | Read more

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.

Webster R, Maxwell S, Spearman H, Tai K, Beckstein O, Sansom M, Beeson D. 2012. A novel congenital myasthenic syndrome due to decreased acetylcholine receptor ion-channel conductance Brain, 135 (4), pp. 1070-1080. | Show Abstract | Read more

Muscle acetylcholine receptor ion channels mediate neurotransmission by depolarizing the postsynaptic membrane at the neuromuscular junction. Inherited disorders of neuromuscular transmission, termed congenital myasthenic syndromes, are commonly caused by mutations in genes encoding the five subunits of the acetylcholine receptor that severely reduce endplate acetylcholine receptor numbers and/or cause kinetic abnormalities of acetylcholine receptor function. We tracked the cause of the myasthenic disorder in a female with onset of first symptoms at birth, who displayed mildly progressive bulbar, respiratory and generalized limb weakness with ptosis and ophthalmoplegia. Direct DNA sequencing revealed heteroallelic mutations in exon 8 of the acetylcholine receptor ε-subunit gene. Two alleles were identified: one with the missense substitution p.εP282R, and the second with a deletion, c.798-800delCTT, which result in the loss of a single amino acid, residue F266, within the M2 transmembrane domain. When these acetylcholine receptor mutations were expressed in HEK 293 cells, the p.εP282R mutation caused severely reduced expression on the cell surface, whereas p.εΔF266 gave robust surface expression. Single-channel analysis for p.εΔF266 acetylcholine receptor channels showed the longest burst duration population was not different from wild-type acetylcholine receptor (4.39±0.6ms versus 4.68±0.7ms, n=5 each) but that the amplitude of channel openings was reduced. Channel amplitudes at different holding potentials showed that single-channel conductance was significantly reduced in p.εΔF266 acetylcholine receptor channels (42.7±1.4 pS, n=8, compared with 70.9±1.6 pS for wild-type, n=6). Although a phenylalanine residue at this position within M2 is conserved throughout ligand-gated excitatory cys-loop channel subunits, deletion of equivalent residues in the other subunits of muscle acetylcholine receptor did not have equivalent effects. Modelling the impact of p.εΔF266 revealed only a minor alteration to channel structure. In this study we uncover the novel mechanism of reduced acetylcholine receptor channel conductance as an underlying cause of congenital myasthenic syndrome, with the 'low conductance' phenotype that results from the p.εΔF266 deletion mutation revealed by the coinheritance of the low-expressor mutation p.εP282R. © 2012 The Author.

Lashley D, Palace J, Jayawant S, Robb S, Beeson D. 2010. Ephedrine treatment in congenital myasthenic syndrome due to mutations in DOK7. Neurology, 74 (19), pp. 1517-1523. | Show Abstract | Read more

BACKGROUND: Mutations in the postsynaptic adaptor protein Dok-7 underlie congenital myasthenic syndrome (CMS) with a characteristic limb girdle pattern of muscle weakness. Patients usually do not respond to or worsen with the standard CMS treatments: cholinesterase inhibitors and 3,4-diaminopyridine. However, anecdotal reports suggest they may improve with ephedrine. METHODS: This was an open prospective follow-up study to determine muscle strength in response to ephedrine in Dok-7 CMS. Patients were first evaluated as inpatients for suitability for a trial of treatment with ephedrine. The response was assessed at 2 and 6 to 8 months follow-up clinic visits using a quantitative myasthenia gravis (severity) score (QMG) and mobility measures. RESULTS: Ten out of 12 of the cohort with DOK7 mutations tolerated ephedrine. We noted a progressive response to treatment over the 6 to 8 months assessment period with a significant improvement at the final QMG score (p = 0.009). Mobility scores also improved (p = 0.0006). Improvements in the subcomponents of the QMG score that measured proximal muscle function (those muscle groups most severely affected) were most marked, and in some cases were dramatic. All patients reported enhanced activities of daily living at 6-8 months. CONCLUSION: Ephedrine appears to be an effective treatment for Dok-7 CMS. It is well-tolerated by most patients and improvement in strength can be profound. Determining the long-term response and the most effective dosing regimen will require further research. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that ephedrine given at doses between 15 and 90 mg/day improves muscle strength in patients with documented mutations in DOK7.

Giraud M, Taubert R, Vandiedonck C, Ke X, Lévi-Strauss M, Pagani F, Baralle FE, Eymard B et al. 2007. An IRF8-binding promoter variant and AIRE control CHRNA1 promiscuous expression in thymus. Nature, 448 (7156), pp. 934-937. | Show Abstract | Read more

Promiscuous expression of tissue-restricted auto-antigens in the thymus imposes T-cell tolerance and provides protection from autoimmune diseases. Promiscuous expression of a set of self-antigens occurs in medullary thymic epithelial cells and is partly controlled by the autoimmune regulator (AIRE), a nuclear protein for which loss-of-function mutations cause the type 1 autoimmune polyendocrine syndrome. However, additional factors must be involved in the regulation of this promiscuous expression. Here we describe a mechanism controlling thymic transcription of a prototypic tissue-restricted human auto-antigen gene, CHRNA1. This gene encodes the alpha-subunit of the muscle acetylcholine receptor, which is the main target of pathogenic auto-antibodies in autoimmune myasthenia gravis. On re-sequencing the CHRNA1 gene, we identified a functional bi-allelic variant in the promoter that is associated with early onset of disease in two independent human populations (France and United Kingdom). We show that this variant prevents binding of interferon regulatory factor 8 (IRF8) and abrogates CHRNA1 promoter activity in thymic epithelial cells in vitro. Notably, both the CHRNA1 promoter variant and AIRE modulate CHRNA1 messenger RNA levels in human medullary thymic epithelial cells ex vivo and also in a transactivation assay. These findings reveal a critical function of AIRE and the interferon signalling pathway in regulating quantitative expression of this auto-antigen in the thymus, suggesting that together they set the threshold for self-tolerance versus autoimmunity.

Cossins J, Webster R, Maxwell S, Burke G, Vincent A, Beeson D. 2004. A mouse model of AChR deficiency syndrome with a phenotype reflecting the human condition. Hum Mol Genet, 13 (23), pp. 2947-2957. | Show Abstract | Read more

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.

Abdelgany A, Wood M, Beeson D. 2003. Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference. Hum Mol Genet, 12 (20), pp. 2637-2644. | Show Abstract | Read more

Slow channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular synapse caused by dominantly inherited missense mutations in genes that encode the muscle acetylcholine receptor (AChR) subunits. Here we investigate the potential of post-transcriptional gene silencing using RNA interference (RNAi) for the selective down-regulation of pathogenic mutant AChR. By transfection of both siRNA and shRNA into mammalian cells expressing wild-type or mutant AChR subunits, we show, using 125I-alpha-bungarotoxin binding and immunofluorescence to measure cell surface AChR expression, efficient discrimination between the silencing of alphaS226F AChR mutant RNA transcripts and the wild-type. In this model we find that selectivity between mutant and wild-type transcripts is optimized with the nucleotide mismatch at position 9 in the shRNA complementary sequence. We also find that allele-specific silencing using shRNA has comparable efficiency to that using siRNA, underlining the general potential of stable expression of shRNA molecules as a long term therapeutic approach for allele-specific silencing of mutant transcripts in dominant genetic disorders.

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