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Mutations in DPAGT1 cause a limb-girdle congenital myasthenic syndrome with tubular aggregates
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.
Oxaliplatin induces hyperexcitability at motor and autonomic neuromuscular junctions through effects on voltage-gated sodium channels.
Oxaliplatin, an effective cytotoxic treatment in combination with 5-fluorouracil for colorectal cancer, is associated with sensory, motor and autonomic neurotoxicity. Motor symptoms include hyperexcitability while autonomic effects include urinary retention, but the cause of these side-effects is unknown. We examined the effects on motor nerve function in the mouse hemidiaphragm and on the autonomic system in the vas deferens. In the mouse diaphragm, oxaliplatin (0.5 mM) induced multiple endplate potentials (EPPs) following a single stimulus, and was associated with an increase in spontaneous miniature EPP frequency. In the vas deferens, spontaneous excitatory junction potential frequency was increased after 30 min exposure to oxaliplatin; no changes in resting Ca(2+) concentration in nerve terminal varicosities were observed, and recovery after stimuli trains was unaffected. In both tissues, an oxaliplatin-induced increase in spontaneous activity was prevented by the voltage-gated Na(+) channel blocker tetrodotoxin (TTX). Carbamazepine (0.3 mM) also prevented multiple EPPs and the increase in spontaneous activity in both tissues. In diaphragm, beta-pompilidotoxin (100 microM), which slows Na(+) channel inactivation, induced multiple EPPs similar to oxaliplatin's effect. By contrast, blockers of K(+) channels (4-aminopyridine and apamin) did not replicate oxaliplatin-induced hyperexcitability in the diaphragm. The prevention of hyperexcitability by TTX blockade implies that oxaliplatin acts on nerve conduction rather than by effecting repolarisation. The similarity between beta-pompilidotoxin and oxaliplatin suggests that alteration of voltage-gated Na(+) channel kinetics is likely to underlie the acute neurotoxic actions of oxaliplatin.
Presence and pathogenic relevance of antibodies to clustered acetylcholine receptor in ocular and generalized myasthenia gravis.
BACKGROUND: Clustered acetylcholine receptor antibodies (clustered AChR-Abs) have been detected in a proportion of patients with previously "seronegative" (SN) generalized myasthenia gravis (GMG), but their presence in patients with ocular MG (OMG) and their pathogenicity in vivo are unknown. OBJECTIVE: To test the presence of clustered AChR-Abs and their pathophysiologic properties in patients with SNMG. DESIGN: Screening and diagnostic tests. SETTING: Regional specialist myasthenia center and clinical laboratory. PATIENTS: Serum samples from 16 patients with SN and OMG were tested for binding to clustered AChRs. Results from 28 further SN patients (14 OMG) were correlated with their single fiber electromyography values. MAIN OUTCOME MEASURES: Presence, complement-fixation capacity, correlation with neurophysiologic changes, and in vivo pathogenicity of clustered AChR-Abs. RESULTS: Up to 50% of patients with previous SN-OMG had complement-fixing IgG1 clustered AChR-Abs. IgG binding (n = 28) and complement deposition (n = 21) each correlated with the mean consecutive difference (jitter) on single-fiber electromyography. Injection of purified IgG from 2 patients with clustered AChR-Abs into wild-type or complement regulator-deficient mice reduced miniature end plate potential amplitudes to an extent similar to that found with AChR-Abs, and complement was deposited at the end plates. A trend was noted toward an increase in the number of packets of acetylcholine released (quantal content). CONCLUSIONS: A proportion of patients with SN-GMG or OMG have clustered AChR-Abs that correlate with their electrophysiologic features. Clustered AChR-Abs can passively transfer disease to mice, demonstrating their pathogenicity, and the mechanisms seem similar to those of patients with typical AChR-Abs.
Passive and active immunization models of MuSK-Ab positive myasthenia: electrophysiological evidence for pre and postsynaptic defects.
Antibodies directed against the post-synaptic neuromuscular junction protein, muscle specific kinase (MuSK) are found in a small proportion of generalized myasthenia gravis (MuSK-MG) patients. MuSK is a receptor tyrosine kinase which is essential for clustering of the acetylcholine receptors (AChRs) at the neuromuscular junction, but the mechanisms by which MuSK antibodies (MuSK-Abs) affect neuromuscular transmission are not clear. Experimental models of MuSK-MG have been described but there have been no detailed electrophysiological studies and no comparisons between the MuSK-MG and the typical form with AChR-Abs (AChR-MG). Here we studied the electrophysiology of neuromuscular transmission after immunization against MuSK compared with immunization against AChR, and also after passive transfer of IgG from MuSK-MG or AChR-MG patients. Overt clinical weakness was observed in 6/10 MuSK-immunized and 3/9 AChR-immunized mice but not in those injected with patients' IgG. Miniature endplate potentials (MEPPS) were reduced in all weak mice consistent with the reduction in postsynaptic AChRs that was found. However, whereas there was an increase in the quantal release of acetylcholine (ACh) in the weak AChR-immunized mice, no such increase was found in the weak MuSK-immunized mice. Similar trends were found after the passive transfer of purified IgG antibodies from MuSK-MG or AChR-MG patients. Preliminary results showed that MuSK expression was considerably higher at the neuromuscular junctions of the masseter (facial) than in the gastrocnemius (leg) with no reduction in MuSK immunostaining at the neuromuscular junctions. Overall, these results suggest that MuSK antibodies act in at least two ways. Firstly by indirectly affecting MuSK's ability to maintain the high density of AChRs and secondly by interfering with a compensatory presynaptic mechanism that regulates quantal release and helps to preserve neuromuscular function. These results raise questions about how MuSK is involved in retrograde signaling, and the combination of post-synaptic defects with lack of presynaptic compensation may begin to explain the more severe disease in MuSK-MG patients.
Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient.
We report here the sequence and functional characterization of a recombinantly expressed autoantibody (mAb 131) previously isolated from a myasthenia gravis patient by immortalization of thymic B cells using Epstein-Barr virus and TLR9 activation. The antibody is characterized by a high degree of somatic mutations as well as a 6 amino acid insertion within the VHCDR2. The recombinant mAb 131 is specific for the γ-subunit of the fetal AChR to which it bound with sub-nanomolar apparent affinity, and detected the presence of fetal AChR on a number of rhabdomyosarcoma cell lines. Mab 131 blocked one of the two α-bungarotoxin binding sites on the fetal AChR, and partially blocked the binding of an antibody (mAb 637) to the α-subunit of the AChR, suggesting that both antibodies bind at or near one ACh binding site at the α/γ subunit interface. However, mAb 131 did not reduce fetal AChR ion channel currents in electrophysiological experiments. These results indicate that mAb 131, although generated from an MG patient, is unlikely to be pathogenic and may make it a potentially useful reagent for studies of myasthenia gravis, rhabdomyosarcoma and arthrogryposis multiplex congenita which can be caused by fetal-specific AChR-blocking autoantibodies.
Animal Models of the Neuromuscular Junction, Vitally Informative for Understanding Function and the Molecular Mechanisms of Congenital Myasthenic Syndromes.
The neuromuscular junction is the point of contact between motor nerve and skeletal muscle, its vital role in muscle function is reliant on the precise location and function of many proteins. Congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders of neuromuscular transmission with 30 or more implicated proteins. The use of animal models has been instrumental in determining the specific role of many CMS-related proteins. The mouse neuromuscular junction (NMJ) has been extensively studied in animal models of CMS due to its amenability for detailed electrophysiological and histological investigations and relative similarity to human NMJ. As well as their use to determine the precise molecular mechanisms of CMS variants, where an animal model accurately reflects the human phenotype they become useful tools for study of therapeutic interventions. Many of the animal models that have been important in deconvolving the complexities of neuromuscular transmission and revealing the molecular mechanisms of disease are highlighted.
Thrombocytopenia in the newborn
© 2019 Elsevier Inc. All rights reserved. Neonatal thrombocytopenia remains a common clinical problem although most episodes are mild and resolve spontaneously. Most early-onset thrombocytopenias are due to impaired fetal megakaryocytopoiesis associated with placental insufficiency/fetal hypoxia. Severe early-onset thrombocytopenia (presenting < 72 hours of life) is usually due to congenital or perinatal infections, asphyxia or fetal/neonatal alloimmune thrombocytopenia (FNAIT) while late-onset thrombocytopenia (presenting after 72 hours) is usually due to sepsis and/or necrotizing enterocolitis (NEC). Major hemorrhage is uncommon in stable neonates with severe thrombocytopenia while clinically unstable neonates often have a poor outcome. Currently, the only therapy is platelet transfusion. However, there is wide variation in platelet transfusion thresholds worldwide and frequent deviation from transfusion guidelines. Controlled trials of platelet transfusion for severe neonatal thrombocytopenia, now underway, aim to identify the best regimens to treat and prevent hemorrhage and target therapy to neonates at highest risk. In future, improved understanding of fetal/neonatal megakaryocytopoiesis and TPO mimetics may benefit neonates with prolonged thrombocytopenia.
Calcium channel subtypes contributing to acetylcholine release from normal, 4-aminopyridine-treated and myasthenic syndrome auto-antibodies-affected neuromuscular junctions.
1 Acetylcholine release at the neuromuscular junction relies on rapid, local and transient calcium increase at presynaptic active zones, triggered by the ion influx through voltage-dependent calcium channels (VDCCs) clustered on the presynaptic membrane. Pharmacological investigation of the role of different VDCC subtypes (L-, N-, P/Q- and R-type) in spontaneous and evoked acetylcholine (ACh) release was carried out in adult mouse neuromuscular junctions (NMJs) under normal and pathological conditions. 2 omega-Agatoxin IVA (500 nM), a specific P/Q-type VDCC blocker, abolished end plate potentials (EPPs) in normal NMJs. However, when neurotransmitter release was potentiated by the presence of the K(+) channel blocker 4-aminopyridine (4-AP), an omega-agatoxin IVA- and omega-conotoxin MVIIC-resistant component was detected. This resistant component was only partially sensitive to 1 micro M omega-conotoxin GVIA (N-type VDCC blocker), but insensitive to any other known VDCC blockers. Spontaneous release was dependent only on P/Q-type VDCC in normal NMJs. However, in the presence of 4-AP, it relied on L-type VDCCs too. 3 ACh release from normal NMJs was compared with that of NMJs of mice passively injected with IgGs obtained from patients with Lambert-Eaton myasthenic syndrome (LEMS), a disorder characterized by a compromised neurotransmitter release. Differently from normal NMJs, in LEMS IgGs-treated NMJs an omega-agatoxin IVA-resistant EPP component was detected, which was only partially blocked by calciseptine (1 micro M), a specific L-type VDCC blocker. 4 Altogether, these data demonstrate that multiple VDCC subtypes are present at the mouse NMJ and that a resistant component can be identified under 'pharmacological' and/or 'pathological' conditions.
Brachydactyly type B: linkage to chromosome 9q22 and evidence for genetic heterogeneity.
Brachydactyly type B (BDB), an autosomal dominant disorder, is the most severe of the brachydactylies and is characterized by hypoplasia or absence of the terminal portions of the index to little fingers, usually with absence of the nails. The thumbs may be of normal length but are often flattened and occasionally are bifid. The feet are similarly but less severely affected. We have performed a genomewide linkage analysis of three families with BDB, two English and one Portugese. The two English families show linkage to the same region on chromosome 9 (combined multipoint maximum LOD score 8.69 with marker D9S257). The 16-cM disease interval is defined by recombinations with markers D9S1680 and D9S1786. These two families share an identical disease haplotype over 18 markers, inclusive of D9S278-D9S280. This provides strong evidence that the English families have the same ancestral mutation, which reduces the disease interval to <12.7 cM between markers D9S257 and D9S1851 in chromosome band 9q22. In the Portuguese family, we excluded linkage to this region, a result indicating that BDB is genetically heterogeneous. Reflecting this, there were atypical clinical features in this family, with shortening of the thumbs and absence or hypoplasia of the nails of the thumb and hallux. These results enable a refined classification of BDB and identify a novel locus for digit morphogenesis in 9q22.
Haemoglobin F modulation in childhood sickle cell disease.
While supportive care remains the best option for most well children with sickle cell disease (SCD), increasing awareness of early signs of chronic organ damage in childhood has focused attention on therapy which modulates the natural history of the disease. Since cure by stem cell transplantation is only feasible for a minority and gene therapy remains developmental, pharmacological modification by Haemoglobin F (HbF)-inducers, is the most widely used approach in SCD. Currently, the only HbF modulator with a clear place in the management of childhood SCD is hydroxycarbamide for which the main indications are frequent painful crises and recurrent acute chest syndrome. In the majority of SCD children treated with hydroxycarbamide there is clear evidence of clinical benefit and the drug is well tolerated. The main disadvantages are the need for frequent monitoring and uncertainity about long-term risks of carcinogenicity and impaired fertility, although these risks appear to be very low. The role of hydroxycarbamide in sickle-associated central nervous system disease remains to be established. Decitabine and butyrate derivatives show some promise although robust data in children with SCD are lacking. A number of other drugs are currently under investigation for their effects on HbF production including thalidomide and lenolidamide.