Cause Of Congenital Myasthenic Syndromes Research Articles

Calcitriol ameliorates motor deficits and prolongs survival of Chrne-deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2

Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including congenital myasthenic syndromes (CMS). Germline mutations in CHRNE encoding the acetylcholine receptor (AChR) ε subunit are the most common cause of CMS. An active form of vitamin D, calcitriol, binds to vitamin D receptor (VDR) and regulates gene expressions. We found that calcitriol enhanced MuSK phosphorylation, AChR clustering, and myotube twitching in co-cultured C2C12 myotubes and NSC34 motor neurons. RNA-seq analysis of co-cultured cells showed that calcitriol increased the expressions of Rspo2, Rapsn, and Dusp6. ChIP-seq of VDR revealed that VDR binds to a region approximately 15 ​kbp upstream to Rspo2. Biallelic deletion of the VDR-binding site of Rspo2 by CRISPR/Cas9 in C2C12 myoblasts/myotubes nullified the calcitriol-mediated induction of Rspo2 expression and MuSK phosphorylation. We generated Chrne knockout (Chrne KO) mouse by CRISPR/Cas9. Intraperitoneal administration of calcitriol markedly increased the number of AChR clusters, as well as the area, the intensity, and the number of synaptophysin-positive synaptic vesicles, in Chrne KO mice. In addition, calcitriol ameliorated motor deficits and prolonged survival of Chrne KO mice. In the skeletal muscle, calcitriol increased the gene expressions of Rspo2, Rapsn, and Dusp6. We propose that calcitriol is a potential therapeutic agent for CMS and other diseases with defective neuromuscular signal transmission.

Novel DPAGT1 Gene Mutation in Two Twins with Congenital Myasthenic Syndrome and a Review of the Literature.

Congenital myasthenic syndromes (CMS) are rare diseases caused by mutation in genes coding for proteins involved in neuromuscular junction structure and function. DPAGT1 gene mutations are a rare cause of CMS whose clinical evolution and pathophysiological mechanisms have not been clarified completely. We present the case of two twins displaying an infancy-onset predominant limb-girdle phenotype and carrying a novel DPAGT1 mutation associated with unusual histological and clinical findings. CMS can mimic paediatric and adult limb-girdle phenotype, hence neurophysiology plays a fundamental role in the differential diagnosis.

Generation and characterization of an induced pluripotent stem cell line SDQLCHi018-A from a congenital myasthenic syndrome patient carrying compound heterozygote mutations in RAPSN gene

Mutations in RAPSN are an important cause of congenital myasthenic syndrome (CMS). In this study, we generated an induced pluripotent stem cell line (iPSC) derived from a 14-day-old male CMS patient carrying compound heterozygote mutations (c.532-2A>G and c.264C>A/p.Asn88Lys) in RAPSN gene. The established iPSC line harboring the original mutations, possessing a normal karyotype, is able to differentiate into all three germ layers in vitro and expresses pluripotency markers.

Congenital myasthenic syndromes

Congenital myasthenic syndromes (CMS) are a group of genetically heterogeneous disorders of the neuromuscular transmission, clinically characterized by muscle weakness and fatigability. Mutations in several proteins affecting structure, development and function of the neuromuscular junction result in CMS by compromising the safety margin of neuromuscular transmission through a variety of mechanisms. Defects in postsynaptic proteins are the most frequent cause of CMS, but presynaptic proteins have been increasingly recognized as causative of these disorders. In addition, it has become evident that myasthenia can be a part of a more complex phenotype featured also by central nervous system involvement. Disease-onset ranges from prenatal life to adulthood while the clinical phenotype spans from severe generalized weakness to mild predominant cranial muscle weakness. Low frequency repetitive nerve stimulation often demonstrates a decremental response. Single-fiber EMG or conditioning with exercise or high frequency nerve stimulation may be needed to unmask the neuromuscular synaptic defect in some cases. Only some presynaptic CMS show facilitation (hallmark of the autoimmune presynaptic defect of neuromuscular transmission), depending on the characteristics of the underlying quantal release defect. The molecular diagnosis and the understanding of the disease mechanism of a specific CMS are crucial to individualize patient care. Cholinergic drugs (such as pyridostigmine or 3,4-diamynopyridine) improve the myasthenic symptoms in certain CMS subtypes patients but worsen the symptoms in others, who respond positively to beta-adrenergic agonists (such as salbutamol) or long-lived acetylcholine receptor channel blockers (such as fluoxetine). The combination of a cholinergic drug and a beta-adrenergic agonist can further optimize the pharmacological treatment of certain CMS subtypes.

Molecular characterization of congenital myasthenic syndromes in Spain

Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetic disorders, all of which impair neuromuscular transmission. Epidemiological data and frequencies of gene mutations are scarce in the literature. Here we describe the molecular genetic and clinical findings of sixty-four genetically confirmed CMS patients from Spain. Thirty-six mutations in the CHRNE, RAPSN, COLQ, GFPT1, DOK7, CHRNG, GMPPB, CHAT, CHRNA1, and CHRNB1 genes were identified in our patients, with five of them not reported so far. These data provide an overview on the relative frequencies of the different CMS subtypes in a large Spanish population. CHRNE mutations are the most common cause of CMS in Spain, accounting for 27% of the total. The second most common are RAPSN mutations. We found a higher rate of GFPT1 mutations in comparison with other populations. Remarkably, several founder mutations made a large contribution to CMS in Spain: RAPSN c.264C > A (p.Asn88Lys), CHRNE c.130insG (Glu44Glyfs*3), CHRNE c.1353insG (p.Asn542Gluf*4), DOK7 c.1124_1127dup (p.Ala378Serfs*30), and particularly frequent in Spain in comparison with other populations, COLQ c.1289A > C (p.Tyr430Ser). Furthermore, we describe phenotypes and distinguishing clinical signs associated with the various CMS genes which might help to identify specific CMS subtypes to guide diagnosis and management.

Congenital Myasthenic Syndrome Caused by Isolated PREPL Deficiency

Background: Mutations in PREPL are a rare cause of congenital myasthenic syndrome. PREPL encodes the prolyl endopeptidase-like enzyme, which plays an important role for vesicular transport of the acetylcholine receptor. So far, only a single case of isolated PREPL deficiency has been reported in the literature. The child presented as a floppy baby and showed developmental delay.

PP09.8 – 2377: Novel DOK7 mutation in a Greek patient with congenital myasthenic syndrome

Introduction Congenital myasthenic syndromes (CMS) are rare genetic syndromes resulting from presynaptic, synaptic or postsynaptic defects of the neuromuscular junction. In 2006 mutations in DOK7, a gene encoding a muscle-intrinsic activator of MUSK, were proven a postsynaptic cause of CMS. Until today 65 DOK7 mutations have been described. Case report A 16 year old girl, daughter to healthy, non-consanguineous parents has been followed up at our clinic since birth. History includes decreased fetal movements, hypotonia at birth and slightly delayed motor milestones with sitting at 8 months and walking at 15 months. Neurological examination discloses facial and proximal muscle weakness, bulbar and respiratory weakness, ptosis, joint laxity, massive kyphoscoliosis, feeding difficulty and failure to thrive. The patient is currently ambulant, able to climb stairs and walk approximately 50 meters, before getting tired. Under nocturnal noninvasive ventilation (BiPAP) she maintains oxygen saturation levels >98%. Creatin kinase is 200 U/l (normal: 150 U/l). Electrophysiologic studies revealed normal NCV's and a mildly myopathic EMG. Repetitive nerve stimulation demonstrated mild decrement at infancy, but now is normal. Muscle biopsy showed predominance of type 1 fibers and decreased oxidative activity. Since the clinical features were consistent with CMS, molecular analysis of CHAT, RAPSN, CHRNE and DOK7 was undertaken, which in turn revealed a compound heterozygous mutation in the DOK7 gene. Conclusion To the best of our knowledge this is the first Greek patient with confirmed DOK7 myasthenic syndrome. One of the mutations, the c.472C>T (p.Arg158Trp) has not been previously described. Accurate diagnosis of DOK7 myasthenic syndromes bears important clinical implications, since ephedrine or salbutamol rather than acetylcholinesterase inhibitors are the treatment of choice.

Salbutamol-responsive limb-girdle congenital myasthenic syndrome due to a novel missense mutation and heteroallelic deletion in MUSK

Congenital myasthenic syndromes (CMS) are clinically and genetically heterogeneous disorders characterized by a neuromuscular transmission defect. In recent years, causative mutations have been identified in atleast 15 genes encoding proteins of the neuromuscular junction. Mutations in MUSK are known as a very rare genetic cause of CMS and have been described in only three families, world-wide. Consequently, the knowledge about efficient drug therapy is very limited. We identified a novel missense mutation (p.Asp38Glu) heteroallelic to a genomic deletion affecting exons 2–3 of MUSK as cause of a limb-girdle CMS in two brothers of Turkish origin. Clinical symptoms included fatigable limb weakness from early childhood on. Upon diagnosis of a MUSK-related CMS at the age of 16 and 13years, respectively, treatment with salbutamol was initiated leading to an impressive improvement of clinical symptoms, while treatment with esterase inhibitors did not show any benefit. Our findings highlight the importance of a molecular diagnosis in CMS and demonstrate considerable similarities between patients with MUSK and DOK7-related CMS in terms of clinical phenotype and treatment options.

Clinical features in a large Iranian family with a limb-girdle congenital myasthenic syndrome due to a mutation in DPAGT1

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.

Congenital myasthenic syndromes due to mutations in ALG2 and ALG14.

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.

A novel congenital myasthenic syndrome due to decreased acetylcholine receptor ion-channel conductance

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.

186th ENMC International Workshop: Congenital myasthenic syndromes 24–26 June 2011, Naarden, The Netherlands

The ENMC hosted a group of 22 participants including parents, clinicians and scientists involved in the care or research of congenital myasthenic syndromes (CMS) patients. These represented different groups and centres from the United Kingdom, Germany, Spain, France, Bulgaria, Switzerland, and the United States. CMS are rare inherited disorders in which the safety margin of the neuromuscular transmission is compromised [1–4]. Currently, full genetic testing for CMS is only available in 4 major centres (Rochester, Paris, Oxford, Munich) who have collectively diagnosed more than 1000 patients (Table 1). The objectives of the meeting were to provide an update on CMS both in the clinical and non-clinical domains and to promote more collaborative work. Since the last ENMC workshop on CMS in 2004, 4 novel genes have been discovered: DOK7 [5,6], AGRN [7], LAMB2 [8] and GFPT1 [9,10]. Mutations in the DOK7 gene have emerged as a very common cause of CMS with a limb-girdle phenotype, usually refractory to esterase inhibitors. By contrast, mutations in GFPT1 have recently been found in pyridostigmine-responsive limb-girdle myasthenia with tubular aggregates. During the Naarden workshop, a number of key topics were explored, including recent advances in understanding

Beneficial effect of albuterol in congenital myasthenic syndrome with epsilon‐subunit mutations

Mutations in the epsilon subunit of the acetylcholine receptor (AChR) are a common cause of congenital myasthenic syndrome (CMS). Patients are usually treated with acetylcholinesterase inhibitors and 3,4-diaminopyridine with modest clinical benefit. We report 2 patients with CMS due to mutations in the AChR epsilon subunit. The first patient carries two heterozygous frameshift mutations, ε127ins5 and ε1293insG. The second patient is homozygous for the εC142Y mutation that curtails AChR expression to 22% of wild-type in HEK cells. Treatment with pyridostigmine and 3,4-diaminopyridine had a limited beneficial effect in the first patient, and the second patient became wheelchair-bound during therapy. The additional use of albuterol produced dramatic improvement in strength and in activities of daily living in both patients. The efficacy and safety of albuterol in patients who harbor identified low-expressor or null mutations in the epsilon or other subunits of AChR merits a well-designed clinical trial.

Hexosamine Biosynthetic Pathway Mutations Cause Neuromuscular Transmission Defect

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

Congenital Myasthenic Syndromes and the Formation of the Neuromuscular Junction

The congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders affecting neuromuscular transmission. Underlying mutations have been identified in at least 11 different genes. The majority of CMS patients have disorders due to mutations in postsynaptic proteins. Initial studies focused on dysfunction of the acetylcholine receptor (AChR) itself as the major cause of CMS. However, it is becoming apparent that mutations of proteins involved in clustering the AChR and maintaining neuromuscular junction structure form important subgroups. Analysis of the mutations in the AChR-clustering protein, rapsyn, show diverse causes for defective AChR localization and suggest that the common mutation rapsyn-N88K results in AChR clusters that are less stable than those generated by wild-type rapsyn. More recently, mutations in the newly identified endplate protein Dok-7 have been shown to affect AChR clustering and the generation and maintenance of specialized structures at the endplate. Dok-7 binds MuSK and many of the mutations of DOK7 impair the MuSK signaling pathway. Components of this pathway will provide attractive gene candidates for additional forms of CMS. The phenotypic characteristics of the different CMS in which muscle groups may be differentially affected not only provide clues for targeted genetic screening, but also pose further intriguing questions about underlying molecular mechanisms.

Phenotypical spectrum of DOK7 mutations in congenital myasthenic syndromes

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.

Dok-7 Mutations Underlie a Neuromuscular Junction Synaptopathy

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

Mechanistic diversity underlying fast channel congenital myasthenic syndromes.

A host of missense mutations in muscle nicotinic receptor subunits have been identified as the cause of congenital myasthenic syndromes (CMS). Two classes of CMS phenotypes have been identified: slow channel myasthenic syndromes (SCCMSs) and fast channel myasthenic syndromes (FCCMSs). Although both have similar phenotypic consequences, they are physiologic opposites. Expression of the FCCMS phenotype requires the missense mutation to be accompanied by a second mutation, either a null or a missense mutation, in the second allele encoding the same receptor subunit. This seemingly rare scenario has arisen with surprisingly high incidence over the past few years, and analyses of the syndromes have revealed a diverse array of mechanisms underlying the pathology. This review focuses on new mechanisms underlying the FCCMS.

Rapsyn N88K is a frequent cause of congenital myasthenic syndromes in European patients.

Mutations in various genes of the neuromuscular junction may cause congenital myasthenic syndromes (CMS). Most mutations identified to date affect the epsilon-subunit gene of the acetylcholine receptor (AChR), leading to end-plate AChR deficiency. Recently, three different mutations in the RAPSN gene have been identified in four CMS patients with AChR deficiency. To perform mutation analysis of the RAPSN gene in patients with sporadic or autosomal recessive CMS. One hundred twenty CMS patients from 110 unrelated families were analyzed for the RAPSN mutation N88K by restriction fragment length polymorphism and sequence analysis. In 12 CMS patients from 10 independent families, RAPSN N88K was identified either homozygous or heteroallelic to another missense mutation. Symptoms usually started perinatally or in the first years of life. However, one patient did not show any myasthenic symptoms before the third decade. Clinical symptoms typically included bilateral ptosis, weakness of facial, bulbar, and limb muscles, and a favorable response to anticholinesterase treatment. Crisis-like exacerbations with respiratory insufficiency provoked by stress, fever, or infections in early childhood were frequent. All RAPSN N88K families originate from Central or Western European countries. Genotype analysis indicated that they derive from a common ancestor (founder). The RAPSN mutation N88K is a frequent cause of rapsyn-related CMS in European patients. In general, patients (RAPSN N88K) were characterized by mild to moderate myasthenic symptoms with favorable response to anticholinesterase treatment. However, severity and onset of symptoms may vary to a great extent.

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

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