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Background Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous neuromuscular disorder characterized by muscle weakness and caused by mutations in more than 35 different genes. This condition should not be overlooked as a subset of patients with CMS are treatable. However, the diagnosis of CMS is often difficult due to the broad variability in disease severity and course. Case report A five-year-old boy without remarkable family history was born with marked general muscle hypotonia and weakness, respiratory insufficiency, anomalies, and multiple joint contractures. Congenital myopathy was suspected based upon type 1 fiber predominance on muscle biopsy. However, he was diagnosed with CMS at age 4 years when his ptosis and ophthalmoplegia were found to be improved by edrophonium chloride and repetitive nerve stimulation showed attenuation of compound muscle action potentials. An exome sequencing identified a compound heterozygous missense variant of c.737C > T (p.A246V) and a novel intronic insertion c.1166 + 4_1166 + 5insAAGCCCACCAC in RAPSN. RT-PCR analysis which showed the skipping of exon 7 in a skeletal muscle sample confirmed that the intronic insertion was pathogenic. His myasthenic symptoms were remarkably improved by pyridostigmine. Conclusion The patient’s diagnosis of CMS was confirmed by exome sequencing, and RT-PCR revealed that the skipping of exon 7 in RAPSN was caused by a novel intronic insertion. The genetic information uncovered in this case should therefore be added to the collection of tools for diagnosing and treating CMS.

DNA methylation in peripheral blood is associated with breast cancer (BC) but has mainly been studied in Caucasian populations. We investigated the association between blood-based methylation of receptor-associated protein of the synapse (RAPSN) and BC in Chinese population. The methylation levels of 12 RAPSN CpG sites were quantitatively evaluated by mass spectrometry in two case-control studies with 283 sporadic BC cases and 331 controls totally. The association was analyzed by logistic regression adjusted for covariants. The RAPSN methylation levels in patients with variant clinical characteristics were investigated by non-parametric tests. We found a significant association between BC and altered RAPSN methylation in blood in women at premenopausal and perimenopausal (age < 50 years old), but not in the elder women. This was approved by two independent case-control studies as well as by combining the subjects of the two studies (taken all subjects together, age < 50 years old, per 5% of methylation, odds ratio (OR) range from 1.17 to 1.30 for two CpG sites; OR = 0.75 for one CpG site; all p values < 0.02). This age-related RAPSN methylation was further modified by human epidermal growth factor receptor 2 (HER2) status (age < 50 years old, HER2 negative, per 5% of methylation, OR range from 1.27 to 1.48 for two CpG sites; OR = 0.76 for one CpG site; all p values < 0.02). We elucidated an association between BC and blood-based RAPSN methylation influenced by age and the status of HER2 in Chinese population.

Background Early detection is essential to improve the survival and life quality of lung cancer (LC) patients. Changes of peripheral blood DNA methylation could be associated with malignancy but were mostly studied in Caucasians. Methods Here, in a Chinese population, we performed mass spectrometry assays to investigate the association between very early stage LC and methylation levels of RAPSN in the peripheral blood by a case-control cohort using of 221 LC patients (93.2% LC at stage I) and 285 unrelated cancer free control individuals. Results The odds ratios (ORs) of all CpG sites were evaluated for their risk to LC using inter-quartile analyses by logistic regression. In general, we observed an association between very early LC and decreased methylation of RAPSN_CpG_1.15 and RAPSN_CpG_3.4 (referring to Q4, OR range from 1.64 to 1.81, p

Congenital myasthenic syndromes (CMS) comprise a heterogeneous group of genetic disorders of the neuromuscular junction. Next generation sequencing has been increasingly used for molecular diagnosis in CMS patients. This study aimed to identify the disease-causing variants in Thai patients. We recruited patients with a diagnosis of CMS based on clinical and electrophysiologic findings, and whole exome sequencing was performed. Thirteen patients aged from 2 to 54 years (median: 8 years) from 12 families were enrolled. Variants were identified in 9 of 13 patients (69%). Five novel variants and two previously reported variant were found in the COLQ, RAPSN and CHRND gene. The previously reported c.393+1G>A splice site variant in the COLQ gene was found in a majority of patients. Five patients harbor the homozygous splice site c.393+1G>A variant, and two patients carry compound heterozygous c.393+1G>A, c.718-1G>T, and c.393+1G>A, c.865G>T (p.Gly289Ter) variants. The novel variants were also found in RAPSN (p.Cys251del, p.Arg282Cys) and CHRND (p.Met481del). Molecular diagnosis in CMS patients can guide treatment decisions and may be life changing, especially in patients with COLQ mutations.

Aim Congenital myasthenic syndromes (CMS) are rare, genetically and phenotypically diverse disorders of neuromuscular transmission. Data on prevalence among children are scarce. Whole exome sequencing facilitated discovery of novel CMS mutations and enabled targeted treatment. Our aim was to identify the prevalence, genetic subtypes and clinical characteristics of CMS in pediatric population of Slovenia. Methods In this observational, national, cross-sectional study, medical records were retrospectively reviewed. Children with genetically confirmed CMS, referred over a 19 – year period (2000–2018) to the University Medical Centre, Ljubljana, Slovenia, were included in the study. Genetic and phenotypic characteristics were collected and prevalence of CMS in children was calculated. Results Eight children with a confirmed genetic mutation in 5 different genes (CHRNE, CHRND, RAPSN, CHAT, MUSK) causative of the CMS were identified. Calculated prevalence of genetically confirmed CMS was 22.2 cases per 1.000.000 children at the end of 2018. Interpretation The prevalence of genetically confirmed CMS in Slovenian children at the end of 2018 exceeds previously reported prevalence by more than two-fold, which suggests that prevalence in the literature is likely to be underestimated. Two extremely rarely detected mutations in MUSK and CHRND gene were detected and patient's clinical descriptions add important information on genotype-phenotype correlation.

In this issue of Neuron, Xing et al. (2021) demonstrate that the multidomain scaffold protein Rapsn can form dense molecular condensates in vitro and in vivo via phase separation. The formation of Rapsn condensates is essential for clustering acetylcholine receptors on muscle membranes and for forming neuromuscular junctions.

Objectives To present phenotype features of a large cohort of congenital myasthenic syndromes (CMS) and correlate them with their molecular diagnosis. Methods Suspected CMS patients were divided into three groups: group A (limb, bulbar or axial weakness, with or without ocular impairment, and all the following: clinical fatigability, electrophysiology compatible with neuromuscular junction involvement and anticholinesterase agents response), group B (limb, bulbar or axial weakness, with or without ocular impairment, and at least one of additional characteristics noted in group A) and group C (pure ocular syndrome). Individual clinical findings and the clinical groups were compared between the group with a confirmed molecular diagnosis of CMS and the group without molecular diagnosis or with a non-CMS molecular diagnosis. Results Seventy-nine patients (68 families) were included in the cohort: 48 in group A, 23 in group B and 8 in group C. Fifty-one were considered confirmed CMS (30 CHRNE, 5 RAPSN, 4 COL13A1, 3 DOK7, 3 COLQ, 2 GFPT1, 1 CHAT, 1 SCN4A, 1 GMPPB, 1 CHRNA1), 7 probable CMS, 5 non-CMS and 16 unsolved. The chance of a confirmed molecular diagnosis of CMS was significantly higher for group A and lower for group C. Some individual clinical features, alterations on biopsy and electrophysiology enhanced specificity for CMS. Muscle imaging showed at least mild alterations in the majority of confirmed cases, with preferential involvement of soleus, especially in CHRNE CMS. Conclusions Stricter clinical criteria increase the chance of confirming a CMS diagnosis, but may lose sensitivity, especially for some specific genes.

We present a case of lethal fetal akinesia deformation sequence (FADS) caused by a frameshift variant in trans with a 148 kbp deletion encompassing 3-36 exons of AGRN. Pathogenic variants in AGRN have been described in families with a form of congenital myasthenic syndrome (CMS), manifesting in the early childhood with variable fatigable muscle weakness. To the best of our knowledge, this is the first case of FADS caused by defects in AGRN gene. FADS has been reported to be caused by pathogenic variants in genes previously associated with CMS including these involved in endplate development and maintenance: MuSK, DOK7, and RAPSN. FADS seems to be the most severe form of CMS. None of the reported in the literature CMS cases associated with AGRN had two null variants, like the case presented herein. This indicates a strong genotype-phenotype correlation.

Objectives Congenital myasthenic syndromes (CMSs) are a genotypically and phenotypically heterogeneous group of neuromuscular disorders, which have in common an impaired neuromuscular transmission. Since the field of CMSs is steadily expanding, the present review aimed at summarizing and discussing current knowledge and recent advances concerning the etiology, clinical presentation, diagnosis, and treatment of CMSs. Methods Systematic literature review. Results Currently, mutations in 32 genes are made responsible for autosomal dominant or autosomal recessive CMSs. These mutations concern 8 presynaptic, 4 synaptic, 15 post-synaptic, and 5 glycosilation proteins. These proteins function as ion-channels, enzymes, or structural, signalling, sensor, or transporter proteins. The most common causative genes are CHAT, COLQ, RAPSN, CHRNE, DOK7, and GFPT1. Phenotypically, these mutations manifest as abnormal fatigability or permanent or fluctuating weakness of extra-ocular, facial, bulbar, axial, respiratory, or limb muscles, hypotonia, or developmental delay. Cognitive disability, dysmorphism, neuropathy, or epilepsy are rare. Low- or high-frequency repetitive nerve stimulation may show an abnormal increment or decrement, and SF-EMG an increased jitter or blockings. Most CMSs respond favourably to acetylcholine-esterase inhibitors, 3,4-diamino-pyridine, salbutamol, albuterol, ephedrine, fluoxetine, or atracurium. Conclusions CMSs are an increasingly recognised group of genetically transmitted defects, which usually respond favorably to drugs enhancing the neuromuscular transmission. CMSs need to be differentiated from neuromuscular disorders due to muscle or nerve dysfunction.

Introduction Nonimmune hydrops fetalis (NIHF) has varied etiology. We assessed the etiological spectrum and evaluated the utility of fetal whole exome sequencing (fWES) for the diagnosis of NIHF. Methods In this prospective cohort study, we evaluated antenatally diagnosed fetuses with NIHF between July 2018 and December 2019 according to the routine diagnostic algorithm. Fetuses that remained undiagnosed after routine NIHF workup were subjected to fetal chromosomal microarray and/or WES. Pregnancies were followed up for clinical outcomes. Results Of the 45 fetuses, consanguinity and recurrent hydrops fetalis were observed in 13.3% (6/45) and 28.8% (13/45), respectively. Overall, an etiological diagnosis was possible in 75.5% (34/45) of fetuses, while the cause remained unknown in 24.4% (11/45). A genetic etiology was identified in 46.6% (21/45): aneuploidy and monogenic disorders in 28.8% (13/45) and 17.8% (8/45), respectively. fWES on 19 fetuses detected disease-causing variants in 42.1% (8/19). Nine novel variants were detected in RAPSN, ASCC1, NEB, PKD1L1, GUSB, and PIEZO1. Only 8.8% (4/45) of the cohort survived without morbidity. Conclusions This study describes the etiological spectrum and the disease-causing variants in an Indian cohort of hydropic fetuses.

Our daily life depends on muscle contraction, a process that is controlled by the neuromuscular junction (NMJ). However, the mechanisms of NMJ assembly remain unclear. Here we show that Rapsn, a protein critical for NMJ formation, undergoes liquid-liquid phase separation (LLPS) and condensates into liquid-like assemblies. Such assemblies can recruit acetylcholine receptors (AChRs), cytoskeletal proteins, and signaling proteins for postsynaptic differentiation. Rapsn LLPS requires multivalent binding of tetratricopeptide repeats (TPRs) and is increased by Musk signaling. The capacity of Rapsn to condensate and co-condensate with interaction proteins is compromised by mutations of congenital myasthenic syndromes (CMSs). NMJ formation is impaired in mutant mice carrying a CMS-associated, LLPS-deficient mutation. These results reveal a critical role of Rapsn LLPS in forming a synaptic semi-membraneless compartment for NMJ formation.

The purpose of the study was to use exome sequencing (ES) to study the contribution of single-gene disorders to recurrent non-immune hydrops fetalis (NIHF) and retrospectively evaluate the value of genetic diagnosis on prenatal management and pregnancy outcome. From January 2012 to October 2018, a cohort of 28 fetuses with recurrent NIHF was analyzed by trio ES. Fetuses with immune hydrops, non-genetic factors (including infection, etc.), karyotype, or CNV abnormalities were excluded. Variants were interpreted based on ACMG/AMP guidelines. Fetal therapy was performed on seven fetuses. Of the 28 fetuses, 10 (36%) were found to carry causal genetic variants (pathogenic or likely pathogenic) in eight genes (GBA, GUSB, GBE1, RAPSN, FOXC2, PIEZO1, LZTR1, and FOXP3). Five (18%) fetuses had variant(s) of uncertain significance (VUS). Of the 10 fetuses with definitive molecular diagnosis, five (50%) were diagnosed with inborn errors of metabolism. Among the seven fetuses who received fetal therapy, two had definitive molecular diagnosis and resulted in neonatal death. Among the remaining five fetuses with negative results, four had newborn survival and one had intrauterine fetal death. Trio ES could facilitate genetic diagnosis of recurrent NIHF and improve the prenatal management and pregnancy outcome.

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.

Central corneal thickness (CCT) is one of the most heritable human traits, with broad-sense heritability estimates ranging between 0.68 to 0.95. Despite the high heritability and numerous previous association studies, only 8.5% of CCT variance is currently explained. Here, we report the results of a multiethnic meta-analysis of available genome-wide association studies in which we find association between CCT and 98 genomic loci, of which 41 are novel. Among these loci, 20 were significantly associated with keratoconus, and one (RAPSN rs3740685) was significantly associated with glaucoma after Bonferroni correction. Two-sample Mendelian randomization analysis suggests that thinner CCT does not causally increase the risk of primary open-angle glaucoma. This large CCT study explains up to 14.2% of CCT variance and increases substantially our understanding of the etiology of CCT variation. This may open new avenues of investigation into human ocular traits and their relationship to the risk of vision disorders., Hélène Choquet et al. report the largest genome-wide analysis of central corneal thickness (CCT) to date, finding novel associations at 41 loci. The study, which includes individuals from 4 ethnic groups, including African Americans and Hispanic/Latino individuals, increases the variance explained for CCT from 8.5% to 14.2%. Study findings also suggest that thinner CCT does not causally increase the risk of primary open-angle glaucoma.

ObjectiveTo investigate the diagnostic challenges of congenital myasthenic syndromes (CMS) in adult neuromuscular practice.MethodsWe searched the Mayo Clinic database for patients with CMS diagnosed in adulthood in the neuromuscular clinic between 2000 and 2016. Clinical, laboratory, and electrodiagnostic data were reviewed.ResultsWe identified 34 patients with CMS, 30 of whom had a molecular diagnosis (14 DOK7, 6 RAPSN, 2 LRP4, 2 COLQ, 2 slow-channel syndrome, 1 primary acetylcholine receptor deficiency, 1 AGRN, 1 GFPT1, and 1 SCN4A). Ophthalmoparesis was often mild and present in 13 patients. Predominant limb-girdle weakness occurred in 19 patients. Two patients had only ptosis. Age at onset ranged from birth to 39 years (median 5 years). The median time from onset to diagnosis was 26 years (range 4–56 years). Thirteen patients had affected family members. Fatigable weakness was present when examined. Creatine kinase was elevated in 4 of 23 patients (range 1.2–4.2 times the upper limit of normal). Repetitive nerve stimulation revealed a decrement in 30 patients. Thirty-two patients were previously misdiagnosed with seronegative myasthenia gravis (n = 16), muscle diseases (n = 15), weakness of undetermined cause (n = 8), and others (n = 4). Fifteen patients received immunotherapy or thymectomy without benefits. Fourteen of the 25 patients receiving pyridostigmine did not improve or worsen.ConclusionMisdiagnosis occurred in 94% of the adult patients with CMS and causes a median diagnostic delay of nearly 3 decades from symptom onset. Seronegative myasthenia gravis and muscle diseases were the 2 most common misdiagnoses, which led to treatment delay and unnecessary exposure to immunotherapy, thymectomy, or muscle biopsy.

Background Congenital myasthenic syndrome with episodic apnoea (CMS-EA) is a rare but potentially treatable cause of apparent life-threatening events in infancy. The underlying mechanisms for sudden and recurrent episodes of respiratory arrest in these patients are unclear. Whilst CMS-EA is most commonly caused by mutations in CHAT, the list of associated genotypes is expanding. Methods We reviewed clinical information from 19 patients with CMS-EA, including patients with mutations in CHAT, SLC5A7 and RAPSN, and patients lacking a genetic diagnosis. Results Lack of genetic diagnosis was more common in CMS-EA than in CMS without EA (56% n = 18, compared to 7% n = 97). Most patients manifested intermittent apnoea in the first 4 months of life (74%, n = 14). A degree of clinical improvement with medication was observed in most patients (74%, n = 14), but the majority of cases also showed a tendency towards complete remission of apnoeic events with age (mean age of resolution 2 years 5 months). Signs of impaired neuromuscular transmission were detected on neurophysiology studies in 79% (n = 15) of cases, but in six cases, this was only apparent following specific neurophysiological testing protocols (prolonged high-frequency stimulation). Conclusions A relatively large proportion of CMS-EA remains genetically undiagnosed, which suggests the existence of novel causative CMS genes which remain uncharacterised. In light of the potential for recurrent life-threatening apnoeas in early life and the positive response to therapy, early diagnostic consideration of CMS-EA is critical, but without specific neurophysiology tests, it may go overlooked.

// Qi-Shuai Zhuang 1,* , Hao Zheng 1,* , Xiao-Dan Gu 1 , Liang Shen 1 and Hong-Fang Ji 1 1 Shandong Provincial Research Center for Bioinformatic Engineering and Technique, School of Life Sciences, Shandong University of Technology, Zibo, P. R. China * These authors have contributed equally to the work Correspondence to: Liang Shen, email: // Hong-Fang Ji, email: // Keywords : Alzheimer’s disease, obesity, bioinformatics, genome-wide association studies, single nucleotide polymorphisms, Gerotarget Received : April 07, 2017 Accepted : June 18, 2017 Published : July 08, 2017 Abstract Alzheimer’s disease (AD) represents the major form of dementia in the elderly. In recent years, accumulating evidence indicate that obesity may act as a risk factor for AD, while the genetic link between the two conditions remains unclear. This bioinformatics analysis aimed to detect the genetic link between AD and obesity on single nucleotide polymorphisms (SNPs), gene, and pathway levels based on genome-wide association studies data. A total of 31 SNPs were found to be shared by AD and obesity, which were linked to 7 genes. These genes included PSMC3, CELF1, MYBPC3, SPI1, APOE, MTCH2 and RAPSN. Further functional enrichment analysis of these genes revealed the following biological pathways, including proteasome, osteoclast differentiation, hypertrophic cardiomyopathy, dilated cardiomyopathy, Epstein-Barr virus and TLV-I infection, as well as several cancer associated pathways, to be common among AD and obesity. The findings deepened our understanding on the genetic basis linking obesity and AD and may help shape possible prevention and treatment strategies.

Though dysfunction of neuromuscular junction (NMJ) is associated with congenital myasthenic syndrome (CMS), the proteins involved in neuromuscular transmission have not been completely identified. In this study, we aimed to identify a novel CMS gene in a consanguineous family with limb-girdle type CMS. Homozygosity mapping of the novel CMS gene was performed using high-density single-nucleotide polymorphism microarrays. The variants in CMS gene were identified by whole-exome sequencing (WES) and Sanger sequencing. A 20 MB-region of homozygosity (ROH) was mapped on chromosome 6q15–21. This was the only ROH that present in all clinically affected siblings and absent in all clinically unaffected siblings. WES showed a novel variant of AK9 gene located in this ROH. This variant was a start-gain mutation and introduced a cryptic 5′-UTR signal in intron 5 of the AK9 gene. The normal splicing signal would be interfered by the cryptic translation signal leading to defective splicing. Another 25 MB-ROH was found on chromosome 11p13–q12 in all siblings. WES showed a homozygous RAPSN pathogenic variant in this ROH. Since RAPSN-associated limb-girdle type CMS was only manifested in AK9 homozygous variant carriers, the disease phenotype was of digenic inheritance, and was determined by the novel disease modifier AK9 which provides NTPs for N-glycosylation. This is the first time that this specific genotype–phenotype correlation is reported. Importantly, the AK9-associated nucleotide deficiency may replete by dietary supplements. Since AK9 is a disease modifier, enhancing N-glycosylation by increasing dietary nucleotides may be a new therapeutic option for CMS patients.

DNA methylation changes in peripheral blood DNA have been shown to be associated with solid tumors. We sought to identify methylation alterations in whole blood DNA that are associated with breast cancer (BC). Epigenome-wide DNA methylation profiling on blood DNA from BC cases and healthy controls was performed by applying Infinium HumanMethylation450K BeadChips. Promising CpG sites were selected and validated in three independent larger sample cohorts via MassARRAY EpiTyper assays. CpG sites located in three genes (cg06418238 in RPTOR, cg00736299 in MGRN1 and cg27466532 in RAPSN), which showed significant hypomethylation in BC patients compared to healthy controls in the discovery cohort (p < 1.00 x 10-6) were selected and successfully validated in three independent cohorts (validation I, n =211; validation II, n=378; validation III, n=520). The observed methylation differences are likely not cell-type specific, as the differences were only seen in whole blood, but not in specific sub cell-types of leucocytes. Moreover, we observed in quartile analysis that women in the lower methylation quartiles of these three loci had higher ORs than women in the higher quartiles. The combined AUC of three loci was 0.79 (95%CI 0.73-0.85) in validation cohort I, and was 0.60 (95%CI 0.54-0.66) and 0.62 (95%CI 0.57-0.67) in validation cohort II and III, respectively. Our study suggests that hypomethylation of CpG sites in RPTOR, MGRN1 and RAPSN in blood is associated with BC and might serve as blood-based marker supplements for BC if these could be verified in prospective studies.

Introduction: Fluoxetine is a selective serotonin reuptake inhibitor and long-lived open channel blocker of the acetylcholine receptor often used in the treatment of slow-channel congenital myasthenic syndromes (CMS). Methods: We report a 42-year woman who had a history of episodic limb weakness that worsened after initiation of fluoxetine for treatment of depression. Genetic testing for CMS revealed a homozygous pathogenic mutation in the rapsyn (RAPSN) gene (p.Asn88Lys). Electrodiagnostic testing was performed prior to and 1 month after discontinuation of fluoxetine. Results: Two-Hz repetitive nerve stimulation of the fibular and spinal accessory nerves showed a baseline decrement of 36% and 14%, respectively. One month after discontinuing fluoxetine, the spinal accessory nerve decrement was no longer present, and the decrement in the fibular nerve was improved at 17%. Conclusion: This case demonstrates worsening of both clinical and electrophysiologic findings in a patient with CMS secondary to a RAPSN mutation treated with fluoxetine. This article is protected by copyright. All rights reserved.

Congenital myasthenic syndromes (CMS) are heterogeneous disorders caused by germline mutations in genes expressed at the neuromuscular junction. Mutations have been identified in 24 genes encoding acetylcholine receptor subunits (CHRNA1, CHRNB1, CHRND, CHRNE and CHRNG), skeletal muscle sodium channel (SCN4A), signaling molecules driving acetylcholine receptor subunits clustering (AGRN, LRP4, MUSK and DOK7), synaptic structural proteins (COLQ, LAMB2 and COL13A1), postsynaptic structural proteins (RAPSN and PLEC), presynaptic molecules (CHAT, SYT2), glycosylation enzymes (GFPT1, DPAGT1, ALG2, ALG14 and GMPPB), and other less characterized molecules (PREPL and SCL25A1). CMS are recessive disorders, except for slow channel CMS and synaptotagmin 2 (SYT2)-CMS. Onsets are largely less than 2 years, but adult-onset is not rare, especially in slow-channel CMS and limb-girdle type CMS caused by glycosylation defects and by DOK7 mutations. Clinical features include fatigable muscle weakness, amyotrophy and minor facial anomalies. Eye, facial and bulbar muscles are frequently affected, but sparing of these muscles is observed, especially in limb-girdle type CMS. Serum creatine kinase levels are frequently elevated in slow-channel CMS, glutamine-fructose-6-phosphate aminotransferase 1 (GFPT1)-CMS, and guanosine diphosphate mannose pyrophosphorylase B (GMPPB)-CMS. Electrophysiological findings supporting compromised neuromuscular signal transmission are a prerequisite for diagnosing CMS. Most CMS patients are likely to be underdiagnosed, and recognition of CMS in undiagnosed muscle weakness and/or amyotrophy is critical for diagnosing CMS.

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

Mutations in RAPSN are an important cause of congenital myasthenic syndrome (CMS), leading to endplate acetylcholine receptor deficiency. We present three RAPSN early-onset CMS patients (from a Brazilian cohort of 61 CMS patients). Patient 1 and patient 2 harbor the mutation p.N88K in homozygosity, while patient 3 harbors p.N88K in compound heterozygosity with another pathogenic variant (p.V165M; c.493G ≥ A). At onset, patient 3 presented with more severe symptoms compared to the other two, showing generalized weakness and repeated episodes of respiratory failure in the first years of life. During adolescence, she became gradually less symptomatic and does not require medication anymore, presenting better long-term outcomes than patients 1 and 2. This case series illustrates the variability of RAPSN early-onset CMS, with patient 3, despite severe onset, revealing an almost complete reversal of myasthenic symptoms, not limited to apneic episodes. Moreover, it suggests that RAPSN CMS may be underdiagnosed in non-European countries.

Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. FADS can result from mutations in CHRNG, CHRNA1, CHRND, DOK7 and RAPSN; however, these genes only account for a minority of cases. Here we identify MUSK as a novel cause of lethal FADS. Fourteen affected fetuses from a Dutch genetic isolate were traced back to common ancestors 11 generations ago. Homozygosity mapping in two fetuses revealed MUSK as a candidate gene. All tested cases carried an identical homozygous variant c.1724T>C; p.(Ile575Thr) in the intracellular domain of MUSK. The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers. Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings. A functional assay in myocytes derived from human fetuses confirmed that the variant blocks MUSK-dependent motor endplate formation. Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.

DNA methylation changes in peripheral blood DNA have been shown to be associated with solid tumors. We sought to identify methylation alterations in whole blood DNA that are associated with breast cancer (BC). Epigenome-wide DNA methylation profiling on blood DNA from BC cases and healthy controls was performed by applying Infinium HumanMethylation450K BeadChips. Promising CpG sites were selected and validated in three independent larger sample cohorts via MassARRAY EpiTyper assays. CpG sites located in three genes (cg06418238 in RPTOR, cg00736299 in MGRN1 and cg27466532 in RAPSN), which showed significant hypomethylation in BC patients compared to healthy controls in the discovery cohort (p < 1.00 × 10−6) were selected and successfully validated in three independent cohorts (validation I, n =211; validation II, n=378; validation III, n=520). The observed methylation differences are likely not cell-type specific, as the differences were only seen in whole blood, but not in specific sub cell-types of leucocytes. Moreover, we observed in quartile analysis that women in the lower methylation quartiles of these three loci had higher ORs than women in the higher quartiles. The combined AUC of three loci was 0.79 (95%CI 0.73-0.85) in validation cohort I, and was 0.60 (95%CI 0.54-0.66) and 0.62 (95%CI 0.57-0.67) in validation cohort II and III, respectively. Our study suggests that hypomethylation of CpG sites in RPTOR, MGRN1 and RAPSN in blood is associated with BC and might serve as blood-based marker supplements for BC if these could be verified in prospective studies.

Background and objectives Infants with congenital myasthenic syndrome (CMS) are at risk of brief resolved unexplained event (BRUE) and sleep-disordered breathing. The aim of the study was to explore sleep in infants with CMS with a particular focus on heart rate (HR) variability. Methods Overnight polygraphy was performed and HR variations associated with respiratory events were analysed. Bradycardia and tachycardia were defined as a variation of HR of ±10 bpm from baseline and analysed as events/hour. Results The data of 5 infants with CMS were analysed. Two patients had known mutations (COLQ and RAPSN). One patient had a tracheostomy. The apnoea-hypopnoea index (AHI) was abnormal in all the patients (range 2.8–47.7 events/h), with the highest AHI being observed in the 3 youngest infants. Nocturnal transcutaneous gas exchange was normal in all patients except the tracheostomised patient. Mean HR was 114 ± 23 bpm with a mean HR index of 4.5 ± 4.3 events/h. The amplitudes of HR variations (bradycardia or tachycardia) were around 15–20 bpm, regardless of the type of respiratory event, and comparable between patients. No correlations were found between HR indexes or variations and the type and mean duration of respiratory events. Ventilatory support was initiated in 3 infants immediately after the sleep study because of a high AHI and/or nocturnal hypoventilation. Conclusions All 5 infants had an abnormal AHI with younger infants having the highest AHI. Three infants required ventilatory support after the polygraphy, underlining its clinical usefulness. No significant abnormalities of HR were observed during the sleep studies.

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. (C) 2017 Elsevier B.V. All rights reserved.

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.

Congenital myasthenic syndromes are heterogeneous genetic disorders characterized by compromised neuromuscular transmission for which defects in multiple mechanisms are responsible. Clinical manifestations vary by syndrome subtype, but generally affected individuals present with fatigable muscle weakness, which can affect ocular, bulbar, limb and respiratory muscles. The onset of disease is usually, but not invariably, in infancy or childhood. The severity of disease varies greatly from death in early childhood to mild muscular weakness throughout life (Engel, 2012). To date, mutations in 16 different genes have been implicated in the development of congenital myasthenic syndromes. These genes encode the presynaptic protein (CHAT), synaptic components (COLQ, AGRN, LAMB2), and postsynaptic proteins (CHRNA1, CHRNB1, CHRNG, CHRND, CHRNE, RAPSN, MUSK, DOK7, GFPT1, SCN4A, PLEC and DPAGT1) (Chaouch et al., 2012; Engel, 2012; Lorenzoni et al., 2012). The functional properties of these genes have highlighted a number of mechanisms that involve the synthesis or packaging of acetylcholine load into synaptic vesicles, the Ca2p-dependent evoked release of acetylcholine from nerve terminals, and the efficiency of released load in generating a postsynaptic depolarization. The functional efficiency of neurotransmission depends on the endplate architecture, density and state of acetylcholinesterase (AChE) in the synaptic space, as well as the density, affinity for acetylcholine and kinetic properties of the acetylcholine receptor. However, for many disease-associated proteins, their role at the neuromuscular junction and contribution to the development of these syndromes remains unknown (Chaouch et al., 2012; Engel, 2012; Lorenzoni et al., 2012). Diagnostic precision is important in assessing the prognosis and managing these disorders and genetic testing is now available for a number of genes implicated in these disorders. The current technology is slow and expensive but research groups and some diagnostic laboratories are moving towards more efficient, cost-effective screening methods, such as next generation sequencing in targeted gene panels and subsequent exome sequencing in the negative families. There are still a number of genes to be identified as those identified to date account for

Congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders caused by genetic defects affecting neuromuscular transmission and leading to muscle weakness accentuated by exertion. Three different aspects have been investigated by members of the national French CMS Network: the difficulties in making a proper diagnosis; the course and long-term prognosis; and the response to therapy, especially for CMS that do not respond to cholinesterase inhibitors. CMS diagnosis is late in most cases because of confusion with other entities such as: congenital myopathies, due to the frequent presentation in patients of myopathies such as permanent muscle weakness, atrophy and scoliosis, and the abnormalities of internal structure, diameter and distribution of fibers (type I predominance, type II atrophy) seen on biopsy; seronegative autoimmune myasthenia gravis, when CMS is of late onset; and metabolic myopathy, with the presence of lipidosis in muscle. The long-term prognosis of CMS was studied in a series of 79 patients recruited with the following gene mutations: CHRNA; CHRNE; DOK7; COLQ; RAPSN; AGRN; and MUSK. Disease-course patterns (progressive worsening, exacerbation, stability, improvement) could be variable throughout life in a given patient. DOK7 patients had the most severe disease course with progressive worsening: of the eight wheelchair-bound and ventilated patients, six had mutations of this gene. Pregnancy was a frequent cause of exacerbation. Anticholinesterase agents are the first-line therapy for CMS patients, except for cases of slow-channel CMS, COLQ and DOK7. In our experience, 3,4-DAP was a useful complement for several patients harboring CMS with AChR loss or RAPSN gene mutations. Ephedrine was given to 18 patients (eight DOK7, five COLQ, four AGRN and one RAPSN). Tolerability was good. Therapeutic responses were encouraging even in the most severely affected patients, particularly with DOK7 and COLQ. Salbutamol was a good alternative in one patient who was allergic to ephedrine.

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.

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.

The objective of the study was to evaluate the epidemiology of patients with congenital myasthenic syndrome (CMS) in Israel. Targeted mutation analysis was performed based on the clinical symptoms and electrophysiological findings for known CMS. Additional specific tests were performed in patients of Iranian and/or Iraqi Jewish origin. All medical records were reviewed and clinical data, genetic mutations and outcomes were recorded. Forty-five patients with genetic mutations in known CMS genes from 35 families were identified. Mutations in RAPSN were identified in 13 kinships in Israel. The most common mutation was c.-38A>G detected in 8 patients of Iranian and/or Iraqi Jewish origin. Four different recessive mutations in COLQ were identified in 11 kinships, 10 of which were of Muslim-Arab descent. Mutations in CHRNE were identified in 7 kinships. Less commonly detected mutations were in CHRND, CHAT, GFPT1 and DOK7. In conclusion, mutations in RAPSN and COLQ are the most common causes of CMS in our cohort. Specific mutations in COLQ, RAPSN, and CHRNE occur in specific ethnic populations and should be taken into account when the diagnosis of a CMS is suspected.

Background: Congenital myasthenic syndromes (CMS) form a heterogeneous group of disorders caused by defects of the neuromuscular transmission. The clinical picture of CMS varies widely between mild and life-limiting forms. Until now, data of long-term follow-up are rare. Patients and Methods: A retrospective analysis (clinical, genetic, and treatment data) of 31 patients with CMS was performed. Additionally, a standardized, not yet validated test to examine 21 patients has been used. Results: We identified 31 patients with 9 different genetic defects of CMS. We formed different phenotypic groups in consideration of clinical symptoms; 18/31 patients presented ocular problems; 21/31 patients had bulbar symptoms at the beginning. Transient respiratory problems were found in 19/31 patients at onset; 6 had persistent respiratory problems; 18/31 patients showed first symptoms of neonatal (CHRNE [3/7], RAPSN [7/8], CHAT [3/5], COLQ [2/3], CHRND [1/1], CHRNB1 [1/1] mutations and “fast-channel” syndrome [1/1]); 12/18 patients later (between 2 months and 6 years). Despite an early and appropriate treatment, following long-term problems were identified: 12/18 patients suffered from difficulties in eating, 11/31 used a wheelchair, scoliosis developed in 9/31 patients, and 5/31 patients needed respiratory support. Conclusion: In patients with CMS, there is no direct genotype-phenotype correlation; nonetheless, in this cohort we identified specific phenotypic groups. Not all patients with CMS benefit in the same way from treatment. Some patients developed irrespective of genetic background and despite an appropriate and early therapy long-term problems. The applied test highlighted problems in ocular, bulbar, cervical, and limb muscle additional to neurological examination.

Rapsyn (RAPSN) mutations are a common cause of postsynaptic congenital myasthenic syndromes. We present a comprehensive description of the clinical and molecular findings of ten patients with CMS due to mutations in RAPSN, mostly with a long-term follow-up. Two patients were homozygous and eight were heterozygous for the common p.Asn88Lys mutation. In three of the heterozygous patients we have identified three novel mutations (c.869T > C; p.Leu290Pro, c.1185delG; p.Thr396Profs*12, and c.358delC; p.G1n120Serfs*8). In our cohort, the RAPSN mutations lead to a relatively homogeneous phenotype, characterized by fluctuating ptosis, occasional bulbar symptoms, neck muscle weakness, and mild proximal muscle weakness with exacerbations precipitated by minor infections. Interestingly, episodic exacerbations continue to occur during adulthood. These were characterized by proximal limb girdle weakness and ptosis, and not so much by respiratory insufficiency after age 6. All patients presented during neonatal period and responded to cholinergic agonists. In most of the affected patients, additional use of 3,4-diaminopyridine resulted in significant clinical benefit. The disease course is stable except for intermittent worsening. (C) 2015 Elsevier B.V. All rights reserved.

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

Congenital myasthenic syndromes (CMS) are rare genetic disorders characterized by impaired neuromuscular transmission. They are caused by mutations in synaptic, presynaptic and post synaptic proteins. Rapsyn is a postsynaptic peripheral membrane protein that anchors the nicotinic acetylcholine receptor to the motor endplate. CMS patients of Iraqi and Persian Jewish origin, carry a common founder mutation in the E box of the RAPSN promoter region (− 38A-G) that causes impaired transcriptional activities of the promoter region. We describe a Persian Jewish family with two siblings affected with typical CMS, harboring the common heterozygous (− 38A-G) E-box mutation associated with a previously unreported heterozygous p.224 insT causing an insertion of Threonine in the TPR6 domain. To the best of our knowledge, this is the first mutation in the TPR6 domain and might give supportive evidence to the role of this domain in rapsyn self association and consequently co-clustering with AchR in the post synaptic membrane.

Introduction: Myasthenia gravis (MG) is an autoimmune disease. Patients without detectable antibodies against the nicotinic acetylcholine receptor or the muscle-specific tyrosine kinase are referred to as seronegative MG (SNMG). Because late-onset congenital myasthenic syndromes (CMSs) due to RAPSN or DOK7 mutations may be mistaken for SNMG, we investigated their frequency in a nationwide SNMG cohort. Methods: We performed sequencing of RAPSN and DOK7 in all Norwegian SNMG patients (n = 74) and 37 healthy controls, examining for the N88K and c.1124_1127dupTGCC mutations, respectively. Results: We found 1 patient homozygous for N88K and 2 carriers of the N88K mutation. Sequencing of DOK7 revealed no mutations. Conclusions: This study confirms that rapsn CMS can be mistaken for SNMG. In addition, the frequency of rapsn CMS in our nationwide SNMG cohort was found to be low. SNMG patients with an atypical clinical presentation and pediatric cases should be tested for the N88K mutation before initiation of immunosuppressive drug treatment or thymectomy. Muscle Nerve, 2011

A workshop on the respiratory management of congenital myasthenic syndromes (CMS) in childhood was held on 8 December 2009 at the UCL Institute of Neurology, UK. The workshop was sponsored by the Muscular Dystrophy Campaign and attended by 20 participants from 9 centres (8 in UK and 1 in Europe). The aim of the workshop was to review respiratory management of childhood CMS in major UK specialist centres and develop recommendations for standards of care to help anticipate and treat respiratory complications. The congenital myasthenic syndromes are a heterogeneous group of genetic disorders which result in impaired neuromuscular transmission and fatiguable weakness. Advances in molecular genetic diagnosis have lead to the identification of distinct genotypes [1] which are associated with particular patterns of respiratory decompensation. Many CMS children present with respiratory difficulties at birth and most are at risk of ventilatory failure with intercurrent respiratory illnesses. However, recurrent, life-threatening episodic apnoea in infancy and childhood (often precipitated by infection or even stress) is a particular feature of the presynaptic CMS caused by mutations in CHAT and postsynaptic CMS due to mutations in RAPSN. Other phenotypes, such as the synaptic CMS due to mutations in COLQ and the slow channel syndromes caused by mutations in the acetylcholine receptor subunits, cause an end plate myopathy which results in progressive respiratory muscle weakness and respiratory failure requiring long term ventilatory support. The fast channel syndromes are associated with recurrent and particularly severe respiratory crises in infancy and childhood. Other genotypes have diverse respiratory presentations, for example the post-synaptic CMS caused by mutations in DOK7 may be associated, in severe cases, with respiratory failure requiring ventilation at birth, or congenital stridor due to vocal cord palsy often necessitating tracheostomy, without any evidence of the limb gir

Objective To perform genetic testing of patients with congenital myasthenic syndromes (CMS) from the Southern Brazilian state of Parana. Patients and methods Twenty-five CMS patients from 18 independent families were included in the study. Known CMS genes were sequenced and restriction digest for the mutation RAPSN p.N88K was performed in all patients. Results We identified recessive mutations of CHRNE in ten families, mutations in DOK7 in three families and mutations in COLQ, CHRNA1 and CHRNB1 in one family each. The mutation CHRNE c.70insG was found in six families. We have repeatedly identified this mutation in patients from Spain and Portugal and haplotype studies indicate that CHRNE c.70insG derives from a common ancestor. Conclusions Recessive mutations in CHRNE are the major cause of CMS in Southern Brazil with a common mutation introduced by Hispanic settlers. The second most common cause is mutations in DOK7. The minimum prevalence of CMS in Parana is 0.18/100000.

Contains fulltext : 70779.pdf (Publisher’s version ) (Closed access) 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.

The congenital myasthenic syndromes (CMS) are a diverse group of genetic disorders caused by abnormal signal transmission at the motor endplate, a special synaptic contact between motor axons and each skeletal muscle fibre. Most CMS stem from molecular defects in the muscle nicotinic acetylcholine receptor, but they can also be caused by mutations in presynaptic proteins, mutations in proteins associated with the synaptic basal lamina, defects in endplate development and maintenance, or defects in protein glycosylation. The specific diagnosis of some CMS can sometimes be reached by phenotypic clues pointing to the mutated gene. In the absence of such clues, exome sequencing is a useful technique for finding the disease gene. Greater understanding of the mechanisms of CMS have been obtained from structural and electrophysiological studies of the endplate, and from biochemical studies. Present therapies for the CMS include cholinergic agonists, long-lived open-channel blockers of the acetylcholine receptor ion channel, and adrenergic agonists. Although most CMS are treatable, caution should be exercised as some drugs that are beneficial in one syndrome can be detrimental in another.

The ENMC hosted a group of 18 experts on Congenital Myasthenic Syndromes (CMS). CMS are inherited disorders in which the safety margin of the neuromuscular transmission is compromised by one or more specific mechanism(s). CMS are caused by various genetic defects. The objectives of the workshop included progress in deciphering the molecular basis of CMS (sessions 1–4) and clinical conclusions for epidemiology, diagnosis and therapy (sessions 5–7). To date, genes known to cause CMS if mutated are the presynaptic choline acetyltransferase gene CHAT, the gene COLQ encoding the synaptic protein ColQ, the genes encoding the different subunits of the postsynaptic acetylcholine receptor (CHRNA1, CHRNB1, CHRND, CHRNE), the genes for the postsynaptic proteins rapsyn (RAPSN), muscle-specific receptor tyrosine kinase (MUSK) and the postsynaptic sodium channel (SCN4A). Since the last ENMC workshop on CMS in October 1999, four novel CMS genes have been identified, namely CHAT, RAPSN, SCN4A and MUSK. As a consequence, several new patients presenting with varying phenotypes of CMS have been described worldwide. In particular, mutations in RAPSN and CHAT turned out to be of high clinical relevance, on one hand because of their apparent worldwide frequency, on the other hand because of their specific clinical phenotype with the occurrence of sudden apneic episodes. Furthermore, considerable progress has been made using a variety of

The objective is mutation analysis of the RAPSN gene in a patient with sporadic congenital myasthenic syndrome (CMS). Mutations in various genes encoding proteins expressed at the neuromuscular junction may cause CMS. Most mutations affect the epsilon subunit gene of the acetylcholine receptor (AChR) leading to endplate AChR deficiency. Recently, mutations in the RAPSN gene have been identified in several CMS patients with AChR deficiency. In most patients, RAPSN N88K was identified, either homozygously or heteroallelic to a second missense mutation. A sporadic CMS patient from Germany was analyzed for RAPSN mutations by RFLP, long-range PCR and sequence analysis. Clinically, the patient presents with an early onset CMS, associated with arthrogryposis multiplex congenita, recurrent episodes of respiratory insufficiency provoked by infections, and a moderate general weakness, responsive to anticholinesterase treatment. The mutation RAPSN N88K was found heterozygously to a large deletion of about 4.5 kb disrupting the RAPSN gene. Interestingly, an Alu-mediated unequal homologous recombination may have caused the deletion. We hypothesize that numerous interspersed Alu elements may predispose the RAPSN locus for genetic rearrangements.

Background: Mutations in various genes of the neuromuscular junction may cause congenital myasthenic syndromes (CMS). Most mutations identified to date affect the e-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. Objective: To perform mutation analysis of the RAPSN gene in patients with sporadic or autosomal recessive CMS. Methods: One hundred twenty CMS patients from 110 unrelated families were analyzed for the RAPSN mutation N88K by restriction fragment length polymorphism and sequence analysis. Results: 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). Conclusions: 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.

Myogenic determination factors are basic helix-loop-helix proteins that govern specification and differentiation of muscle cells, and bind to the E-box consensus sequence CANNTG in promoter regions of muscle-specific genes. No E-box mutation has been reported to date. RAPSN encodes rapsyn, a 43 kDa postsynaptic peripheral membrane protein that clusters the nicotinic acetylcholine receptor at the motor endplate. Transcriptional regulation mechanisms of RAPSN have not been studied. We here report two novel E-box mutations in the RAPSN promoter region in eight congenital myasthenic syndrome patients. Patient 1 carries -27C-->G that changes an E-box at -27 to -22 from CAGCTG to GAGCTG. An allele harboring -27C-->G is not transcribed in patient's muscle. Patients 2-8 are of Oriental Jewish stock of Iraqi or Iranian origin with facial malformations, and harbor -38A-->G that changes another E-box at -40 to -35 from CAACTG to CAGCTG, which does not affect the consensus CANNTG sequence. Haplotype analysis shows that -38A-->G arises from a common founder. For each mutation, position +1 represents the major transcriptional start site that we determine to be 172 nucleotides upstream of the translational start site. Electrophoretic mobility shift assays reveal that -38A-->G gains, and -27C-->G looses, binding affinity for different components of nuclear extracts of C2C12 myotubes. Luciferase reporter assays show that both -38A-->G and -27C-->G attenuate reporter gene expression in C2C12 myotubes, and that -27C-->G additionally attenuates reporter gene expression in MyoD- or myogenin-transfected HEK cells. The -27C-->G mutation also markedly attenuates the enhancer activity of an E-box on an SV40 promoter. Impaired transcriptional activities of the RAPSN promoter region predict reduced rapsyn expression and endplate acetylcholine receptor deficiency.

Congenital myasthenic syndromes (CMS) are heterogeneous disorders caused by congenital defects of molecules expressed at the neuromuscular junctions. Clinical features include fatigable muscle weakness, amyotrophy and minor facial anomalies. Mutations have been identified in 18 genes encoding acetylcholine receptor (AChR) subunits (CHRNA1, CHRNB1, CHRND and CHRNE); skeletal muscle sodium channel (SCN4A); signalling molecules driving AChR clustering and subserving maintenance and differentiation of the postsynaptic region (AGRN, LRP4, MUSK and DOK7); postsynaptic structural proteins (RAPSN and PLEC); synaptic β2 laminin, which promotes presynaptic differentiation, and synaptic collagen Q; presynaptic choline acetyltransferase and enzymes in subserving protein glycosylation (GFPT1, DPAGT1, ALG14, and ALG2). The CMS are caused by recessive mutations except for the slow-channel CMS. The recent development of the exome sequencing has speeded identification of causative mutations. Mutations in glycosylation genes were recently discovered, but the mechanisms by which they impair neuromuscular signal transmission have not been fully elucidated. Key Concepts: Congenital myasthenic syndromes are caused by germline mutations in molecules expressed at the neuromuscular junction (NMJ). Muscle nicotinic acetylcholine receptor (AChR) is a pentameric ligand-gated ion channel in the stoichiometry of α2βδϵ subunits. Missense mutations in AChR subunit genes can cause abnormally long and brief ion channel openings resulting in slow- and fast-channel myasthenic syndromes, respectively. Primary endplate AChR deficiency can be due to low-expressor or null mutations in the AChR ϵ subunit. The phenotype in case of biallelic low-expressor or null mutations in the ϵ subunit is rescued by expression of the foetal γ subunit. Biallelic null mutations in non-ϵ are embryonic lethal mutations. A second group of endplate AChR deficiency is caused by mutations in signalling molecules including agrin, LRP4, MuSK, Dok-7, which drive AChR clustering. The third group of endplate AChR deficiency stems from mutations in the postsynaptic structural proteins of rapsyn or plectin. Mutations in enzymes subserving the N-glycosylation pathway of GFPT1, DPAGT1, ALG14 and ALG2 cause limb-girdle CMS with tubular aggregates. Endplate acetylcholinesterase (AChE) deficiency is caused by mutations in collagen Q (ColQ), which anchors AChE to the synaptic basal lamina. Protein-anchoring therapy, in which ectopically expressed AChE/ColQ complex is specifically anchored to the neuromuscular junction using the proprietary binding motifs, markedly ameliorates myasthenic symptoms of Colq-knockout mice. Mutations in choline acetyltransferase (ChAT) cause defective resynthesis of ACh at the nerve terminal and a CMS associated with frequent episodic apnoea. Keywords: congenital myasthenic syndromes; neuromuscular junction; muscle nicotinic acetylcholine receptor; skeletal muscle sodium channel; acetylcholinesterase; choline acetyltransferase; protein glycosylation

MuSK myasthenia gravis is a rare, severe autoimmune disease of the neuromuscular junction, only identified in 2001, with unclear pathogenic mechanisms. In this review we describe the clinical aspects that distinguish MuSK MG from AChR MG, review what is known about the role of MuSK in the development and function of the neuromuscular junction, and discuss the data that address how the antibodies to MuSK lead to neuromuscular transmission failure. © 2013 Anatomical Society.