Rapsyn Mutations Research Articles

VP.42 Clinical characteristics, molecular genetics and long-term clinical outcomes in 43 patients with congenital myasthenia syndrome due to RAPSYN mutation

Congenital myasthenic syndromes (CMS) are a group of rare inherited disorders of neuromuscular transmission. Rapsyn (receptor-associated protein of the synapse) is a post-synaptic protein that binds to the acetylcholine receptor (AChR) subunits and has a crucial role in AChR clustering at the post synaptic membrane. We describe the clinical characteristics, molecular genetic findings, and long-term clinical outcomes in 43 patients with CMS due to <i>RAPSYN</i> mutations seen at three UK specialist neuromuscular centres. 43 patients (20 females) with confirmed <i>RAPSYN</i> mutations and at least one follow-up visit were included. Median age at last follow up was 18 years (range 4-73 years). Symptoms at onset, included arthrogryposis 41%, ptosis 44%, respiratory involvement 48%, limb weakness/motor delay 67% and feeding difficulties 62%. Recurrent apnoea was the commonest respiratory symptom, 46% required short term and 7% required long-term respiratory support for apnoea management. 32% patients required nasogastric or gastrostomy feeding. Typical pattern of weakness was ptosis, facial weakness, axial, generalised or proximal weakness in limbs. 95% patients had ptosis, 62% patients had a divergent/ convergent squint and ophthalmoplegia was absent in all but one. All patients achieved independent ambulation, median age in 27 patients was 16 months (range 12- 48 months). At last follow up, all but one patient was independently ambulant. At last follow up 25% patients had no CMS features, 37% only ptosis and remainder had mild limb weakness. All patients were on normal oral diet except one who remained on mixture of oral and gastrostomy feeds since childhood. Patients who required long-term respiratory support in childhood were weaned off it successfully and no patient had respiratory crisis after 7 years of age. Except for 2, all patients had N88K mutation (homozygous in 14 patients). All but one patient was on treatment. Pyridostigmine was first line with good response in all treated patients, 28% required additional 3,4 diaminopyridine and 2% salbutamol. Two patients were able to reduce treatment in 2nd/3rd decade. In summary, this is the largest cohort of RAPSYN patients with clinical and genetic characteristics and also long-term outcome which was of consistent clinical improvements after 1st decade and stability in adult life in majority of patients, a crucial information for treating clinicians, patients and families.

A mechanism in agrin signaling revealed by a prevalent Rapsyn mutation in congenital myasthenic syndrome.

Neuromuscular junction is a synapse between motoneurons and skeletal muscles, where acetylcholine receptors (AChRs) are concentrated to control muscle contraction. Studies of this synapse have contributed to our understanding of synapse assembly and pathological mechanisms of neuromuscular disorders. Nevertheless, underlying mechanisms of NMJ formation was not well understood. To this end, we took a novel approach - studying mutant genes implicated in congenital myasthenic syndrome (CMS). We showed that knock-in mice carrying N88K, a prevalent CMS mutation of Rapsyn (Rapsn), died soon after birth with profound NMJ deficits. Rapsn is an adapter protein that bridges AChRs to the cytoskeleton and possesses E3 ligase activity. In investigating how N88K impairs the NMJ, we uncovered a novel signaling pathway by which Agrin-LRP4-MuSK induces tyrosine phosphorylation of Rapsn, which is required for its self-association and E3 ligase activity. Our results also provide insight into pathological mechanisms of CMS.

Long-term follow-up in patients with congenital myasthenic syndrome due to RAPSN mutations

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.Gln120Serfs*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.

G.P.83 - Congenital myasthenic syndromes in Israel: Genetic and clinical characterization

The genetic and clinical characteristics of Israeli patients with congenital myasthenic syndromes (CMS) evaluated in four medical centers are presented. Forty-one patients with CMS from thirty two independent families in whom genetic mutation was detected are described. Genetic evaluation was initially performed depending on patients' clinical symptoms and known CMS genes were sequenced. All patients' medical records were reviewed and their clinical data, electrophysiological studies, genetic mutation and outcome were recorded. Mutations in Rapsyn were detected in 15 patients of 13 families. The most common mutation was the Rap-38A>G, identified in patients of Iran–Iraq Jewish origin. Mutations in COLQ were confirmed in 11 patients of 8 families. Four different recessive mutations of COLQ were identified mostly in Arab patients. CHRNE mutations were confirmed in 15 patients of 13 families. The less common detected mutations were: CHRND, ChAT, GFTP1 and dok-7. The vast majority of children had clinical symptoms in infancy. Ptosis, ophthalmoplegia and feeding problems were the most common symptoms. RAPSYN, COLQ and CHRNE were the most common causes of CMS in our cohort. Specific mutations in these genes are detected in unique ethnic populations in Israel.

P164 – 2586: Congenital myasthenic syndrome in Israel: Genetic and clinical characterization

Objective Congenital myasthenic syndromes (CMS) are a rare heterogeneous group of inherited neuromuscular disorders associated with distinctive clinical, electrophysiological and genetic abnormalities. Mutations in various genes of the neuromuscular junction may cause presynaptic, synaptic or postsynaptic defects. In this study, the genetic and clinical characteristics of Israeli patients with CMS were evaluated. Methods Twentysix patients with CMS from 16 independent families in whom genetic mutation was detected were diagnosed in two Israeli medical centers. Genetic evaluation was initially performed depending on patients' clinical symptoms and known CMS genes were sequenced. Patients of Jewish Iranian or Iraqi ethnic origin were screened directly for a known 38 A-G mutation in RAPSYN. In two patients, whole exome sequencing was analyzed. All patients' medical records were reviewed and their clinical data, electrophysiological studies and outcome were recorded. Results The diagnosis of CMS was confirmed in 26 patients of 16 families. Among these patients, 8 patients of 4 families had recessive mutations in COLQ, eight patients of 5 families, mutations in CHRNE, and eight patients of 5 families, mutations in RAPSN. CHRND was diagnosed in 2 patients from one family. The mean age of onset of clinical symptoms was 6 months. Ptosis, ophthalmoplegia and feeding problems were the most common symptoms. Conclusions RAPSYN and CHRNE are the most common causes of CMS in our cohort. The second most common cause is COLQ. Specific mutations in RAPSYN, CHRNE and COLQ are detected in unique ethnic populations in Israel.

P.12.3 Do congenital myasthenic syndromes in childhood have a common face? Clinical profile of slow channel, CHRNE and RAPSYN mutations

Congenital myasthenic syndromes (CMSs) form a group of hereditary disorders, which are clinically and genetically heterogeneous. The neuromuscular transmission cascade is disrupted at presynaptic, synaptic or postsynaptic level, the latter is the most common. As a large part of CMSs have their onset in childhood, performing diagnostic studies leading to diagnosis can be very challenging. CMSs are highly treatable but treatment depends on the mutation type, so clinicians are urged to perform tailored genetic testing guided by clinical features. This systematic review of the literature shows the clinical neurological features of published cases with CMS due to a slow channel syndrome, RAPSYN deficiency or acetylcholine receptor (AChR) deficiency. English and Dutch case reports and series describing patients with a genetically proven CHRNE, RAPSYN or slow channel CMS from 1980 to 2012 were collected. Clinical features were evaluated. Findings were mostly in line with clinical features mentioned in previous literature. Minor differences were encountered. Very common were ocular, bulbar and limb weakness for AChR deficiency (N = 100). Cervical and distal weakness was often seen in slow channel syndromes (N = 51). Bulbar symptoms, arthrogryposis multiplex congenita, respiratory difficulties and the N88K founder mutation occurred frequently in the RAPSYN deficient patients (N = 98). Opposed to earlier reports no early- and late-onset phenotype was identified in the latter group and ophthalmoparesis was rare. In slow channel syndromes bulbar (25%) and ocular weakness (50%) was more common than previously reported. Ethnicity was not always in line with previous literature but could still be helpful in more targeted genetic testing. Overall we think that detailed descriptions of clinical features could guide the clinician in the diagnostic process.

Myasthenic syndrome due to defects in rapsyn: Clinical and molecular findings in 39 patients.

Pathogenic mutations in rapsyn result in endplate acetylcholine receptor (AChR) deficiency and are a common cause of postsynaptic congenital myasthenic syndromes. Clinical, electrophysiologic, pathologic, and molecular studies were done in 39 patients. In all but one patient, the disease presented in the first 2 years of life. In 9 patients, the myasthenic symptoms included constant or episodic ophthalmoparesis, and 1 patient had a pure limb-girdle phenotype. More than one-half of the patients experienced intermittent exacerbations. Long-term follow-up was available in 25 patients after start of cholinergic therapy: 21 became stable or were improved and 2 of these became asymptomatic; 3 had a progressive course; and 1 died in infancy. In 7 patients who had endplate studies, the average counts of AChR per endplate and the synaptic response to ACh were less reduced than in patients harboring low AChR expressor mutations. Eight patients were homozygous and 23 heterozygous for the common p.N88K mutation. Six mutations, comprising 3 missense mutations, an in-frame deletion, a splice-site mutation, and a nonsense mutation, are novel. Homozygosity for p.N88K was associated with varying grades of severity. No genotype-phenotype correlations were observed except in 8 Near-Eastern patients homozygous for the promoter mutation (c.-38A>G), who had a mild course. All but 1 patient presented early in life and most responded to cholinergic agonists. With early diagnosis and therapy, rapsyn deficiency has a benign course in most patients. There was no consistent phenotype-genotype correlation except for an E-box mutation associated with jaw deformities.

M.P.1.05 Successful long-term acethylcholinesterase inhibitor therapy in patients affected by Rapsyn (RAPSN) mutation early onset phenotype

M.P.1.05 Successful long-term acethylcholinesterase inhibitor therapy in patients affected by Rapsyn (RAPSN) mutation early onset phenotype

Late-onset congenital myasthenic syndrome due to rapsyn mutation

Late-onset congenital myasthenic syndrome due to rapsyn mutation

Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations

Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations

Congenital myasthenic syndrome caused by two non‐N88K rapsyn mutations

Congenital myasthenic syndrome caused by two non‐N88K rapsyn mutations

The Dynamics of the Rapsyn Scaffolding Protein at Individual Acetylcholine Receptor Clusters

Rapsyn, a cytoplasmic receptor-associated protein, is required for the clustering of acetylcholine receptors (AChRs). Although AChR dynamics have been extensively studied, little is known about the dynamics of rapsyn. Here, we used a rapsyn-green fluorescent protein (GFP) fusion protein and quantitative fluorescent imaging to study the dynamics of rapsyn in transfected C2C12 myotubes. First, we found that rapsyn-GFP expression at clusters did not alter AChR aggregation, function, or turnover. Quantification of rapsyn immunofluorescence indicated that the expression of rapsyn-GFP proteins at clusters does not increase the overall rapsyn density compared with untransfected myotube clusters. Using time lapse imaging and fluorescence recovery after photobleaching, we demonstrated that the recovery of rapsyn-GFP fluorescence at clusters was very fast, with a halftime of about approximately 1.5 h (approximately 3 times faster than AChRs). Inhibition of protein kinase C significantly altered receptor insertion, but it had no effect on rapsyn insertion. When cells were treated with the broad spectrum kinase inhibitor staurosporine, receptor insertion was decreased even further. However, inhibition of protein kinase A had no effect on insertion of either rapsyn or receptors. Finally, when cells were treated with neural agrin, rapsyn and AChRs were both directed away from preexisting clusters and accumulated together in new small clusters. These results demonstrate the remarkable dynamism of rapsyn, which may underlie the stability and maintenance of the postsynaptic scaffold and suggest that the insertion of different postsynaptic proteins may be operating independently.

Overexpression of Rapsyn in Rat Muscle Increases Acetylcholine Receptor Levels in Chronic Experimental Autoimmune Myasthenia Gravis

The primary autoantigen in myasthenia gravis, the acetylcholine receptor (AChR), is clustered and anchored in the postsynaptic membrane of the neuromuscular junction by rapsyn. Previously, we found that overexpression of rapsyn by cDNA transfection protects AChRs in rat muscles from antibody-mediated loss in passive transfer experimental autoimmune myasthenia gravis (EAMG). Here, we determined whether rapsyn overexpression can reduce or even reverse AChR loss in muscles that are already damaged by chronic EAMG, which mimics the human disease. Active immunization against purified AChR was performed in female Lewis rats. Rapsyn overexpression resulted in an increase in total muscle membrane AChR levels, with some AChR at neuromuscular junctions but much of it in extrasynaptic membrane regions. At the ultrastructural level, most endplates in rapsyn-treated chronic EAMG muscles showed increased damage to the postsynaptic membrane. Although rapsyn overexpression stabilized AChRs in intact or mildly damaged endplates, the rapsyn-induced increase of membrane AChR enhanced autoantibody binding and membrane damage in severe ongoing disease. Thus, these results show the complexity of synaptic stabilization of AChR during the autoantibody attack. They also indicate that the expression of receptor-associated proteins may determine the severity of autoimmune diseases caused by anti-receptor antibodies.

Unusual features in a boy with the rapsyn N88K mutation

Fluctuating or episodic weakness starting in infancy is seen in congenital myasthenic syndromes (CMSs), periodic paralyses, and a disorder precipitated by febrile illness mapped to Xp22.3.1 Mutations in rapsyn ( RAPSN ) cause congenital myasthenia and more than 20 mutations with three different clinical phenotypes have been identified.2-4 The patient, now 19 years old, developed cyanotic spells at age 3 days. He was hypotonic with a weak cry, but had no persisting respiratory difficulties. He received nasogastric feeding and antibiotics, despite normal infection parameters, and was discharged well after 3 days. At age 6 months, fever developed again, possibly due to a respiratory infection; he also had problems swallowing saliva and one episode in which he became cyanotic and increasingly hypotonic. His leukocyte count fell from 36.8 to 9.2 (normal 3.5 to 11.0 × 109/L); chest x-ray, cerebral ultrasound, and EEG were normal. He was treated with oxygen and ephedrine and discharged well 5 days later. During the next 5 years, he had five more attacks, all preceded by fever and characterized by generalized weakness, including facial and tongue muscles, ptosis, and diplopia. He became briefly respirator dependent on …

Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations

Congenital myasthenic syndromes are inherited disorders of neuromuscular transmission characterized by fatigable muscle weakness. Autosomal recessive acetylcholine receptor (AChR) deficiency syndromes, in which levels of this receptor at the neuromuscular junction are severely reduced, may be caused by mutations within genes encoding the AChR or the AChR-clustering protein, rapsyn. Most patients have mutations within the rapsyn coding region and are either homozygous for N88K or heteroallelic for N88K and a second mutation. In some cases the second allele carries a null mutation but in many the mutations are missense, and are located in different functional domains. Little is known about the functional effects of these mutations, but we hypothesize that they would have an effect on AChR clustering by a variety of mechanisms that might correlate with disease severity. Here we expressed RAPSN mutations A25V, N88K, R91L, L361R and K373del in TE671 cells and in rapsyn-/- myotubes to determine their pathogenic mechanisms. The A25Vmutation impaired colocalization of rapsyn with AChR and prevented agrin-induced AChR clusters in rapsyn-/- myotubes. In TE671 cells, R91L reduced the ability of rapsyn to self-associate, and K373del-rapsyn was significantly less stable than wild-type. The effects of mutations L361R and N88K were more subtle: in TE671 cells, in comparison with wild-type rapsyn, L361R-rapsyn showed reduced expression/stability, and both N88K-rapsyn and L361R-rapsyn showed significantly reduced co-localization with AChR. N88K-rapsyn and L361R-rapsyn could effectively mediate agrin-induced AChR clusters, but these were reduced in number and were less stable than with wild-type rapsyn. The disease severity of patients harbouring the compound allelic mutations was greater than that of patients with homozygous rapsyn mutation N88K, suggesting that the second mutant allele may largely determine severity.

CHRND mutation causes a congenital myasthenic syndrome by impairing co-clustering of the acetylcholine receptor with rapsyn

The objective of this study was to analyse the mutations of the acetylcholine receptor (AChR) delta subunit gene (CHRND) in a patient with sporadic congenital myasthenic syndrome (CMS). Mutations in various genes encoding proteins expressed at the neuromuscular junction may cause CMS. Mutations of AChR subunit genes lead to end-plate AChR deficiency or to altered kinetic properties of the receptor. Mutations in the alpha, beta and delta subunits of the AChR are less frequent than mutations of the epsilon subunit; mutations in these subunits leading to AChR deficiency are often associated with a severe phenotype. A sporadic patient from Germany was studied, who presented with an early onset CMS associated with feeding difficulties, ptosis, a moderate general weakness responsive to anticholinesterase treatment and recurrent episodes of respiratory insufficiency provoked by infections. The CHRND gene was screened for mutations by RFLP, long-range PCR and sequence analysis. Subsequently, we conducted functional studies of AChR mutants co-transfected with rapsyn in HEK 293 cells. Heterozygously to a 2.2 kb microdeletion disrupting the CHRND gene, we identified a novel point mutation in the long cytoplasmic loop, CHRND E381K. The cytoplasmic loop of the AChR subunits is known to be essential for AChR-rapsyn co-clustering. We therefore studied the interaction of AChR containing the CHRND E381K mutation with rapsyn by evaluating expression and co-localization of rapsyn and mutated AChR subunits in co-transfected HEK 293 cells. Interestingly, the mutated receptor showed severely reduced cluster formation compared with the wild-type receptor. In contrast, the corresponding amino acid substitution in the cytoplasmic loop of the AChR epsilon (CHRNE E376K) as well as a recently reported CMS mutation affecting this domain (CHRNE N436del) had no impact on cluster formation. CHRND mutations are a rare cause for CMS but should be considered in patients with a severe, early onset disease form, clinically resembling a rapsyn phenotype with recurrent episodic apnoeas. Our results suggest that impairment of AChR-rapsyn co-clustering--a well-known molecular mechanism for rapsyn mutations--could also result from mutations in the delta subunit. Introduction of the same mutation in the epsilon subunit had no effect on AChR clustering indicating a special role of the delta subunit in AChR-rapsyn interactions.

Distinct phenotypes of congenital acetylcholine receptor deficiency

<h2>Abstract</h2> We contrast the phenotypes associated with hereditary acetylcholine receptor deficiency arising from mutations in either the acetylcholine receptor ε subunit or the endplate acetylcholine receptor clustering protein rapsyn. Mutational screening was performed by amplification of promoter and coding regions by PCR and direct DNA sequencing. We identified mutations in 37 acetylcholine receptor deficiency patients; 18 had acetylcholine receptor-ε mutations, 19 had rapsyn mutations. Mutated acetylcholine receptor-ε associated with bulbar symptoms, ptosis and ophthalmoplegia at birth, and generalized weakness. Mutated rapsyn caused either an <i>early onset</i> (rapsyn-EO) <i>or late onset</i> (rapsyn-LO) phenotype. Rapsyn-EO associated with arthrogryposis and life-threatening exacerbations during early childhood. Rapsyn-LO presented with limb weakness in adolescence or adulthood resembling seronegative myasthenia gravis. Awareness of distinct phenotypic features of acetylcholine receptor deficiency resulting from acetylcholine receptor-ε or rapsyn mutations should facilitate targeted genetic diagnosis, avoid inappropriate immunological therapy and, in some infants, prompt the rapid introduction of treatment that could be life saving.

Distinct phenotypes of congenital acetylcholine receptor deficiency

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

Electrophysiological and morphological characterization of a case of autosomal recessive congenital myasthenic syndrome with acetylcholine receptor deficiency due to a N88K rapsyn homozygous mutation

Congenital myasthenic syndromes are rare heterogeneous hereditary disorders, which lead to defective neuromuscular transmission resulting in fatigable muscle weakness. Post-synaptic congenital myasthenic syndromes are caused by acetylcholine receptor kinetic abnormalities or by acetylcholine receptor deficiency. Most of the congenital myasthenic syndromes with acetylcholine receptor deficiency are due to mutations in acetylcholine receptor subunit genes. Some have recently been attributed to mutations in the rapsyn gene. Here, we report the case of a 28-year-old French congenital myasthenic syndrome patient who had mild diplopia and fatigability from the age of 5 years. His muscle biopsy revealed a marked reduction in rapsyn and acetylcholine receptor at neuromuscular junctions together with a simplification of the subneural apparatus structure. In this patient, we excluded mutations in the acetylcholine receptor subunit genes and identified the homozygous N88K rapsyn mutation, which has already been shown by cell expression to impair rapsyn and acetylcholine receptor aggregation at the neuromuscular junction. The detection of the N88K mutation at the heterozygous state in five of 300 unrelated control subjects shows that this mutation is not infrequent in the healthy population. Electrophysiological measurements on biopsied intercostal muscle from this patient showed that his rapsyn mutation-induced fatigable weakness is expressed not only in a diminution in acetylcholine receptor membrane density but also in a decline of endplate potentials evoked at low frequency.

Rapsyn mutations in hereditary myasthenia: distinct early- and late-onset phenotypes.

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