Doi:10.1016/j.nmd.2005.05.00

Neuromuscular Disorders 15 (2005) 498-512 126th International Workshop: Congenital Myasthenic Syndromes, 24-26 September 2004, Naarden, The Netherlands David Beesona, Daniel Hantai¨b, Hanns Lochmu¨llerc,*, Andrew G. Engeld aNeurosciences Group, Weatherall Institute of Molecular Medicine, The John Radcliff, Oxford, UK bINSERM U582, Institut de Myologie, Hoˆpital de la Salpeˆtrie`re, Paris, France cDepartment of Neurology, Friedrich-Baur-Institute, Ludwig-Maximilians-University of Munich, Munich, Germany dDepartment of Neurology, Mayo Clinic, Rochester, MN, USA Keywords: Slow-channel syndrome; Fast-channel syndrome; Congenital Myasthenic Syndromes The ENMC hosted a group of 18 experts on Congenital approaches towards understanding CMS. Agreement was Myasthenic Syndromes (CMS). CMS are inherited dis- reached on the usefulness of coordinated research into orders in which the safety margin of the neuromuscular genetic causes of CMS, phenotype-genotype correlations, transmission is compromised by one or more specific mechanism(s). CMS are caused by various genetic defects.
The objectives of the workshop included progress indeciphering the molecular basis of CMS (sessions 1-4) 1. Session 1: Introduction to CMS and the and clinical conclusions for epidemiology, diagnosis and therapy (sessions 5-7). To date, genes known to cause CMSif mutated are the presynaptic choline acetyltransferase geneCHAT, the gene COLQ encoding the synaptic protein ColQ, 1.1. Functional organization of the neuromuscular the genes encoding the different subunits of the postsynaptic acetylcholine receptor (CHRNA1, CHRNB1, CHRND, The functional organization of the NMJ was introduced CHRNE), the genes for the postsynaptic proteins rapsyn by C. Slater. Structural features of the NMJ have an (RAPSN), muscle-specific receptor tyrosine kinase (MUSK) important influence on its functional efficacy. On the and the postsynaptic sodium channel (SCN4A). Since the presynaptic side, junctions from a variety of species release last ENMC workshop on CMS in October 1999, four novel quite different numbers of acetylcholine (ACh) quanta, and CMS genes have been identified, namely CHAT, RAPSN, this seems to be related to their size. There is thus a relative SCN4A and MUSK. As a consequence, several new patients constancy of quanta released per unit area of synaptic presenting with varying phenotypes of CMS have been contact. For a small synaptic bouton 2-3 mm in diameter, described worldwide. In particular, mutations in RAPSN and about 1 quantum is released per nerve impulse. Human CHAT turned out to be of high clinical relevance, on one NMJs are relatively small and therefore release fewer hand because of their apparent worldwide frequency, on the quanta than some other species. On the postsynaptic side, a other hand because of their specific clinical phenotype with single quantum exerts its effect over a region about 1 mm in the occurrence of sudden apneic episodes. Furthermore, diameter. If more than one quantum were released by a considerable progress has been made using a variety of single bouton at a human NMJ, their postsynaptic domainswould overlap and there would be a loss of efficacy.
* Corresponding author. Tel.: C49 89 2180 78180; fax: C49 89 2180 Voltage-gated sodium channels play a central part in converting the local depolarization of the endplate potential E-mail address: hanns.lochmueller@med.uni-muenchen.de (H. Lochmu¨ller).
into a propagated action potential that can activatecontraction. These channels are concentrated in the folds, 0960-8966/$ - see front matter q 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.nmd.2005.05.001 which lower the effective threshold for action potential D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 generation and, in a sense, amplify the effects of each 2. Session 2: Update on genes previously linked to quantum of ACh. Human NMJs have extensive folds, which may help to counteract the relatively small amount oftransmitter released from the nerve. NMJs are capable of 2.1. Mutations of AChR subunit genes (CHRNA1, extensive adaptive plasticity, seen in old age, after muscle damage, and after botulinum intoxication. This allows theformation of new synaptic contacts, assisting the recovery Mutations involving subunits of acetylcholine receptor of neuromuscular transmission after it has been impaired.
(AChR) fall into two major classes: kinetic mutations with The extensive plasticity of normal NMJs raises an important or without minor acetylcholine receptor deficiency, and question: why similar plasticity does not allow the recovery low-expressor mutations with or without minor kinetic effects. The kinetic mutations fall into two classes accordingto whether they cause slow-channel or fast-channel syndromes. The two syndromes have reciprocal propertiesbut there are some exceptions in the pattern of inheritance Congenital myasthenic syndromes are currently classi- fied as presynaptic, synaptic basal lamina-associated, andpostsynaptic (The percentage of patients having different types of CMS shown in is based on 205 The name slow-channel syndrome originates from the index patients investigated at the Mayo Clinic. One hundred abnormally slow decay of synaptic currents caused by and eight patients of these had intercostal muscle biopsies abnormally prolonged opening events of the AChR channel.
that correlated in vitro parameters of neuromuscular The prolonged EP potential outlasts the refractory period of transmission, ultrastructural and cytochemical studies of the muscle fiber action potential so that single nerve stimuli endplate (EPs), and genetic analysis; 97 were investigated evoke a characteristic repetitive compound muscle action using DNA isolated from blood. Similar observations have potential (CMAP). The prolonged as well as spontaneous been made in three other referral centers (Paris, Oxford, channel openings overload the postsynaptic region with Munich), where classification is mainly based on genetic cations, including calcium, to cause an EP myopathy results. indicates that the most frequent CMSs are manifested by degeneration of the junctional folds, loss of postsynaptic and the least frequent ones are presynaptic in AChR from the folds, widening of the synaptic space to all centers. This classification is useful but still incomplete alter EP geometry, and vacuolar change and apoptosis under because additional types of CMS likely exist, and because in the EPs. The safety margin of neuromuscular transmission is at least one type of CMS, namely limb-girdle CMS, the site compromised by AChR loss, staircase summation of Table 1Classification of the CMS based on site of defect Presynaptic defectsCholine acetyltransferase deficiencyc Paucity of synaptic vesicles and reduced quantal release Synaptic basal lamina-associated defectsEndplate AChE deficiencyc Postsynaptic defectsPrimary kinetic abnormality of AChR with/without AChR deficiencyc AChR deficiency with/without kinetic abnormalityc Classification based on cohort of CMS patients investigated at the Mayo Clinic between 1988 and 2004. AChE, acetylcholinesterase; AChR, acetylcholinereceptor; ChAT, choline acetyltransferase; MuSK, muscle-specific receptor tyrosine kinase.
a No intercostal muscle biopsies and patch-clamp analyses available.
b Based upon functional studies of identified gene mutations.
D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 negative effect. We believe that the dominant-negative effect is due to the mutation residing close to the ion-channel gate and thus results in severely reduced ion-channel function in around 75% of AChR. As a consequence neuromuscular transmission is mediated through the remaining 25% of functional receptors. In most cases, the fast-channel mutation in one allele is accompanied by a null mutation in the second allele so that the fast-channel mutation determines the phenotype. A single report describes two heteroallelic fast-channel mutations (eD175N and eN182Y) in one patient ).
The dE59K mutation is of further interest as it results in multiple joint contractures at birth The aV132L mutation in the signature cys-loop and positioned near the M2-M3 linker markedly reduces affinity for ACh and CMS with severe EP AChR deficiency result from different types of homozygous or more frequently fromheterozygous recessive mutations in AChR subunit genes.
b, channel opening rate; a, channel closing rate.
These mutations are concentrated in the 3-subunit. There are Recessive inheritance can occur with small gain of function (e.g.
eP245L), or for unknown reasons (e.g. eL78P ).
two possible reasons for this: (1) expression of the fetal type b The F256L fast-channel mutation in the a-subunit of the M2 domain g-subunit, although at a low level, can partially compensate for absence of the 3-subunit whereas patients harboring c Different combinations of mechanisms operate in the individual slow- null mutations in non-e-subunit genes might not survive for lack of a substituting subunit; (2) the gene encoding the depolarization block at physiologic rates of stimulation, and 3-subunit, and especially exons coding for the long an increased propensity of the receptor to become cytoplasmic loop, has a high GC content that likely Not fewer than 18 slow-channel mutations have been Different types of recessive low-expressor mutations have been identified. Some cause premature termination of reported to date (see Gene Table in Neuromuscular the translational chain; these mutations are frameshifting Disorders and a recent review The slow-channel occur at a splice site , or produce a stop codon syndrome results from 'gain of function' mutations that directly. An important mutation in this group is the cause prolonged activations of the AChR ion channel either 1369delG in the e-subunit that results in loss of a C- by enhancing affinity for ACh, gating efficiency, or both.
terminal cysteine, C470, crucial to both maturation and Typically the syndrome shows dominant inheritance.
surface expression of the adult receptor Thus any Conversely, AChR deficiency or fast-channel mutations mutation that truncates the e-subunit upstream of C470 is cause a 'loss of function' and typically show recessive predicted to inhibit expression of the e-subunit.
inheritance. However, there are some exceptions. Analysis A second type of recessive mutations are point mutations of kinships harboring the slow-channel mutation 3L221F in the Ets binding site, or N-box, of the promoter region of shows there is variable penetrance with respect to the e-subunit gene: e-154G/A , e-155G/A , and expression of myasthenic symptoms, and in a consangui- e-156C/T. The N-box represents the end point of neous kinship harboring the slow-channel mutation 3L78P, a signaling cascade driven by neuregulin through ErbB clinical symptoms were only apparent in individuals receptors. ErbB receptors phosphorylate mitogen-activated In addition to the above mutations, there are also missense mutations in a signal peptide region (eG-8R and The name fast-channel syndrome originates from eV-13D ), and missense mutations involving residues abnormally fast decay of the synaptic response caused by essential for assembly of the pentameric receptor. Finally, abnormally brief channel opening events due to decreased some missense mutations causing severe AChR deficiency affinity for ACh, decreased gating efficiency, or because they corrupt the fidelity of gating. Fast-channel For a full list of AChR subunit gene mutations and the mutations cause loss of function and are typically recessive, appropriate references, the reader is referred to a recently but an a256L mutation in the M2 domain has a dominant D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 AChE deficiency do not prevent expression of asymmetricforms of the enzyme in COS cells Subsequently, three In the absence of AChE, ACh disappears from the of these mutations (D342E, Q371X, R410Q) were shown to synaptic cleft slowly and only after activating a series of generate insertion-incompetent asymmetric AChE. The AChRs in tandem, thereby prolonging the duration of the nucleotide change predicting E415G was shown to cause synaptic current. As in the slow-channel syndrome, the skipping of the exon in which it is located and a frameshift prolonged EP potential evokes a repetitive CMAP, but instead of E415G. The reason why C444Y fails to insert unlike in the slow-channel syndrome, the AChR channel into the mammalian synaptic basal lamina is still not openings are of normal length. As in the slow-channel syndrome, the prolonged EP currents instigate an EPmyopathy. Concomitantly, and attesting to trophic inter-actions at the synapse, quantal release by nerve impulse is curtailed by smallness of the nerve terminals and theirencasement by Schwann cells. The safety margin of 3.1. Mutations in choline acetyl transferase (CHAT) transmission is compromised by the reduced quantalrelease, the EP myopathy, and depolarization block and This CMS is associated with episodes of severe desensitization of AChR during physiologic activity.
respiratory distress and bulbar weakness leading to apnea.
The endplate species of AChE is a heteromeric These attacks are precipitated by infections, fever, excite- asymmetric enzyme composed of 1, 2, or 3 homotetramers ment, or no known cause and occur against a background of of globular catalytic subunits (AChET encoded by ACHET) variable interictal myasthenic symptoms. In some patients, attached to a triple-stranded collagenic tail (ColQ, encoded the disease presents at birth with respiratory distress and by COLQ). ColQ has a N-terminal proline-rich region apnea that improves but is followed by apneic attacks later in attachment domain (PRAD), a collagenic central domain, life; other patients are normal at birth but develop apneic and a globular C-terminal region enriched in charged attacks during infancy or childhood. Most apneic attacks residues and cysteines. Each ColQ strand can bind an resolve rapidly but some patients remain respirator-depen- AChET tetramer to its PRAD giving rise to A4, A8, and A12 dent for days or even weeks Between attacks, 2-Hz species of asymmetric AChE. Two groups of charged stimulation of rested muscles may not elicit a decremental residues in the collagen domain (heparan sulfate proteogly- EMG response, but a decremental response appears after a can binding domains, or HSPBD) plus other residues in the conditioning train of 10-Hz stimuli for 5 min. The miniature C-terminal region assure that the asymmetric EP potential and the EP potential are normal in rested muscle enzyme is inserted into the synaptic basal lamina. The C- but decrease abnormally during 10-Hz stimulation for 5 min, terminal region is also required for initiating the triple and then recover slowly over the next 10-15 min while the helical assembly of ColQ that proceeds from a C- to a N- quantal content of the EP potential remains stable. A terminal direction in a zipper-like manner. Expression of defect in neuromuscular transmission be induced by globular and asymmetric forms of AChE in muscle or in increasing neuronal impulse flow has pointed to a defect in COS cells transfected with ACHET cDNA plus COLQ vesicular packaging or resynthesis of ACh and this, in cDNA is readily monitored by density gradient centrifu- combination with the clinical history, lead to discovery of 10 gation of muscle or COS cell extracts. The density gradient recessive mutations in CHAT in five CMS patients. One profiles reveal distinct peaks corresponding to globular mutation caused a frameshift and nine were missense.
AChET unattached to the collagenic tail and to the A4, A8, Kinetic studies of the nine bacterially expressed missense mutants showed that one lacked catalytic activity and eight In 1998, human COLQ cDNA was cloned, the genomic others significantly impaired the catalytic efficiency of the structure of COLQ determined, and the molecular basis of enzyme. Five additional CHAT mutations were endplate AChE deficiency traced to recessive mutations in recently detected in CMS patients with unexpected episodes COLQ. Twenty-four COLQ mutations in 25 kinships have of sudden apnea. The identified mutations are listed in the been identified to date (see recent reviews and Gene Gene Table for CMS in Neuromuscular Disorders.
Table in Neuromuscular Disorders ). The mutations are No ChAT-deficient patients reported to date was noted to of three major types: (i) PRAD mutations prevent attach- have significant symptoms referable to the central or ment of AChET to ColQ; (ii) collagen domain mutations autonomic nervous system. The reason for this is not produce a short, single-stranded ColQ that binds a single known, but a likely explanation is that ChAT is rate limiting AChET tetramer and is insertion-incompetent; (iii) C- for ACh synthesis at the nerve-muscle synapse but not at terminal mutations hinder the triple helical assembly of other cholinergic synapses. However, apneic episodes in the collagen domain or produce an asymmetric species of infants are sometimes difficult to distinguish from epileptic AChE that is insertion-incompetent, or both.
seizures, and prolonged hypoxia may result in develop- Some mutations in the C-terminal domain of ColQ mental delay and permanent brain damage. Impor- (D342E, Q371X, R410Q, E45G, and C444Y) causing EP tantly, patients with rapsyn deficiency or Na-channel D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 myasthenia can also experience sudden respiratory crises, but have different EMG and in vitro electrophysiologic Features of 29 patients with rapsyn deficiency Mild course and strong facial deformities with homozygous E-box mutations in Near-Eastern Jews.
Increased frequency of arthrogryposis with truncatingmutations. No phenotype correlation for othermutations Rapsyn, under the influence of agrin and MuSK, plays a At birth, 17; early infancy, 8; childhood, 2; third critical role in concentrating AChR in the postsynaptic membrane and linking it to the subsynaptic cytoskeleton Relatively mild (20-50% of normal mean) through dystroglycan. Rapsyn carries a myristoylation Arthrogryposis at birth, 7; transient torticollis at birth, signal at the N-terminus required for membrane association, 1; facial deformities, 9 (8 carry E-box mutation) has seven tetratrico peptide repeats (TPRs) that subserve Worsening in 15, including respiratory insufficiency self-association, a coiled-coil domain that interacts with ACh, a RING-H2 domain that binds to the cytoplasmic All respond to AChE inhibitor; 3 derived further domains of b-dystroglycan and mediates the MuSK-induced phosphorylation of AChR and a serine phos-phorylation site at codon 406. Transcription of rapsyn inmuscle is under the control of helix-loop-helix myogenic with arthrogryposis had truncating mutations, but among 22 determination factors that bind to the cis-acting E-box patients without arthrogryposis only one had a truncating mutation, indicating a significant association between The clue to discovery of mutations in RAPSN came from truncating mutations and arthrogryposis (P!0.001 by CMS patients with demonstrated EP AChR deficiency who Fisher test). This conclusion also holds when the 13 cases carried no mutation in any subunit of AChR. Muscle investigated by Burke and co-workers are pooled with the biopsy reveals a marked reduction in rapsyn and AChR at neuromuscular junctions together with a simplification ofthe subneural apparatus structure. Electrophysiological 3.3. Sodium-channel myasthenia-mutations in SCN4A measurements on biopsied intercostal muscle showed thathis rapsyn mutation-induced fatigable weakness is This CMS is the only one in which the reduced safety expressed not only in a diminution in acetylcholine receptor margin of neuromuscular transmission occurs in the setting membrane density but also in a decline of endplate of a normal endplate potential. One patient with this type potentials evoked at low frequency No fewer than 21 of CMS has been observed to date This was the case mutations in rapsyn have been identified to date (see Gene of a 20-year-old normokalemic woman with eyelid ptosis, Table for CMS in Neuromuscular Disorders and a recent marked generalized fatigable weakness, and recurrent review RAPSN mutations are likely one of the most attacks of respiratory and bulbar paralysis since birth that common causes for CMS in patients of Indo-European caused anoxic brain injury. Nerve stimulation at physio- ethnic origin All patients with mutations in the logic rates rapidly decremented the CMAP. Intercostal translated region of RAPSN carry the N88K mutation in at muscle studies revealed no abnormality of the resting least one allele; other mutations in the translated region are membrane potential, evoked quantal release, synaptic dispersed over different rapsyn domains. There is evidence potentials, AChR channel kinetics, or endplate ultrastruc- for an ancient Indo-European founder for N88K but ture, but endplate potentials depolarizing the resting not all patients with the N88K mutation carry the same membrane potential to K40 mV failed to excite action haplotype. Recently, a patient with a chromosomal potentials. This clue pointed to a defect in the action microdeletion of the RAPSN gene has been reported that potential mechanism and to SCN4A encoding the NaV1.4 may be caused by an Alu-mediated unequal homologous skeletal muscle sodium channel. SCN4A harbored two recombination Mutations also occur in the E-box of the heteroallelic mutations involving conserved residues not RAPSN promoter. One of these mutations, -38A/G, was present in 400 normal alleles: S246L in the S4/S5 observed at homozygosity and shown to arise from a cytoplasmic linker in domain I, and V1442E in the S3/S4 common founder in Near-Eastern Jews with marked jaw and extracellular linker in domain IV. Expression studies on the other facial malformations. Another E-box mutation, observed mutations in HEK cells revealed that V1442E -27C/G, was heterozygous with the N88K mutation Na-channel showed marked enhancement of fast inacti- summarizes clinical features of 29 patients with vation close to the resting potential, and enhanced user- rapsyn mutations investigated at the Mayo Clinic. In this dependent inactivation on high frequency stimulation; series, as well as in the series reported by Burke and co- workers , arthrogryposis was common in patients suggesting that it is a benign polymorphism. Nav1.4 presenting at birth. In the Mayo series, 7 of the 17 patients expression at the EPs and over the sarcolemma was normal presenting at birth had arthrogryposis. Five of the seven D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 The inheritance pattern for this CMS remains uncertain.
agrin that lacks this activity. In a first part, Ruegg presented The more severe V1442E mutation may be dominant, but experiments where gene knock-down techniques (RNA this could not be proved since this mutation derived from interference) were used to study the role of MuSK in the the patient's father who could not be evaluated, and because maintenance of the NMJ In muscle electroporated with V1442E was observed only in combination with the plasmids encoding short hairpin RNA (shRNA) to MuSK, heteroallelic S246L mutation. The S246L mutation alone NMJs were frequently disassembled as evidenced by the was clinically silent; therefore, it is either a rare poly- fragmentation of postsynaptic acetylcholine receptor morphism with detectable biophysical changes but no (AChR) clusters. In severe cases, postsynaptic nerve clinical or EMG phenotype, or is a recessive mutation.
terminals began to sprout indicative of overt denervationof skeletal muscle. No such phenomena were observed in 3.4. Mutations in muscle-specific kinase (MUSK) skeletal muscle electroporated with shRNA encoding thecontrol gene CD4. Based on these results, Ruegg raised the The first case of a CMS due to mutations in the gene hypothesis that a similar disassembly of NMJs may occur in encoding the muscle-specific receptor tyrosine kinase patients suffering from CMS due to mutations in MuSK (see (MuSK) was presented. Two heteroallelic mutations, a above). In a second part, Ruegg presented a project where frameshift mutation (c.220insC) and a missense mutation transcripts were identified whose expression is altered (V790M), were identified. In vitro and in vivo expression during the formation of postsynaptic structures in skeletal experiments were performed using mutant MuSK reprodu- muscle. To this end, recombinant 'neural' agrin was injected cing the human mutations. The frameshift mutation led to into soleus muscle. Seven days later, many postsynaptic the absence of MuSK expression. The missense mutation structures were formed in non-synaptic regions that were did not affect MuSK catalytic kinase activity but diminished indistinguishable from postsynaptic regions of NMJs.
expression and stability of MuSK leading to decreased Using the Affymetrix GeneChip technology, genes were agrin-dependent AChR aggregation, a critical step in the identified that were upregulated at these ectopic postsyn- formation of the neuromuscular junction. In electroporated aptic structures. Among them was the AChR-3-subunit that mouse muscle, overexpression of the missense mutation is known to be regulated by innervation, and early growth induced, within a week, a phenotype similar to the patient response-1 (Egr-1) that has recently been shown to bind to a muscle biopsy: a severe decrease in synaptic AChR and an promoter element in the AChR-3 gene and to contribute to aberrant axonal outgrowth. Furthermore, the question expression of this subunit at NMJs. Other genes was discussed whether polymorphisms of the MUSK gene identified belong to the mitogen-activated protein (MAP) may predispose certain individuals to develop autoimmune kinase pathway. Ruegg presented evidence that this path- myasthenia gravis and whether anti-MuSK antibodies way might indeed be important for NMJ formation and suggested that some of the genes identified in this screenmight be new candidates involved in CMS. Finally, Rueggpresented evidence that the transgenic overexpression of a 4. Session 4: New phenotypes and candidate genes mini-gene encoding 'non-neuronal' agrin, which has beenshown to ameliorate disease in an animal model of MDC1A Presently, genetic defects in the above described genes , also influences the NMJ. The NMJs of such mini-agrin can be identified in more than 50% of all CMS kinships.
transgenic mice resemble the structure of immature NMJs However, there are additional clinical phenotypes (limb- indicated by the rather diffuse appearance of postsynaptic girdle myasthenia; presynaptic CMS) where the underlying AChRs. This appearance remains for several months and genetic defects remained elusive, so far. New clues for CMS interestingly, concentrations of AChRs remain high at sites candidate genes may come from recently described animal where presynaptic nerve terminals have retracted as revealed by electron microscopy. In addition, the musclefibers in nerve-free regions with high concentration of 4.1. Genes at the neuromuscular synapse and animal AChRs also had postsynaptic folds. The same synaptic phenotype was also observed in mice that overexpressed adesigned mini-gene encoding a fusion protein between the M. Ruegg summarized the developmental steps required laminin-binding part of agrin and the region in perlecan that to assemble a functional neuromuscular junction (NMJ).
binds to dystroglycan. These studies thus indicate that tight The emphasis of his presentations was on the role of the linkage of extracellular matrix to dystroglycan can serve as extracellular matrix molecule agrin that, by activating the an anchor to stabilize postsynaptic structures. It remains to receptor tyrosine kinase MuSK, plays a critical role in the be seen whether any of the factors used in these studies organization of postsynaptic differentiation. He reminded could also be useful in stabilizing NMJs in CMS patients the audience of the fact that agrin undergoes an alternative and thereby prevent some of the deterioration.
mRNA splicing to generate 'neural' agrin that is active in L. Schaeffer presented experimental evidence that ErbB4 the induction of postsynaptic structures, and 'non-neuronal' is required at the NMJ and might be a good candidate to D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 cause CMS. Neuregulins are concentrated at the NMJ, more than 9 months without severe deterioration, but where they activate synaptic gene expression upon binding display fatigable muscle weakness, reduced miniature to their postsynaptic membrane tyrosine kinase receptors.
endplate potentials and endplate currents, reduced endplate The neuregulin receptor family comprises three members, AChR number and altered endplate morphology. The results ErbB-2, -3 or -4, which homo- and heterodimerize. In suggest that up-regulation of g-subunit expression is skeletal muscle, ErbB-2, -3 and -4 are all accumulated at the potentially a beneficial therapeutic strategy for severe NMJ. However, the composition of the functional neur- cases of AChR deficiency due to 3-subunit mutations.
egulin receptor remains unknown since no ErbB isoform Moreover, these transgenic mice will be helpful in the study was shown to be essential for synapse-specific gene of present and future treatments of AChR deficiency and expression yet. Schaeffer and coworkers developed a technique allowing muscle-specific inhibition of geneexpression by combining the RNAi and electroporation 4.2. Presynaptic CMS not caused by ChAT mutations technologies. When electroporated into skeletal muscle,RNA polymerase III directed siRNA expression vectors M. Milone reported that except for the CMS caused by trigger a long-term inhibition of ErbB2 and ErbB4 mutations in CHAT, presynaptic CMSs have not been well expression. These results suggest a direct demonstration of characterized. In few CMS patients, the safety margin of the requirement of the tyrosine kinase receptor ErbB4 in the neuromuscular transmission is compromised by decrease in regulation of synaptic gene expression at the NMJ. In the number of ACh quanta released by nerve impulse (m). A addition, the analysis of synaptic markers in a CMS patient defect in the quantal release mechanisms can stem from a revealed a strong decrease of ErbB4 accumulation at the reduced number of quanta (n) available for immediate NMJ, whereas the other markers were unaffected.
release or a reduced probability (P) of quantal release.
Altogether these results suggest that defects in ErbB4accumulation at the NMJ could cause CMS.
J. Koenig presented data derived from denervation/rein- A Lambert-Eaton like CMS was described in 1987 by nervation experiments and gene inactivation in rodents that Bady and colleagues. A second patient was studied at the affect acetylcholine release and neuromuscular junction Mayo Clinic. No mutations were found in the gene encoding (NMJ) structure. First, Koenig compared ColQ knockout the a1-subunit of the P/Q-type calcium channel (CACNA1A) mice (ColQ K/K) and COLQ mutations in human CMS. In normal mice, inhibition of acetyl- and butyrylcholines-terases modifies acetylcholine release The acetyl- 4.2.2. Presynaptic CMS associated with paucity choline release decreases in ColQ K/K mice lacking acetyl- and butyrylcholinesterases as in CMS One patient with this form of CMS had been observed at Moreover, similar synaptic gutter abnormalities are the Mayo Clinic and was reported in 1990. She was a observed in ColQ K/K mouse as in CMS patients 23-year-old woman with generalized myasthenic symptoms Second, Koenig compared AChR 3-subunit knockout since birth and a decremental EMG response that did not mice (3 -subunit K/K) with 3-subunit gene (CHRNE) facilitate at higher frequencies of stimulation. In vitro mutations in human CMS: In the 3-subunit K/K mouse, the electrophysiologic studies showed normal amplitude amplitude and decay time of miniature endplate potentials MEPPs, but an abnormally low m due to low n.
(MEPPs) are diminished. The g-subunit persists at adult Ultrastructural studies showed a decrease of synaptic NMJs and the synaptic gutters are abnormal. Similar vesicle density that was proportionate to the decrease in n.
features were observed in a CMS patient who carries twoframeshift mutations in CHRNE.
4.2.3. Presynaptic CMS with reduced quantal release Very recently, an animal model of AChR deficiency syndrome has been generated. Many cases of AChR Maselli and colleagues reported three unrelated patients deficiency are due to homozygous or compound hetero- with myasthenic symptoms and mild ataxia. One patient zygous null mutations of the 3-subunit gene. It appears also had nystagmus. All three had decremental EMG likely that in these cases neuromuscular transmission is response at 2 Hz and showed no facilitation at high- partially rescued through AChR pentamers harboring the g frequency nerve stimulation. They had decreased m due to (fetal)-subunit. In contrast to humans, mice with ablated 3- low n, and one also had a slightly low P. No mutations subunit alleles die between 8 and 14 weeks after birth. To provide a better animal model of AChR deficiency Milone and Engel studied two patients, 7 and 48 years of syndrome, transgenic mice that constitutively express the age (Pts 1 and 2) who had severe generalized weakness human AChR g-subunit were generated, and the g-subunit since early infancy. Both were wheelchair-bound and had transgene bred into mice with an AChR 3-subunit gene multiple skeletal deformities; Pt 1 cannot speak or swallow K/K background. These mice show striking similarities to and was respirator-dependent. They had a 30-50% EMG human AChR deficiency syndrome. They have survived for decrement on 2-Hz stimulation and no facilitation at higher D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 stimulation frequencies. Both responded initially and then samples from vastus lateralis from eight of these patients.
became refractory to pyridostigmine and 3,4-DAP. EP In these samples, muscle fiber diameter was slightly studies revealed no AChR or AChE deficiency. The quantal increased and one of the eight patients had tubular content of the EP potential (m) was markedly reduced due to aggregates. Compared to controls, they had approximately low n and P. On electron microscopy, most EPs were normal quantal size but the quantal content was reduced normal in Pt 1; in Pt 2, many nerve terminals were small and roughly in proportion to a reduction in the overall area of were encased by Schwann cells, and some postsynaptic synaptic contact of the NMJ. They also had reduced regions were degenerating or simplified. Direct sequencing postsynaptic folding. The reductions in quantal release of genes encoding proteins involved in synaptic vesicle and postsynaptic folding would both tend to lower the safety docking and fusion (syntaxin-1, SNAP-25, synaptobrevin, factor of neuromuscular transmission and probably account complexin, and RAB3A), in regulation of vesicle exocytosis for the clinical symptoms. Investigations showed no (Munc-18, synaptotagmin), and in vesicle distribution abnormality of function of AChR or AChE. DNA analysis (synapsin-1) revealed no mutations. No mutations were failed to detect mutations in the genes for AChR subunits found in the a1-subunit of the P/Q calcium channel, and rapsyn. It seems that mutations of as yet unidentified cysteine-string protein, the NaC-Ca2C exchanger, the genes cause an impairment of neuromuscular transmission catalytic subunit of AChE, b2-laminin, NCAM, and primarily as a consequence of altered NMJ structure. Most neurotrophin 3. The EMG in these patients gave no of the patients responded positively to treatment with indication that the defect was presynaptic. The genetic anticholinesterases, although in some the benefits of this basis of the two syndromes remains elusive.
treatment were not maintained on a long-term basis.
5. Session 5: Genotype-phenotype correlations C. Rodolico summarized current knowledge about a clinically distinct form of CMS. Familial LGM is likely tobe inherited by autosomal recessive traits, but gene defects 5.1. Genotype-phenotype correlations, clinical clues remain to be identified. Clinically, LGM is characterized byproximal weakness and wasting starting in childhood. In U. Schara reported on clinical findings of 17 CMS- contrast to other forms of CMS, extra-ocular muscles are patients (2 female, 15 male) out of 15 unrelated families; spared. CK levels are often increased. Electrophysiological age at examination varied from 3 to 25 years. Mutations in studies are consistent with a post-synaptic NMJ defect.
CHRNE (nZ6), CHRND (nZ1), RAPSN (nZ3) and CHAT Patients generally respond to acetylcholinesterase inhibi- (nZ1) as well as in COLQ (nZ1) were disclosed. Patients tors. Muscle biopsy is characterized by the presence of with mutations in CHRNE showed pronounced facial tubular aggregates. Tubular aggregates, derived from the symptoms, no or only mild generalized skeletal muscle sarcoplasmic reticulum, are thought to represent an adaptive involvement and absence of crises; they improved to mechanism aiming at regulating an increased level of pyridostigmine bromide therapy, but often symptoms did calcium, in order to prevent muscle fibers from hyper- not resolve entirely. In contrast, the patient with mutations contraction and necrosis. Rodolico observed five cases with in CHRND showed a more generalized involvement of the familial form of LGM in Southern Italy over a 15-year- skeletal muscles and crises including respiratory insuffi- period. Each patient had consanguineous parents All ciency. Symptoms improved to pyridostigmine therapy.
known CMS genes (see above) were excluded by direct or Patients with mutations in RAPSN presented with sudden indirect genetic analysis. In cooperation with Dr R. Krahe episodes including generalized muscle weakness and (Houston, TX) linkage analysis is underway to determine hypotonia as well as respiratory insufficiency requiring the location of the LGM gene. Additional LGM families of assisted ventilation or even leading to death (occasionally different ethnic background may be helpful to validate the diagnosed as SIDS). Between crises, the clinical course was often stable; under medication, normal development was C. Slater presented in vitro studies of NMJs from LGM achieved. In general, rapsyn-deficient patients showed a patients in Northern England. Over a period of 20 years, 10 patients with myasthenia with a predominantly limb-girdle J. Verschuuren referred to the clinical observations distribution have been seen. These patients have a very that asymmetric ptosis is most often associated with characteristic 'waddling' gait and little or no muscle acquired and autoimmune myasthenia gravis (MG) in wasting. They lack oculomotor signs and have at most contrast to CMS and other inherited disorders. He and mild facial weakness or ptosis. The severity of their his coworkers studied the diagnostic value of this symptoms typically fluctuates on a time scale of weeks or observation by comparing a group of patients with months. They all show decrementing CMAP during autoimmune MG or Lambert-Eaton myasthenic syn- repetitive stimulation and SFEMG studies reveal increased drome (LEMS) with a group of patients with hereditary jitter and blocking. Slater studied motor point biopsy myopathy. Studying the files of 250 MG or LEMS D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 patients and 83 hereditary myopathy or CMS patients mutations located in either the AChR 3-subunit (82 patients) (dystrophic myotonia, progressive external ophthalmople- or in the endplate AChR clustering protein, rapsyn (30 gia, oculopharyngeal muscle dystrophy, CMS) revealed a patients). Bulbar symptoms, ptosis and ophthalmo- similar frequency of ptosis in both groups (76 and 78%, plegia are prominent as early features associated with respectively). Asymmetry was noted in 80% of the MG mutations in the AChR 3-subunit. Mutated rapsyn, harbour- or LEMS patients and in 30% of the myopathy or CMS ing at least one copy of N88K, may cause either an early patients. To verify these findings, a prospective study onset (rapsyn-EO) or late onset (rapsyn-LO) phenotype was started in which the ptosis was quantified using . Rapsyn-EO associated with arthrogryposis and life- digital photography. So far, 52% of 23 MG patients and threatening exacerbations during early childhood whereas none of 20 myopathy/CMS patients were found to have a rapsyn-LO presented with limb weakness in adolescence or significant asymmetric ptosis. The results of these adulthood resembling AChR-antibody-negative myasthenia ongoing studies suggest that asymmetric ptosis points gravis. Awareness of these distinguishing key features is to an underlying autoimmune disease and less likely clinically important to facilitate targeted genetic analysis towards a hereditary myopathy or CMS.
enabling a more rapid diagnosis and introduction of B. Eymard reported on the experience of the French appropriate treatment. However, more specific genotype- CMS network. He and his coworkers observed patients of phenotype correlations were not observed. For example, two Gypsy families homozygous for the common marked differences in severity occur in patients harbouring CHRNE 1267delG mutation. CMS was very severe in the same CHRNE mutations, and both rapsyn-EO and the first family with episodic apnea in all three affected rapsyn-LO phenotypes, of varying severity, were seen in cases. In the second family, there was one case with patients homozygous for the RAPSN N88K mutation.
intermediate severity and two mild cases. Variable In general, the following genotype-phenotype corre- severity was also found in a family with slow-channel lations were drawn: In the slow-channel syndromes, syndrome (CHRNA1 G153S mutation). With colleagues mutations in the channel domain have more severe from Algeria and Tunisia, Eymard studied 27 CMS- phenotypic consequences than those in the extracellular patients of Northern Africanan origin homozygous for domain, but there are variations in phenotypic expressivity the CHRNE 1293insG mutation. Main features were: between and within kinships harboring the same mutation.
neonatal onset 16/27, within the first year 3/27; In the fast-channel syndromes, the aV132L mutations in the ophthalmoplegia in all patients, bulbar symptoms in signature cys-loop were extremely disabling in that the 15/27; no ventilation required; decrement in 19/27; patient could not speak or swallow or hold her head erect benefit of anti-AChE drugs in all but two cases. Seven and was wheelchair-bound Mutations in the extracellu- patients with COLQ mutations were observed. Age at lar domain of AChR cause moderately severe to severe onset, severity and course were variable; absence of anti- symptoms, while the aV285I mutation in M3 domain is only AChE effect and repetitive motor response were moderately disabling. Among patients with low-expressor constantly found, but oculomotor involvement and slow AChR mutations, those harboring mutations in the e-subunit pupillary response was inconstant. Seven different are generally less affected than those with mutations in mutations, all along the gene, were found in the other subunits, but the spectrum can range from mild to following domains: C terminal (3), collagen domain moderately severe. Patients with low-expressor mutations in (2), PRAD (2). Three were homozygous, four hetero- both alleles of genes encoding non-e-subunits of AChR allelic, five nonsense, two missense. Except for a double are generally severely affected with severe ocular, missense mutation in the anchoring region (Y430S) that bulbar, and respiratory muscle weakness from birth, was associated with the mildest cases, no clear and some survive only with respiratory support and correlation between severity and the localization or the type of mutation was obtained. Features of eight patients Most patients with mutations in COLQ are severely from seven families harboring RAPSN gene mutations disabled from an early age. Some patients with missense were as follows: severe cases (four patients) with bulbar mutations in the C terminal domain of ColQ present later paresis and respiratory failure, all with early onset (foetal and have a milder clinical course. Variations in phenotypic onset with arthrogryposis in two cases, at birth in two expressivity between and within kinships harboring the cases); mild cases (four patients) with late onset (child- same mutation can also occur All patients with hood in three, adulthood in one). N88K mutation was mutations in CHAT are at risk of apneic episodes and sudden present in all patients, homozygous in three mild cases death. The interictal symptoms vary in severity and and heteroallelic in five cases (four severe and one mild); the crises vary in frequency, and neither feature can be AChE inhibitors were effective in all patients.
correlated with the two heterozygous mutations in individ- G. Burke reported on genotype-phenotype correlations ual patients except that one patient with a catalytically in 112 patients with hereditary acetylcholine receptor inefficient mutation on one allele plus a mutation that (AChR) deficiency identified through the Oxford Myasthe- abrogates the catalytic activity of ChAT on the other allele nia Centre. Distinct phenotypes were associated with was more severely affected than the other patients. There are D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 no phenotype correlations for most rapsyn mutations.
However, patients carrying a truncating RAPSN mutation Frequency of mutations in AChR subunits observed in more than twoindependent kinships are at a higher risk of arthrogryposis at birth, and Near-Eastern Jews homozygous for the RAPSN -38A/G E-box mutation have strong facial malformations and a generally 5.2. Frequency of underlying mutations and founder P. Richard reported that CHRNE and RAPSN are the two most frequently mutated genes in recessive CMS investigated by the French network. In CMS-patients Missense resulting in AChR deficiency with/without kinetic abnormality with rapsyn deficiency, the RAPSN N88K mutation was always found either at homozygous state or heteroallelic with another variant. An evaluation of the general population showed a carrier rate of 1% for RAPSN N88K. In accordance with this apparent frequency of healthy mutation carriers, two families with a pseudodo- minant mode of inheritance were identified. The CHRNE 1293insG mutation was found with a rate of 20% in CMS patients originating from Maghreb (especially Algeria and Tunisia). Analysis of a founder effect by haplotyping with microsatellite markers and intragenic polymorphisms showed strong linkage disequilibrium and allowed to define a founder haplotype suggesting anancient founder effect for this mutation.
J. Mu¨ller reported on founder mutations and the 6. Session 6: Diagnostic proceedings-contribution frequency of underlying mutations in 200 CMS patients of muscle biopsy versus molecular genetics and investigated in Munich. In 60% of the patients, an underlying mutation in one of the known CMS genes hasbeen detected. The CHRNE 1267delG mutation is the most P. Richard and J. Koenig reported on the current practice common single CMS mutation and is due to a founder effect of the French CMS network. The diagnostic strategy for in the Gypsy population that can be traced back to the exodus molecular analysis of CMS is based on the association of of the ancestral Gypsy population out of India approximately clinical signs, electromyography and muscle biopsy.
900 years ago. Carrier rates in the general Gypsy Absence of acetylcholinesterase on muscle biopsy as well population are as high as 5% and pseudodominant as a repetitive response on EMG after single stimulation inheritance has been observed Further recurrent orientate the testing of COLQ gene. A dominant mode of mutations were observed in CHRNE (70insG in CMS inheritance or a slow-channel syndrome leads to test patients of Portuguese and Spanish origin; 1293insG in CHRNE and CHRNA1. Recessive forms of postsynaptic North-African, and also in Portuguese and Spanish patients).
CMS are the most frequently encountered, and frequently For the mutation RAPSN N88K, an ancient founder allele clear clues orienting the analysis towards a single gene are was demonstrated by SNP haplotyping of 41 mutated alleles.
missing. Thus, all genes should be tested with a priority for All analyzed alleles share a common 360 kb fragment on CHRNE and RAPSN which are mutated in at least 30% of chromosome 11p11 surrounding the RAPSN gene (core patients. For numerous patients without identified mutation founder allele). All but one patient shares a common 1.4 MB and to orientate towards candidate genes in small families in fragment (extended founder allele). It was hypothesized that which linkage analysis is not feasible, the analysis of the the mutation derives from an ancient Indo-European structure of the NMJ and of the expression of synaptic founder, which would explain that the mutation is widely markers using immunofluorescence may help to propose distributed in Europe and North America, but not in Africa new candidate genes (as it was the case for MuSK).
A.G. Engel and M. Milone reviewed the usefulness of muscle biopsies in the diagnosis of CMS.
mutations that occurred more than twice among kinships Biopsies obtained intact from origin to insertion from investigated in one of the participating centers who carried intercostal or anconeus muscles have served an important purpose in the past. Detailed in vitro analysis of D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 neuromuscular transmission (e.g. amplitudes of the minia- † Some CMS cannot be characterized without mRNA ture EP potentials and currents, parameters of quantal analysis and hence a muscle biopsy. Also, only EP- release, in vitro response to agents that block or facilitate enriched muscle specimens are a good source for neuromuscular transmission, and analysis of currents flow- ing through single AChR channels), cytochemical and † Genetic analysis by itself does not prove EP AChR immunocytochemical localization of EP-specific proteins deficiency unless both mutations can be predicted to (e.g. AChE, AChR, b2-laminin, MuSK, agrin, rapsyn, cause premature termination of the translational chain.
utrophin, and Nav1.4), quantitation of the number of Well designed expression studies give an indication of AChRs per EP, and electron microscopy analysis of the the effect of the mutation on the surface expression of neuromuscular junction defined factors that impaired the AChR in heterologous cells, but again these studies may safety margin of transmission in different forms of CMS.
not correspond to the level of AChR expressed at the EP.
Between 1975 and early 1990s, this type of analysis revealedCMSs caused by AChE deficiency, AChR deficiency, slow- We consider the following guidelines useful in investi- channel kinetics, fast-channel kinetics, and putative defects gating CMS patients when a facility for mutation analysis is in ACh resynthesis. Since 1994, molecular genetic studies identified mutations in the initially recognized syndromesand carefully designed expression studies yielded mechan- 1. Targeted genetic analysis is done if a phenotypic clue istic insights into how pathogenic mutations deranged points to one or limited number of genes. For example, a neuromuscular transmission. A few years later, clues derived repetitive CMAP and refractoriness to AChE inhibitors from correlation of clinical data, EMG tests, and results of point to a mutation in COLQ. Acute respiratory crises or previous mutation analyses opened the door to targeted worsening with a febrile illness suggest mutations in genetic analysis of different types of CMS; consequently, the CHAT or RAPSN; careful EMG studies can give further intercostal biopsy was no longer required to accurately evidence for or against a mutation in CHAT. Arthro- diagnose all CMS patients. Moreover, genetic analysis, when gryposis is a clue for a mutation in RAPSN or CHRND. A successful, opened the door to genetic counseling and mild CMS with mostly ocular findings could be due to a prenatal diagnosis. Still, some syndromes would not have been discovered if the intercostal biopsy approach had been 2. In the absence of phenotypic clues, we first sequence abandoned. It was the combination of EP AChR deficiency CHRNE and also look for the common N88K mutation in identified in the biopsy specimen and absence of mutations in RAPSN, because mutations in these two genes account AChR subunits that resulted in discovery of mutations in rapsyn; it was the presence of a normally depolarizing EP 3. If the clinical and EMG findings point to a slow-channel potential failing to trigger an action potential that leads to syndrome, we first sequence CHRNA1 and then CHRNE, discovery of sodium-channel myasthenia and mutations in SCN4A. Also, patch-clamp recordings from EP AChRs 4. If the above genetic analyses fail to identify a mutation, provided crucial proof that single channel currents are we proceed with the intercostal muscle biopsy studies.
abnormally prolonged in the slow-channel syndrome and areabnormally brief in the fast-channel syndrome; and thatpatients with low-expressor mutations in CHRNE are rescued 7. Session 7: Management and therapeutic options by expression of the fetal form of g-AChR.
Currently, the following points argue in favor of obtaining muscle specimens from some CMS patients: P. Richard commented on the fact that genetic counsel- † The biopsy studies remain essential for the characteriz- ing in CMS is not much developed because of the small ation of previously unrecognized CMS.
number of genotyped families. While most CMS are † Mutation analysis does not identify the kinetic properties inherited in recessive traits, particular attention has to be of novel or previously uncharacterized missense attributed to missense mutations of AChR subunit genes that may be dominant because of their possible kinetic effects.
† Fast-channel syndromes cannot be identified without in Siblings of patients with CHAT and RAPSN mutations vitro electrophysiologic analysis, although they can be should be tested even if not symptomatic for early detection identified if mutation analysis is followed by patch-clamp of mutation carriers that are at risk of life-threatening apneic studies of genetically engineered mutants expressed in crisis. However, many questions remain unclear; is the HEK cells. Expression studies in HEK cells, however, do severity of the disease in an affected child a prediction of the not always consistently reflect AChR expression at the EP.
severity of the mutation? An important inter-familial but † Presynaptic defects, with possible exception of the also intra-familial heterogeneity is observed and more Lambert-Eaton-like CMS, cannot be identified without genotype-phenotype correlation studies are required. Can in vitro electrophysiologic studies.
we consider that all CMS are severe diseases without D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 effective treatment? Is the presymptomatic diagnosis in generally cause very severe myasthenic symptoms with newborns or young adults feasible? Prenatal diagnosis can high fatality during infancy or childhood and marked be undertaken if the mutation involved is identified.
disability in those who reach adulthood.
Furthermore, we have to be aware of consanguineous CMS caused by mutations in RAPSN vary in severity couples and families originating from regions in which even in patients who carry identical mutations. Also, those founder mutations are prevalent. Searching for mutations born with arthrogryposis may eventually turn out to have with a high carrier rate should be done even in the absence mild disease; and either mildly or severely affected patients of consanguinity. The final decision is taken in a consensus are often significantly worsened by intercurrent infections.
way between the geneticist, neurologist and psychologist CMS patients with mutations in CHAT are at a high after examination of the severity of the disease in the family risk of anoxic brain injury or death from respiratory arrest during infancy and childhood, but this can be preventedby prophylactic use of AChE inhibitors. The disease becomes milder and episodes of respiratory distress lessfrequent with age.
B. Eymard summarized the French experience as The single patient with a CMS caused by a sodium- follows. There was a wide diversity of severity whatever channel defect and frequent episodes of apnea survived only be the mutated gene. Mild late onset cases and severe early because apnea monitoring was started in the neonatal onset cases (with severe apnoeic episodes) were found with CMS associated with mutations of RAPSN, COLQ andCHRNE. In several patients with RAPSN and COLQ genemutations, the course of the disease changed drastically: 7.3. Approach to pharmacotherapy and standard very severe initial stage and major improvement later. On the other hand, severe late onset progressive motordeterioration in one patient with COLQ gene mutations AChE inhibitors were efficient in most of B. Eymard's was observed, while disease was moderate in childhood. On cases, except in patients with AChE deficiency and slow- the whole, long-term CMS prognosis is very difficult to channel syndrome. In one slow-channel family, a good assess. In Eymard's experience, two general rules are benefit of Quinidine was observed for weakness. 3,4- fundamental for CMS prognosis and therapy: first to Diaminopyridine (3,4-DAP) was given to 19 patients, 15 of consider the diagnosis of CMS; several patients from the them receiving anti-AChE therapy that was maintained.
French series, initially misdiagnosed as myopathy, were Good response to 3,4-DAP was observed in CMS due to wheelchair-bound before AChE inhibitor administration mutations of the following genes: MUSK (one case), which improved the clinical condition drastically; second to CHRNE (four cases with various mutations), COLQ (one recognize and to manage life-threatening complications, case, without AChE drugs), RAPSN (one case), unknown that may occur rapidly (respiratory crises, swallowing (six cases). For one COLQ-mutated patient, positive effect disturbances) in intensive care units.
of 3,4-DAP was found at age of 26 years, with a clear gain A.G. Engel made the following observations on the natural history of the CMS: although the natural history of J. Palace reported on clinical trials to test the efficacy of each type of CMS can vary greatly between and within adding 3,4-DAP to ongoing treatment regimes in 16 patients kinships, the following generalizations are considered with CMS. In a non-blind myometry-based assessment, 13/14 patients improved on treatment when assessed at 7 The slow-channel syndrome is a progressive disease; days and all four patients in double-blind placebo controlled severe cases present at birth, milder cases present in cross-over study showed a good response. The majority of childhood or even early adulthood. Eventually, weakness patients were already on mestinon having found it improved of cervical muscles causes a head drop and secondary their weakness. Subsequent genetic analysis has identified cervical spondylosis; paraspinal muscle weakness even- 11 CHRNE null mutations, one fast-channel, one normal tually results in scoliosis that requires corrective surgery; sibling, one fast-channel and one slow-channel mutation and progressive ventilatory insufficiency eventually and 4 uncharacterized so far. The slow-channel patient was requires assisted ventilation. Fortunately, the progression suspected prior to genetic analysis because of a negative of the disease can be obviated by therapy (see below).
response to mestinon and became slightly weaker on 3,4- The course of the fast-channel syndromes is highly variable, the phenotypes ranging from either mild or to Six patients with CHRNE null or RAPSN mutations have extremely severe (see above, under Section 2.1.2).
had non-blinded functional strength assessments to compare The majority of low-expressor mutations in CHRNE the effects of mestinon with 3,4-DAP and a combination. A cause clinically mild disease that either progresses slowly or similar degree of improvement was seen with either appears to be stationary. In contrast, low-expressor mestinon or 3,4-DAP alone but a combination produced mutations involving both alleles in other AChR subunits the best clinical effect. The exception was in the treatment D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 of ptosis which appeared much more responsive to mestinon Response to ephedrine was assessed in an unblinded ChAT deficiency: AChE inhibitor; oral prophylaxis, parenteral use in crisis fashion in five patients: three with CHRNE null mutations (who were maintained on mestinon), one slow-channel AChE deficiency: avoid AChE inhibitors; try ephedrineSimple AChR deficiency patients, and two unknown (one with a limb-girdle : AChE inhibitor; 3,4-diaminopyridine also helps in phenotype). A small overall improvement was seen in all Slow-channel CMS: quinidine or fluoxetine (long-lived open channel patients. Two siblings who had CHRNE null mutations showed the best response but this was less than with 3,4- Fast-channel CMS: AChE inhibitor and 3,4-diaminopyridine DAP. Interestingly swallowing time improved most Rapsyn deficiency: AChE inhibitor; some benefit further from 3,4-diaminopyridine Na-channel myasthenia: AChE inhibitor and acetazolamide In addition to the commonly encountered side effects, Limb-girdle myasthenia: AChE inhibitor; some benefit from ephedrine two deaths were noted in two children started on 3,4-DAP Presynaptic CMS: empiric trial of AChE inhibitor, 3,4-diaminopyridine, or who had fast-channel mutations. The clinicians are aware that this could be unrelated to the drug but are cautiousabout using it subsequently in young children and in fast- whilst having only a slight effect on expression of the wild channel patients. Additionally quinidine has been used in type AChR a-subunit. The maximum difference for two families with slow-channel mutations. Of three patients silencing was obtained when the mismatch was located treated in one of these families, two developed the near the center of the siRNA 21-mer sequence in positions 9 prolonged QT intervals at low plasma levels which limited or 10. Selective down-regulation of mutant a-subunit mRNA was obtained for each of four slow-channel syndrome In conclusion, a combined clinical and EMG approaches mutations, aG153S, aS226F, aT254I and aS269I. The to assess the response of CMS patients to pharmacotherapy results highlight the potential of RNAi for selectively () are recommended. The protocol used at the Mayo controlling expression of synaptic proteins, although an Clinic for initial clinical and EMG evaluation of CMS effective and practical delivery system are yet to be devised.
patients is shown in. Agents used for treatment ofthe individual CMS are listed in This Workshop was made possible thanks to the financial Allele-specific silencing by RNAi may be a mechanism support of the European Neuromuscular Centre (ENMC) for future therapy for dominant neurological disorders. The neuromuscular junction is particularly attractive for thestudy of the therapeutic potential of RNAi to modify - Association Franc¸aise contre les Myopathies (France) synaptic transmission. The slow-channel syndrome was used - Deutsche Gesellschaft fu¨r Muskelkranke (Germany) as a model for investigating allele-specific gene silencing in dominant genetic disorder by RNAi. Transfection of - Muscular Dystrophy Campaign (United Kingdom) HEK 293 cells expressing the AChR with short interfering RNA (siRNA) or hairpin RNAi (shRNAi) resulted in down- - Prinses Beatrix Fonds (The Netherlands) regulation of surface 125I-a-bungarotoxin binding. By - Schweizerische Stiftung fu¨r die Erforschung der Mus- designing siRNA that is exactly complementary to the mutant sequence but has a single mismatch with the wild ¨ sterreichische Muskelforschung (Austria) type sequence, siRNAs were generated that selectively - Vereniging Spierziekten Nederland (The Netherlands) down-regulated expression of the mutant mRNA species Table 5Combined clinical and EMG and approach to pharmacotherapy of the CMS - Asociacion Espan˜ola contra las Enfermedades Neuro- Determine strength of selected muscles, arm elevation time, number of times rising from squatting, etcFind muscle showing significant decrement on slow repetitive nerve List of Participants: Prof. David Beeson, Oxford, UK; Dr Georgina Burke, Oxford, UK; Prof. Andrew G. Engel, Leave stimulating and recording electrodes in placeAdminister edrophonium intravenously Rochester, USA; Prof. Bruno Eymard, Paris, France; Prof.
Monitor decremental response at 1 min intervals for 5-10 min Daniel Hantai¨, Paris, France; Prof. Jeanine Koenig, Paris, France; Prof. Hanns Lochmu¨ller, Munich, Germany; Dr Monitor decremental response at 10 min intervals for 1 h Margherita Milone, Rochester, USA; Juliane S. Mu¨ller, Munich, Germany; Dr Jackie Palace, Oxford, UK; D. Beeson et al. / Neuromuscular Disorders 15 (2005) 498-512 Dr Pascale Richard, Paris, France; Dr Carmelo Rodolico, [18] Kimbell LM, Ohno K, Engel AG, Rotundo RL. C-terminal and Messina, Italy; Prof. Markus A. Ruegg, Basel, Switzerland; heparin-binding domains of collagenic tail subunit are both essential Prof. Laurent Schaeffer, Lyon, France; Dr Ulrike Schara, for anchoring acetylcholinesterase at the synapse. J Biol Chem 2004;279:10997-1005.
Neuss, Germany; Prof. Clarke R. Slater, Newcastle upon [19] Engel AG, Ohno K, Sine SM. Congenital myasthenic syndromes: Tyne, UK; Prof. J. Andoni Urtizberea, Paris, France; Prof.
progress over the past decade. Muscle Nerve 2003;27:251-97.
Jan Verschuuren, Leiden, The Netherlands.
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