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

BACKGROUND: Pathogenic mutations in rapsyn result in endplate acetylcholine receptor (AChR) deficiency and are a common cause of postsynaptic congenital myasthenic syndromes. METHODS: Clinical, electrophysiologic, pathologic, and molecular studies were done in 39 patients. RESULTS: 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. CONCLUSIONS: 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.

Novel congenital myasthenic syndromes associated with defects in quantal release.

BACKGROUND: Most congenital myasthenic syndromes are caused by defects in postsynaptic or synaptic basal lamina-associated proteins; congenital myasthenic syndromes (CMSs) associated with presynaptic defects are uncommon. Here, the authors describe clinical, electrophysiologic, and morphologic features of two novel and highly disabling CMSs, one determined by presynaptic and the other determined by combined presynaptic and postsynaptic defects. METHODS: Microelectrode, single channel patch clamp, immunocytochemical, [(125)I]alpha-bungarotoxin binding, and quantitative electron microscopy studies of endplates were performed. Candidate genes were directly sequenced. RESULTS: Patient 1, a 7-year-old boy, had severe myasthenic symptoms since infancy. Patient 2, a 48-year-old man, had delayed motor milestones and became progressively weaker after age 2 years. Both used wheelchairs and had a 30-50% EMG decrement on 2-Hz stimulation. Evoked quantal release was reduced to approximately 25% of normal in both. In Patient 2, the synaptic response to acetylcholine was further compromised by degeneration of the junctional folds with concomitant loss of the acetylcholine receptor (AChR). A search for mutations in components of the synaptic vesicle release complex and in other candidate proteins failed to identify the molecular basis of the two syndromes. CONCLUSIONS: Combined clinical, morphologic, and in vitro electrophysiologic findings define two novel congenital myasthenic syndromes. The molecular basis of these syndromes awaits discovery.

Congenital myasthenic syndrome caused by low-expressor fast-channel AChR delta subunit mutation.

OBJECTIVE: To determine the molecular basis of a disabling congenital myasthenic syndrome (CMS) observed in two related and one unrelated Arab kinship. BACKGROUND: CMS can arise from defects in presynaptic, synaptic basal lamina-associated, or postsynaptic proteins. Most CMS are postsynaptic, and most reside in the AChR epsilon subunit; only two mutations have been reported in the AChR delta subunit to date. METHODS: Cytochemistry, electron microscopy, alpha-bungarotoxin binding studies, microelectrode and patch-clamp recordings, mutation analysis, mutagenesis, and expression studies in human embryonic kidney cells were employed. RESULTS: Endplate studies showed AChR deficiency, fast decaying, low-amplitude endplate currents, and abnormally brief channel opening events. Mutation analysis revealed a novel homozygous missense mutation (deltaP250Q) of the penultimate proline in the first transmembrane domain (TMD1) of the AChR delta subunit. Expression studies indicate that deltaP250Q (1) hinders delta/alpha subunit association during early AChR assembly; (2) hinders opening of the doubly occupied closed receptor (A(2)R); and (3) speeds the dissociation of acetylcholine from A(2)R. Mutagenesis studies indicate that deltaP250L also has fast-channel effects, whereas epsilon P245L and epsilon P245Q, identical mutations of the corresponding proline in the epsilon subunit, have mild slow-channel effects. CONCLUSIONS: deltaP250Q represents the third mutation observed in the AChR delta subunit. The severe phenotype caused by deltaP250Q is attributed to endplate AChR deficiency, fast decay of the synaptic response, and lack of compensatory factors. That the penultimate prolines in TMD1 of the delta and epsilon subunits exert a reciprocal regulatory effect on the length of the channel opening bursts reveals an unexpected functional asymmetry between the two subunits.

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

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

Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans.

Choline acetyltransferase (ChAT; EC ) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist in Caenorhabditis elegans and Drosophila, but no CHAT mutations have been observed in humans to date. Here we report that mutations in CHAT cause a congenital myasthenic syndrome associated with frequently fatal episodes of apnea (CMS-EA). Studies of the neuromuscular junction in this disease show a stimulation-dependent decrease of the amplitude of the miniature endplate potential and no deficiency of the ACh receptor. These findings point to a defect in ACh resynthesis or vesicular filling and to CHAT as one of the candidate genes. Direct sequencing of CHAT reveals 10 recessive mutations in five patients with CMS-EA. One mutation (523insCC) is a frameshifting null mutation. Three mutations (I305T, R420C, and E441K) markedly reduce ChAT expression in COS cells. Kinetic studies of nine bacterially expressed ChAT mutants demonstrate that one mutant (E441K) lacks catalytic activity, and eight mutants (L210P, P211A, I305T, R420C, R482G, S498L, V506L, and R560H) have significantly impaired catalytic efficiencies.

Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome.

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation epsilonA411P in the amphipathic helix of the acetylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three unrelated families are either homozygous for epsilonA411P or are heterozygous and harbor a null mutation in the second epsilon allele, indicating that epsilonA411P is recessive. We expressed human AChRs containing wild-type or A411P epsilon subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by epsilonA411P. Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.

The spectrum of mutations causing end-plate acetylcholinesterase deficiency.

The end-plate species of acetylcholinesterase (AChE) is an asymmetric enzyme consisting of a collagenic tail subunit composed of three collagenic strands (ColQ), each attached to a tetramer of the T isoform of the catalytic subunit (AChE(T)) via a proline-rich attachment domain. The principal function of the tail subunit is to anchor asymmetric AChE in the synaptic basal lamina. Human end-plate AChE deficiency was recently shown to be caused by mutations in COLQ. We here report nine novel COLQ mutations in 7 patients with end-plate AChE deficiency. We examine the effects of the mutations on the assembly of asymmetric AChE by coexpressing each genetically engineered COLQ mutant with ACHE(T) in COS cells. We classify the newly recognized and previously reported COLQ mutations into four classes according to their position in ColQ and their effect on AChE expression. We find that missense mutations in the proline-rich attachment domain abrogate attachment of catalytic subunits, that truncation mutations in the ColQ collagen domain prevent the assembly of asymmetric AChE, that hydrophobic missense residues in the C-terminal domain prevent triple helical assembly of the ColQ collagen domain, and that other mutations in the C-terminal region produce asymmetric species of AChE that are likely insertion incompetent.

Mutation causing congenital myasthenia reveals acetylcholine receptor beta/delta subunit interaction essential for assembly.

We describe a severe postsynaptic congenital myasthenic syndrome with marked endplate acetylcholine receptor (AChR) deficiency caused by 2 heteroallelic mutations in the beta subunit gene. One mutation causes skipping of exon 8, truncating the beta subunit before its M1 transmembrane domain, and abolishing surface expression of pentameric AChR. The other mutation, a 3-codon deletion (beta426delEQE) in the long cytoplasmic loop between the M3 and M4 domains, curtails but does not abolish expression. By coexpressing beta426delEQE with combinations of wild-type subunits in 293 HEK cells, we demonstrate that beta426delEQE impairs AChR assembly by disrupting a specific interaction between beta and delta subunits. Studies with related deletion and missense mutants indicate that secondary structure in this region of the beta subunit is crucial for interaction with the delta subunit. The findings imply that the mutated residues are positioned at the interface between beta and delta subunits and demonstrate contribution of this local region of the long cytoplasmic loop to AChR assembly.

Chromosome 17p-linked myasthenias stem from defects in the acetylcholine receptor epsilon-subunit gene.

OBJECTIVE: To identify and to characterize functionally the mutational basis of congenital myasthenic syndromes (CMS) linked to chromosome 17p. BACKGROUND: A total of 37 patients belonging to 13 CMS families, 9 of them consanguineous, were investigated. All patients were linked previously to the telomeric region of chromosome 17p. Two candidate genes in this region encode synaptobrevin 2, a presynaptic protein, and the epsilon-subunit of the acetylcholine receptor (AChR). Direct sequencing of the synaptobrevin 2 gene revealed no mutations. The authors thus searched for mutations in the epsilon-subunit gene of AChR. METHODS: Direct sequencing of the AChR epsilon-subunit, restriction analysis, allele-specific PCR, and expression studies in human embryonic kidney cells were performed. RESULTS: The authors identified two previously characterized and five novel epsilon-subunit gene mutations, all homozygous, in the 13 kinships. Two of the novel mutations are truncating (epsilon723delC and epsilon760ins8), one is a missense mutation in the signal peptide region (epsilonV-13D), one is a missense mutation in the N-terminal extracellular domain (epsilonT51P), and one is a splice donor site mutation in intron 10 (epsilonIVS10+2T-->G). Unaffected family members have no mutations or are heterozygous. Expression studies indicate that the four novel mutations in the coding region of the gene and the most likely transcript of the splice-site mutation, which skips exon 10, are low-expressor or null mutations. CONCLUSIONS: Chromosome 17p-linked congenital myasthenic syndromes are caused by low-expressor/null mutations in the AChR epsilon-subunit gene. Mutations in this gene are a common cause of CMS in eastern Mediterranean countries.

Congenital end-plate acetylcholinesterase deficiency caused by a nonsense mutation and an A-->G splice-donor-site mutation at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G at position +3 result in aberrant splicing?

Congenital end-plate acetylcholinesterase (AChE) deficiency (CEAD), the cause of a disabling myasthenic syndrome, arises from defects in the COLQ gene, which encodes the AChE triple-helical collagenlike-tail subunit that anchors catalytic subunits of AChE to the synaptic basal lamina. Here we describe a patient with CEAD with a nonsense mutation (R315X) and a splice-donor-site mutation at position +3 of intron 16 (IVS16+3A-->G) of COLQ. Because both A and G are consensus nucleotides at the +3 position of splice-donor sites, we constructed a minigene that spans exons 15-17 and harbors IVS16+3A-->G for expression in COS cells. We found that the mutation causes skipping of exon 16. The mutant splice-donor site of intron 16 harbors five discordant nucleotides (at -3, -2, +3, +4, and +6) that do not base-pair with U1 small-nuclear RNA (snRNA), the molecule responsible for splice-donor-site recognition. Versions of the minigene harboring, at either +4 or +6, nucleotides complementary to U1 snRNA restore normal splicing. Analysis of 1,801 native splice-donor sites reveals that presence of a G nucleotide at +3 is associated with preferential usage, at positions +4 to +6, of nucleotides concordant to U1 snRNA. Analysis of 11 disease-associated IVS+3A-->G mutations indicates that, on average, two of three nucleotides at positions +4 to +6 fail to base-pair, and that the nucleotide at +4 never base-pairs, with U1 snRNA. We conclude that, with G at +3, normal splicing generally depends on the concordance that residues at +4 to +6 have with U1 snRNA, but other cis-acting elements may also be important in assuring the fidelity of splicing.

Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating.

By defining the functional defect in a congenital myasthenic syndrome (CMS), we show that the third transmembrane domain (M3) of the muscle acetylcholine receptor governs the speed and efficiency of gating of its channel. The clinical phenotype of this CMS results from the mutation V285I in M3 of the alpha subunit, which attenuates endplate currents, accelerates their decay and causes abnormally brief acetylcholine-induced single-channel currents. Kinetic analysis of engineered alpha V285I receptors demonstrated a predominant effect on channel gating, with abnormally slow opening and rapid closing rates. Analysis of site-directed mutations revealed stereochemical and volume-dependent contributions of alpha V285 to channel gating. Thus, we demonstrate a functional role for the M3 domain as a key component of the nicotinic acetylcholine receptor channel-gating mechanism.

Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzyme.

In skeletal muscle, acetylcholinesterase (AChE) exists in homomeric globular forms of type T catalytic subunits (ACHET) and heteromeric asymmetric forms composed of 1, 2, or 3 tetrameric ACHET attached to a collagenic tail (ColQ). Asymmetric AChE is concentrated at the endplate (EP), where its collagenic tail anchors it into the basal lamina. The ACHET gene has been cloned in humans; COLQ cDNA has been cloned in Torpedo and rodents but not in humans. In a disabling congenital myasthenic syndrome, EP AChE deficiency (EAD), the normal asymmetric species of AChE are absent from muscle. EAD could stem from a defect that prevents binding of ColQ to ACHET or the insertion of ColQ into the basal lamina. In six EAD patients, we found no mutations in ACHET. We therefore cloned human COLQ cDNA, determined the genomic structure and chromosomal localization of COLQ, and then searched for mutations in this gene. We identified six recessive truncation mutations of COLQ in six patients. Coexpression of each COLQ mutant with wild-type ACHET in SV40-transformed monkey kidney fibroblast (COS) cells reveals that a mutation proximal to the ColQ attachment domain for ACHET prevents association of ColQ with ACHET; mutations distal to the attachment domain generate a mutant approximately 10.5S species of AChE composed of one ACHET tetramer and a truncated ColQ strand. The approximately 10.5S species lack part of the collagen domain and the entire C-terminal domain of ColQ, or they lack only the C-terminal domain, which is required for formation of the triple collagen helix, and this likely prevents their insertion into the basal lamina.

Myasthenic syndromes in Turkish kinships due to mutations in the acetylcholine receptor.

We report and functionally characterize five new mutations of the acetylcholine receptor (AChR) in 11 Turkish patients with recessive congenital myasthenic syndromes (CMS) belonging to six families. All mutations are in the epsilon-subunit gene. Parental consanguinity is present in three families. The disease cosegregates with homozygous mutations in five families and with two different heteroallelic mutations in one family. Four mutations are frameshifting, predicting truncation of the epsilon subunit, and one occurs at a splice donor site. Expression of each frameshifting mutation and the likely transcripts of the splice-site mutation in human embryonic kidney 293 cells shows that each mutation is a null mutation. The findings support the notion that loss-of-function mutations of the acetylcholine receptor causing CMS are concentrated in the epsilon subunit, and that such mutations are a frequent cause of CMS.

Quinidine normalizes the open duration of slow-channel mutants of the acetylcholine receptor.

Quinidine is a long-lived open-channel blocker of the wild-type endplate acetylcholine receptor (AChR). To test the hypothesis that quinidine can normalize the prolonged channel opening events of slow-channel mutants of human AChR, we expressed wild-type AChR and five well characterized slow-channel mutants of AChR in HEK 293 cells and monitored the effects of quinidine on acetylcholine-induced channel currents. Quinidine shortens the longest component of channel opening burst (tau3b) of both wild-type and mutant AChRs in a concentration-dependent manner, and 5 microM quinidine reduces tau3b of the mutant AChRs to that of wild-type AChRs in the absence of quinidine. Because this concentration of quinidine is attainable in clinical practice, the findings predict a therapeutic effect for quinidine in the slow-channel congenital myasthenic syndrome.

Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor epsilon subunit.

We describe the genetic and kinetic defects in a congenital myasthenic syndrome caused by heteroallelic mutations of the acetylcholine receptor (AChR) epsilon subunit gene. The mutations are an in-frame duplication of six residues in the long cytoplasmic loop (epsilon1254ins18) and a cysteine-loop null mutation (epsilonC128S). The epsilon1254 ins18 mutation causes mode switching in the kinetics of receptor activation in which three modes activate slowly and inactivate rapidly. The epsilon1245ins18-AChR at the endplate shows abnormally brief activation episodes during steady state agonist application and appears electrically silent during the synaptic response to acetylcholine. The phenotypic consequences are endplate AChR deficiency, simplification of the postsynaptic region, and compensatory expression of fetal AChR that restores electrical activity at the endplate and rescues the phenotype.

Congenital myasthenic syndromes due to heteroallelic nonsense/missense mutations in the acetylcholine receptor epsilon subunit gene: identification and functional characterization of six new mutations.

We describe and functionally characterize six mutations of the acetylcholine receptor (AChR) epsilon subunit gene in three congenital myasthenic syndrome patients. Endplate studies demonstrated severe endplate AChR deficiency, dispersed endplate regions and well preserved junctional folds in all three patients. Electrophysiologic studies were consistent with expression of the fetal gamma-AChR at the endplates in one patient, prolongation of some channel events in another and gamma-AChR expression as well as some shorter than normal channel events in still another. Genetic analysis revealed two recessive and heteroallelic epsilon subunit gene mutations in each patient. One mutation in each (epsilonC190T [epsilon R64X], epsilon 127ins5 and epsilon 553del 7) generates a nonsense codon that predicts truncation of the epsilon subunit in its N-terminal, extracellular domain; and one mutation in each generates a missense codon (epsilon R147L, epsilon P245L and epsilon R311W). None of the mutations was detected in 100 controls. Expression studies in HEK cells indicate that the three nonsense mutations are null mutations and that surface expression of AChRs harboring the missense mutations is significantly reduced. Kinetic analysis of AChRs harboring the missense mutations show that epsilon R147L is kinetically benign, epsilon P245L prolongs burst open duration 2-fold by slowing the rate of channel closing and epsilon R311W shortens burst duration 2-fold by slowing the rate of channel opening and speeding the rate of ACh dissociation. The modest changes in activation kinetics are probably overshadowed by reduced expression of the missense mutations. The consequences of the endplate AChR deficiency are mitigated by persistent expression of gamma-AChR, changes in the release of transmitter quanta and appearance of multiple endplate regions on the muscle fiber.