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2.
Neuromuscul Disord ; 28(4): 315-322, 2018 04.
Article in English | MEDLINE | ID: mdl-29395675

ABSTRACT

Congenital myasthenic syndromes (CMS) are a group of hereditary disorders affecting the neuromuscular junction. Here, we present clinical, electrophysiological and genetic findings of 69 patients from 51 unrelated kinships from Turkey. Genetic tests of 60 patients were performed at Mayo Clinic. Median follow-up time was 9.8 years (range 1-22 years). The most common CMS was primary acetylcholine receptor (AChR) deficiency (31/51) and the most common mutations in AChR were c.1219 + 2T > G (12/51) and c.1327delG (6/51) in CHRNE. Four of our 5 kinships with AChE deficiency carried p.W148X that truncates the collagen domain of COLQ, and was previously reported only in patients from Turkey. These were followed by GFPT1 deficiency (4/51), DOK7 deficiency (3/51), slow channel CMS (3/51), fast channel CMS (3/51), choline acetyltransferase deficiency (1/51) and a CMS associated with desmin deficiency (1/51). Distribution of muscle weakness was sometimes useful in giving a clue to the CMS subtype. Presence of repetitive compound muscle action potentials pointed to AChE deficiency or slow channel CMS. Our experience confirms that one needs to be cautious using pyridostigmine, since it can worsen some types of CMS. Ephedrine/salbutamol were very effective in AChE and DOK7 deficiencies and were useful as adjuncts in other types of CMS. Long follow-up gave us a chance to assess progression of the disease, and to witness 12 mainly uneventful pregnancies in 8 patients. In this study, we describe some new phenotypes and detail the clinical features of the well-known CMS.


Subject(s)
Muscle Proteins/genetics , Myasthenic Syndromes, Congenital/genetics , Neuromuscular Junction/metabolism , Acetylcholinesterase/genetics , Adolescent , Collagen/metabolism , Female , Follow-Up Studies , Humans , Male , Mutation/genetics , Phenotype , Prognosis , Receptors, Cholinergic/genetics , Retrospective Studies , Young Adult
3.
JCI Insight ; 3(2)2018 01 25.
Article in English | MEDLINE | ID: mdl-29367459

ABSTRACT

We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) in 3 patients with severe congenital myasthenia: εR218W in the pre-M1 region in 2 patients and εE184K in the ß8-ß9 linker in 1 patient. Arg218 is conserved in all eukaryotic members of the Cys-loop receptor superfamily, while Glu184 is conserved in the α-, δ-, and ε-subunits of AChRs from all species. εR218W reduces channel gating efficiency 338-fold and AChR expression on the cell surface 5-fold, whereas εE184K reduces channel gating efficiency 11-fold but does not alter AChR cell surface expression. Determinations of the effective channel gating rate constants, combined with mutant cycle analyses, demonstrate strong energetic coupling between εR218 and εE184, and between εR218 and εE45 from the ß1-ß2 linker, as also observed for equivalent residues in the principal coupling pathway of the α-subunit. Thus, efficient and rapid gating of the AChR channel is achieved not only by coupling between conserved residues within the principal coupling pathway of the α-subunit, but also between corresponding residues in the ε-subunit.


Subject(s)
Evoked Potentials, Motor/physiology , Myasthenic Syndromes, Congenital/genetics , Receptors, Nicotinic/genetics , Adult , Arginine/genetics , Arginine/metabolism , Consanguinity , DNA Mutational Analysis , Female , Glutamic Acid/genetics , Glutamic Acid/metabolism , HEK293 Cells , Homozygote , Humans , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Mutation , Myasthenic Syndromes, Congenital/pathology , Myasthenic Syndromes, Congenital/physiopathology , Patch-Clamp Techniques , Receptors, Nicotinic/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
4.
Ann Clin Transl Neurol ; 4(2): 130-138, 2017 Feb.
Article in English | MEDLINE | ID: mdl-28168212

ABSTRACT

OBJECTIVE: To identify the molecular basis and elucidate the pathogenesis of a fatal congenital myasthenic syndrome. METHODS: We performed clinical electrophysiology studies, exome and Sanger sequencing, and analyzed functional consequences of the identified mutation. RESULTS: Clinical electrophysiology studies of the patient revealed several-fold potentiation of the evoked muscle action potential by high frequency nerve stimulation pointing to a presynaptic defect. Exome sequencing identified a homozygous c.340delA frameshift mutation in synaptobrevin 1 (SYB1), one of the three SNARE proteins essential for synaptic vesicle exocytosis. Analysis of both human spinal cord gray matter and normal human muscle revealed expression of the SYB1A and SYB1D isoforms, predicting expression of one or both isoforms in the motor nerve terminal. The identified mutation elongates the intravesicular C-terminus of the A isoform from 5 to 71, and of the D isoform from 4 to 31 residues. Transfection of either mutant isoform into bovine chromaffin cells markedly reduces depolarization-evoked exocytosis, and transfection of either mutant isoform into HEK cells significantly decreases expression of either mutant compared to wild type. INTERPRETATION: The mutation is pathogenic because elongation of the intravesicular C-terminus of the A and D isoforms increases the energy required to move their C-terminus into the synaptic vesicle membrane, a key step for fusion of the synaptic vesicle with the presynaptic membrane, and because it is predicted to reduce expression of either isoform in the nerve terminal.

5.
J Biol Chem ; 291(7): 3291-301, 2016 Feb 12.
Article in English | MEDLINE | ID: mdl-26698174

ABSTRACT

We identify two heteroallelic mutations in the acetylcholine receptor δ-subunit from a patient with severe myasthenic symptoms since birth: a novel δD140N mutation in the signature Cys-loop and a mutation in intron 7 of the δ-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that δD140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the α-, ß-, and ϵ-subunits also suppresses apparent channel gating, but the suppression is much greater in the α-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that αAsp-138 is energetically coupled to αArg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved αAsp-138 and αArg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.


Subject(s)
Acetylcholine/metabolism , Models, Molecular , Mutation , Myasthenic Syndromes, Congenital/genetics , Receptors, Nicotinic/genetics , Acetylcholine/chemistry , Amino Acid Substitution , Bungarotoxins/pharmacology , Child , Conserved Sequence , DNA Mutational Analysis , Female , HEK293 Cells , Humans , Introns , Ligands , Muscle Weakness/etiology , Myasthenic Syndromes, Congenital/metabolism , Myasthenic Syndromes, Congenital/physiopathology , Nicotinic Agonists/pharmacology , Protein Conformation , Protein Interaction Domains and Motifs , RNA Splicing , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Severity of Illness Index
6.
Neurology ; 83(24): 2247-55, 2014 Dec 09.
Article in English | MEDLINE | ID: mdl-25381298

ABSTRACT

OBJECTIVE: To identify and characterize the molecular basis of a syndrome associated with myasthenia, cortical hyperexcitability, cerebellar ataxia, and intellectual disability. METHODS: We performed in vitro microelectrode studies of neuromuscular transmission, performed exome and Sanger sequencing, and analyzed functional consequences of the identified mutation in expression studies. RESULTS: Neuromuscular transmission at patient endplates was compromised by reduced evoked quantal release. Exome sequencing identified a dominant de novo variant, p.Ile67Asn, in SNAP25B, a SNARE protein essential for exocytosis of synaptic vesicles from nerve terminals and of dense-core vesicles from endocrine cells. Ca(2+)-triggered exocytosis is initiated when synaptobrevin attached to synaptic vesicles (v-SNARE) assembles with SNAP25B and syntaxin anchored in the presynaptic membrane (t-SNAREs) into an α-helical coiled-coil held together by hydrophobic interactions. Pathogenicity of the Ile67Asn mutation was confirmed by 2 measures. First, the Ca(2+) triggered fusion of liposomes incorporating v-SNARE with liposomes containing t-SNAREs was hindered when t-SNAREs harbored the mutant SNAP25B moiety. Second, depolarization of bovine chromaffin cells transfected with mutant SNAP25B or with mutant plus wild-type SNAP25B markedly reduced depolarization-evoked exocytosis compared with wild-type transfected cells. CONCLUSION: Ile67Asn variant in SNAP25B is pathogenic because it inhibits synaptic vesicle exocytosis. We attribute the deleterious effects of the mutation to disruption of the hydrophobic α-helical coiled-coil structure of the SNARE complex by replacement of a highly hydrophobic isoleucine by a strongly hydrophilic asparagine.


Subject(s)
Ataxia/genetics , Brain Diseases/genetics , Intellectual Disability/genetics , Synaptosomal-Associated Protein 25/genetics , Adolescent , Animals , Ataxia/physiopathology , Brain Diseases/physiopathology , COS Cells , Cattle , Chlorocebus aethiops , Chromaffin Cells/physiology , DNA Mutational Analysis , Exocytosis/physiology , Female , Humans , Intellectual Disability/physiopathology , Microelectrodes , Motor Endplate/physiopathology , Mutation , SNARE Proteins/metabolism , Synaptic Vesicles/physiology , Synaptosomal-Associated Protein 25/metabolism , Syndrome , Transfection
7.
Neurology ; 82(20): 1822-30, 2014 May 20.
Article in English | MEDLINE | ID: mdl-24759841

ABSTRACT

OBJECTIVE: To investigate patients with DPAGT1 (UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase 1)-associated myasthenic syndrome. METHODS: We performed exome and Sanger sequencing, determined glycoprotein expression in patient muscles, assessed pathogenicity of the mutant proteins by examining their expression and enzymatic activity in transfected cells, evaluated structural changes in muscle and the neuromuscular junction, and examined electrophysiologic aspects of neuromuscular transmission in vitro. RESULTS: Patients 1 and 2, 16 and 14 years of age, had progressive fatigable weakness since infancy and are intellectually disabled. Patient 3, a less severely affected brother of patient 1, also has autistic features. Each patient harbors 2 novel heteroallelic mutations in DPAGT1, an enzyme subserving protein N-glycosylation. Patients 1 and 3 harbor Met1Leu, which reduces protein expression, and His375Tyr, which decreases enzyme activity. Patient 2 carries Val264Met, which abolishes enzyme activity, and a synonymous Leu120Leu mutation that markedly augments exon skipping, resulting in some skipped and infrequent nonskipped alleles. Therefore, the nonskipped allele rescues the phenotype. Intracellular microelectrode studies indicate combined pre- and postsynaptic defects of neuromuscular transmission with evidence for somatic mosaicism in patient 2. Structural studies reveal hypoplastic endplates, fiber-type disproportion, tubular aggregates, and degeneration of muscle fiber organelles resulting in autophagocytosis. CONCLUSIONS: DPAGT1 myasthenia affects multiple parameters of neuromuscular transmission, causes fiber-type disproportion and an autophagic myopathy, and can be associated with intellectual disability. We speculate that hypoglycosylation of synapse-specific proteins causes defects in central as well as motor synapses.


Subject(s)
Intellectual Disability/genetics , Muscle Weakness/genetics , Myasthenic Syndromes, Congenital/genetics , N-Acetylglucosaminyltransferases/genetics , Adolescent , Child , Female , Humans , Intellectual Disability/metabolism , Male , Muscle Weakness/metabolism , Muscle, Skeletal/metabolism , Mutation , Myasthenic Syndromes, Congenital/metabolism , N-Acetylglucosaminyltransferases/metabolism , Phenotype , Synapses/genetics , Synapses/metabolism
8.
Neurology ; 81(4): 370-8, 2013 Jul 23.
Article in English | MEDLINE | ID: mdl-23794683

ABSTRACT

OBJECTIVE: To identify patients with GFPT1-related limb-girdle myasthenia and analyze phenotypic consequences of the mutations. METHODS: We performed genetic analysis, histochemical, immunoblot, and ultrastructural studies and in vitro electrophysiologic analysis of neuromuscular transmission. RESULTS: We identified 16 recessive mutations in GFPT1 in 11 patients, of which 12 are novel. Ten patients had slowly progressive limb-girdle weakness responsive to cholinergic agonists with onset between infancy and age 19 years. One patient (no. 6) harbored a nonsense mutation and a second mutation that disrupts the muscle-specific GFPT1 exon. This patient never moved in utero, was apneic and arthrogrypotic at birth, and was bedfast, tube-fed, and barely responded to therapy at age 6 years. Histochemical studies in 9 of 11 patients showed tubular aggregates in 6 and rimmed vacuoles in 3. Microelectrode studies of intercostal muscle endplates in 5 patients indicated reduced synaptic response to acetylcholine in 3 and severely reduced quantal release in patient 6. Endplate acetylcholine receptor content was moderately reduced in only one patient. The synaptic contacts were small and single or grape-like, and quantitative electron microscopy revealed hypoplastic endplate regions. Numerous muscle fibers of patient 6 contained myriad dilated and degenerate vesicular profiles, autophagic vacuoles, and bizarre apoptotic nuclei. Glycoprotein expression in muscle was absent in patient 6 and reduced in 5 others. CONCLUSIONS: GFPT1-myasthenia is more heterogeneous than previously reported. Different parameters of neuromuscular transmission are variably affected. When disruption of muscle-specific isoform determines the phenotype, this has devastating clinical, pathologic, and biochemical consequences.


Subject(s)
Action Potentials/physiology , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/genetics , Muscle, Skeletal/pathology , Myasthenia Gravis/genetics , Myasthenia Gravis/pathology , Myasthenia Gravis/physiopathology , Acetylcholine/pharmacology , Acetylcholinesterase/metabolism , Adolescent , Child , Child, Preschool , DNA Mutational Analysis , Female , Genotype , Humans , In Vitro Techniques , Infant , Male , Microscopy, Electron, Transmission , Muscle, Skeletal/metabolism , Muscle, Skeletal/ultrastructure , Mutation/genetics , Myasthenia Gravis/drug therapy , Neuromuscular Junction/drug effects , Neuromuscular Junction/genetics , Neuromuscular Junction/ultrastructure , Pyridostigmine Bromide/pharmacology , Pyridostigmine Bromide/therapeutic use , Receptors, Cholinergic/metabolism , Receptors, Cholinergic/ultrastructure , Sarcoplasmic Reticulum/pathology , Young Adult
9.
J Clin Invest ; 122(7): 2613-21, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22728938

ABSTRACT

Congenital myasthenic syndromes (CMSs) are neuromuscular disorders that can be caused by defects in ace-tylcholine receptor (AChR) function. Disease-associated point mutants can reveal the unsuspected functional significance of mutated residues. We identified two pathogenic mutations in the extracellular domain of the AChR α subunit (AChRα) in a patient with myasthenic symptoms since birth: a V188M mutation in the C-loop and a heteroallelic G74C mutation in the main immunogenic region. The G74C mutation markedly reduced surface AChR expression in cultured cells, whereas the V188M mutant was expressed robustly but had severely impaired kinetics. Single-channel patch-clamp analysis indicated that V188M markedly decreased the apparent AChR channel opening rate and gating efficiency. Mutant cycle analysis of energetic coupling among conserved residues within or dispersed around the AChRα C-loop revealed that V188 is functionally linked to Y190 in the C-loop and to D200 in ß-strand 10, which connects to the M1 transmembrane domain. Furthermore, V188M weakens inter-residue coupling of K145 in ß-strand 7 with Y190 and with D200. Cumulatively, these results indicate that V188 of AChRα is part of an interdependent tetrad that contributes to rearrangement of the C-loop during the initial coupling of agonist binding to channel gating.


Subject(s)
Mutation, Missense , Myasthenic Syndromes, Congenital/genetics , Receptors, Nicotinic/genetics , Acetylcholine/pharmacology , Adult , Amino Acid Motifs , Amino Acid Sequence , Bungarotoxins/metabolism , Cholinergic Agonists/pharmacology , Conserved Sequence , DNA Mutational Analysis , Female , HEK293 Cells , Humans , Kinetics , Membrane Potentials , Molecular Sequence Data , Mutagenesis , Patch-Clamp Techniques , Protein Binding , Receptors, Nicotinic/metabolism , Receptors, Nicotinic/physiology , Thermodynamics
10.
Neurology ; 79(5): 449-54, 2012 Jul 31.
Article in English | MEDLINE | ID: mdl-22592360

ABSTRACT

OBJECTIVE: To characterize the molecular basis of a novel fast-channel congenital myasthenic syndrome. METHODS: We used the candidate gene approach to identify the pathogenic mutation in the acetylcholine receptor (AChR) ε subunit, genetically engineered the mutant AChR into HEK cells, and evaluated the level of expression and kinetic properties of the mutant receptor. RESULTS: An 8-year-old boy born to consanguineous parents had severe myasthenic symptoms since birth. He is wheelchair bound and pyridostigmine therapy enables him to take only a few steps. Three similarly affected siblings died in infancy. He carries a homozygous p.W55R mutation at the α/ε subunit interface of the AChR agonist binding site. The mutant protein expresses well in HEK cells. Patch-clamp analysis of the mutant receptor expressed in HEK cells reveals 30-fold reduced apparent agonist affinity, 75-fold reduced apparent gating efficiency, and strikingly attenuated channel opening probability (P(open)) over a range agonist concentrations. CONCLUSION: Introduction of a cationic Arg into the anionic environment of α/ε AChR binding site hinders stabilization of cationic ACh by aromatic residues and accounts for the markedly perturbed kinetic properties of the receptor. The very low P(open) explains the poor response to pyridostigmine and the high fatality of the disease.


Subject(s)
Mutation/genetics , Myasthenic Syndromes, Congenital/genetics , Receptors, Nicotinic/genetics , Acetylcholine/pharmacology , Binding Sites/drug effects , Binding Sites/genetics , Biophysical Phenomena/drug effects , Biophysical Phenomena/genetics , Bungarotoxins/pharmacokinetics , Cell Line, Transformed , Child , Cholinergic Agonists/pharmacology , DNA Mutational Analysis , Dose-Response Relationship, Drug , Electric Stimulation , Humans , Iodine Isotopes/pharmacokinetics , Ion Channel Gating/drug effects , Ion Channel Gating/genetics , Male , Membrane Potentials/drug effects , Membrane Potentials/genetics , Patch-Clamp Techniques , Protein Binding/drug effects , Transfection
11.
Hum Mutat ; 32(11): 1259-67, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21786365

ABSTRACT

Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyzes synthesis of acetylcholine from acetyl-CoA (AcCoA) and choline in cholinergic neurons. Mutations in CHAT cause potentially lethal congenital myasthenic syndromes associated with episodic apnea (ChAT-CMS). Here, we analyze the functional consequences of 12 missense and one nonsense mutations of CHAT in 11 patients. Nine of the mutations are novel. We examine expression of the recombinant missense mutants in Bosc 23 cells, determine their kinetic properties and thermal stability, and interpret the functional effects of 11 mutations in the context of the atomic structural model of human ChAT. Five mutations (p.Trp421Ser, p.Ser498Pro, p.Thr553Asn, p.Ala557Thr, and p.Ser572Trp) reduce enzyme expression to less than 50% of wild-type. Mutations with severe kinetic effects are located in the active-site tunnel (p.Met202Arg, p.Thr553Asn, and p.Ala557Thr) or adjacent to the substrate binding site (p.Ser572Trp), or exert their effect allosterically (p.Trp421Ser and p.Ile689Ser). Two mutations with milder kinetic effects (p.Val136Met and p.Ala235Thr) are also predicted to act allosterically. One mutation (p.Thr608Asn) below the nucleotide binding site of CoA enhances dissociation of AcCoA from the enzyme-substrate complex. Two mutations introducing a proline residue into an α-helix (p.Ser498Pro and p.Ser704Pro) impair the thermal stability of ChAT.


Subject(s)
Choline O-Acetyltransferase/genetics , Mutation , Binding Sites , Choline O-Acetyltransferase/chemistry , Choline O-Acetyltransferase/metabolism , Cholinergic Neurons/enzymology , Cholinergic Neurons/metabolism , Genetic Association Studies , Humans , Kinetics , Myasthenic Syndromes, Congenital/genetics , Protein Conformation , Structure-Activity Relationship
12.
Neuromuscul Disord ; 21(3): 214-8, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21316238

ABSTRACT

The slow-channel congenital myasthenic syndrome (SCCMS) is an autosomal dominant neuromuscular disorder caused by mutations in different subunits of the acetylcholine receptor (AChR). We here report our clinical findings in three generations of a large Thai kinship suffering from SCCMS and trace the disease to the p.Gly153Ser mutation in the AChR α subunit. The same mutation had previously been reported only in Caucasian but not in Asian patients. The clinical features include ptosis, ophthalmoparesis, and weakness of the cervical and finger extensor muscles as well as marked phenotypic heterogeneity.


Subject(s)
Family Health , Mutation/genetics , Myasthenic Syndromes, Congenital/genetics , Myasthenic Syndromes, Congenital/physiopathology , Phenotype , Receptors, Nicotinic/genetics , Siblings , Adult , Aged , Child , Child, Preschool , Female , Glycine/genetics , Humans , Male , Middle Aged , Myasthenic Syndromes, Congenital/pathology , Serine/genetics , Thailand , Young Adult
13.
Hum Mol Genet ; 12(7): 739-48, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12651869

ABSTRACT

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


Subject(s)
Muscle Proteins/chemistry , Muscle Proteins/genetics , Mutation , Myasthenic Syndromes, Congenital/genetics , Adolescent , Adult , Alleles , Base Sequence , Cell Line , Cell Membrane/metabolism , Cell Nucleus/metabolism , Child , DNA Mutational Analysis , DNA, Complementary/metabolism , Enhancer Elements, Genetic , Facies , Female , Genes, Reporter , Genetic Vectors , Haplotypes , Humans , Luciferases/metabolism , Male , Microscopy, Fluorescence , Models, Genetic , Molecular Sequence Data , Muscles/metabolism , Pedigree , Phenotype , Promoter Regions, Genetic , Protein Structure, Tertiary , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Transfection
14.
J Clin Invest ; 111(4): 497-505, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12588888

ABSTRACT

We describe a highly disabling congenital myasthenic syndrome (CMS) associated with rapidly decaying, low-amplitude synaptic currents, and trace its cause to a valine to leucine mutation in the signature cystine loop (cys-loop) of the AChR alpha subunit. The recently solved crystal structure of an ACh-binding protein places the cys-loop at the junction between the extracellular ligand-binding and transmembrane domains where it may couple agonist binding to channel gating. We therefore analyzed the kinetics of ACh-induced single-channel currents to identify elementary steps in the receptor activation mechanism altered by the alphaV132L mutation. The analysis reveals that alphaV132L markedly impairs ACh binding to receptors in the resting closed state, decreasing binding affinity for the second binding step 30-fold, but attenuates gating efficiency only about twofold. By contrast, mutation of the equivalent valine residue in the delta subunit impairs channel gating approximately fourfold with little effect on ACh binding, while corresponding mutations in the beta and epsilon subunits are without effect. The unique functional contribution of the alpha subunit cys-loop likely owes to its direct connection via a beta strand to alphaW149 at the center of the ligand-binding domain. The overall findings reveal functional asymmetry between cys-loops of the different AChR subunits in contributing to ACh binding and channel gating.


Subject(s)
Myasthenic Syndromes, Congenital/genetics , Myasthenic Syndromes, Congenital/metabolism , Point Mutation , Receptors, Cholinergic/chemistry , Receptors, Cholinergic/genetics , Acetylcholine/metabolism , Amino Acid Sequence , Case-Control Studies , Cell Line , Child, Preschool , Cysteine/chemistry , Female , Humans , In Vitro Techniques , Ion Channel Gating , Kinetics , Male , Models, Molecular , Molecular Sequence Data , Phenotype , Protein Subunits , Receptors, Cholinergic/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid
15.
Am J Hum Genet ; 70(4): 875-85, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11791205

ABSTRACT

Congenital myasthenic syndromes (CMSs) stem from genetic defects in endplate (EP)-specific presynaptic, synaptic, and postsynaptic proteins. The postsynaptic CMSs identified to date stem from a deficiency or kinetic abnormality of the acetylcholine receptor (AChR). All CMSs with a kinetic abnormality of AChR, as well as many CMSs with a deficiency of AChR, have been traced to mutations in AChR-subunit genes. However, in a subset of patients with EP AChR deficiency, the genetic defect has remained elusive. Rapsyn, a 43-kDa postsynaptic protein, plays an essential role in the clustering of AChR at the EP. Seven tetratricopeptide repeats (TPRs) of rapsyn subserve self-association, a coiled-coil domain binds to AChR, and a RING-H2 domain associates with beta-dystroglycan and links rapsyn to the subsynaptic cytoskeleton. Rapsyn self-association precedes recruitment of AChR to rapsyn clusters. In four patients with EP AChR deficiency but with no mutations in AChR subunits, we identify three recessive rapsyn mutations: one patient carries L14P in TPR1 and N88K in TPR3; two are homozygous for N88K; and one carries N88K and 553ins5, which frameshifts in TPR5. EP studies in each case show decreased staining for rapsyn and AChR, as well as impaired postsynaptic morphological development. Expression studies in HEK cells indicate that none of the mutations hinders rapsyn self-association but that all three diminish coclustering of AChR with rapsyn.


Subject(s)
Motor Endplate/metabolism , Muscle Proteins/genetics , Mutation/genetics , Myasthenic Syndromes, Congenital/genetics , Myasthenic Syndromes, Congenital/metabolism , Receptors, Cholinergic/deficiency , Adult , Amino Acid Sequence , Animals , Binding Sites , Bungarotoxins/metabolism , Cell Line , Child , Child, Preschool , Cholinergic Antagonists/metabolism , DNA Mutational Analysis , Female , Humans , Infant , Male , Microelectrodes , Molecular Sequence Data , Motor Endplate/pathology , Motor Endplate/physiopathology , Motor Endplate/ultrastructure , Muscle Proteins/analysis , Muscle Proteins/chemistry , Muscle Proteins/metabolism , Myasthenic Syndromes, Congenital/pathology , Myasthenic Syndromes, Congenital/physiopathology , Phenotype , Protein Binding , Protein Structure, Tertiary , Receptors, Cholinergic/analysis , Receptors, Cholinergic/metabolism , Sequence Alignment , Synaptic Transmission/physiology
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