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1.  New horizons for congenital myasthenic syndromes 
During the past 5 years an increasing number of patients were diagnosed with congenital myasthenic syndromes (CMS) and a number of novel syndromes were recognized and investigated. This presentation focuses on the CMS caused by defects in choline acetyltransferase, novel fast-channel syndromes that hinder isomerization of the acetylcholine receptor from the closed to the open state, the consequences of deleterious mutations in the intermediate filament linker plectin, altered neuromuscular transmission in a centronuclear myopathy, and two recently identified CMS caused by congenital defects in glycosylation.
doi:10.1111/j.1749-6632.2012.06803.x
PMCID: PMC3546605  PMID: 23278578
congenital myasthenic syndromes; acetylcholine receptor; fast-channel syndromes; choline acetyltransferase; plectin; centronuclear myopathy; GFPT1; DPAGT1
2.  Current Status of the Congenital Myasthenic Syndromes 
Neuromuscular Disorders  2011;22(2):99-111.
Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Clinical, electrophysiologic, and morphologic studies have paved the way for detecting CMS-related mutations in proteins residing in the nerve terminal, the synaptic basal lamina, and in the postsynaptic region of the motor endplate. The disease proteins identified to date include choline acetyltransferase (ChAT), the endplate species of acetylcholinesterase (AChE), β2-laminin, the acetylcholine receptor (AChR), rapsyn, plectin, Nav1.4, the muscle specific protein kinase (MuSK), agrin, downstream of tyrosine kinase 7 (Dok-7), and glutamine-fructose-6-phosphate transaminase 1 (GFPT1). Myasthenic syndromes associated with centronuclear myopathies were recently recognized. Analysis of properties of expressed mutant proteins contributed to finding improved therapy for most CMS. Despite these advances, the molecular basis of some phenotypically characterized CMS remains elusive. Moreover, other types of CMS and disease genes likely exist and await discovery.
doi:10.1016/j.nmd.2011.10.009
PMCID: PMC3269564  PMID: 22104196
Congenital myasthenic syndrome; neuromuscular junction; EMG; choline acetyltransferase; ColQ; β2-laminin; acetylcholine receptor; rapsyn; agrin; MuSK; Dok-7; GFPT1; plectin; fetal akinesia syndrome
3.  BENEFICIAL EFFECTS OF ALBUTEROL IN CONGENITAL ENDPLATE ACETYLCHOLINESTERASE DEFICIENCY AND DOK-7 MYASTHENIA 
Muscle & nerve  2011;44(5):789-794.
Background
Congenital myasthenic syndromes (CMS) are disabling but treatable disorders. Anticholinesterase therapy is effective in most, but is contraindicated in endplate (EP) acetylcholinesterase (AChE) deficiency, the slow-channel syndrome, Dok-7 myasthenia, β2-laminin deficiency, and is not useful in CMS due to defects in MuSK, agrin, and plectin. EP AChE, Dok-7 and β2-laminin deficiencies respond favorably to ephedrine but ephedrine can no longer be prescribed in the US.
Methods
We used albuterol, another sympathomimetic agent, to treat three patients with EP AChE deficiency and 15 with Dok-7 myasthenia. Response to therapy was evaluated by a 9-point questionnaire pertaining to activities of daily life.
Results
Comparison of the pre- and post-treatment responses indicated a beneficial response to albuterol (p values <0.001) in both patient groups. The adverse effects of therapy were like those of ephedrine.
Discussion
Our observations should spur controlled prospective clinical trials of albuterol in these as well as other CMS.
doi:10.1002/mus.22176
PMCID: PMC3196786  PMID: 21952943
Congenital myasthenic syndrome; Dok-7 myasthenia; Endplate AChE deficiency; Albuterol
4.  Further Observations in Congenital Myasthenic Syndromes 
During the past five years many patients suffering from congenital myasthenic syndromes (CMS) have been identified worldwide and novel causative genes and mutations have been discovered. The disease genes now include those encoding each subunit of the acetylcholine receptor (AChR), the ColQ part of acetylcholinesterase (AChE), choline acetyltransferase, Nav 1.4, MuSK, and Dok-7. Moreover, emerging genotype-phenotype correlations are providing clues for targeted mutation analysis. This review focuses on the recent observations in selected CMS.
doi:10.1196/annals.1405.039
PMCID: PMC3478107  PMID: 18567859
congenital myasthenic syndromes; acetylcholinesterase; choline acetyltransferase; acetylcholine receptor; Dok-7
5.  HnRNP L and hnRNP LL antagonistically modulate PTB-mediated splicing suppression of CHRNA1 pre-mRNA 
Scientific Reports  2013;3:2931.
CHRNA1 gene, encoding the muscle nicotinic acetylcholine receptor alpha subunit, harbors an inframe exon P3A. Inclusion of exon P3A disables assembly of the acetylcholine receptor subunits. A single nucleotide mutation in exon P3A identified in congenital myasthenic syndrome causes exclusive inclusion of exon P3A. The mutation gains a de novo binding affinity for a splicing enhancing RNA-binding protein, hnRNP LL, and displaces binding of a splicing suppressing RNA-binding protein, hnRNP L. The hnRNP L binds to another splicing repressor PTB through the proline-rich region and promotes PTB binding to the polypyrimidine tract upstream of exon P3A, whereas hnRNP LL lacking the proline-rich region cannot bind to PTB. Interaction of hnRNP L with PTB inhibits association of U2AF65 and U1 snRNP with the upstream and downstream of P3A, respectively, which causes a defect in exon P3A definition. HnRNP L and hnRNP LL thus antagonistically modulate PTB-mediated splicing suppression of exon P3A.
doi:10.1038/srep02931
PMCID: PMC3796306  PMID: 24121633
6.  Endplate Structure and Parameters of Neuromuscular Transmission in Sporadic Centronuclear Myopathy Associated with Myasthenia 
Neuromuscular disorders : NMD  2011;21(6):387-395.
Centronuclear myopathy is a pathologically diagnosed congenital myopathy. The disease genes encode proteins with membrane modulating properties (MTM1, DNM2, and BIN1) or alter excitation-contraction coupling (RYR1). Some patients also have myasthenic symptoms but electrodiagnostic and endplate studies in these are limited. A sporadic patient had fatigable weakness and a decremental EMG response. Analysis of centronuclear myopathy disease- and candidate- genes identified no mutations. Quantitative endplate structure and in vitro microelectrode studies revealed simplified postsynaptic regions, endplate remodeling with normal nerve terminal size, normal synaptic vesicle density, and mild acetylcholine receptor deficiency. The amplitude of the miniature endplate potential was decreased to 60% of normal. Quantal release by nerve impulse was reduced to 40% of normal due to a decreased number of releasable quanta. The safety margin of neuromuscular transmission is compromised by decreased quantal release by nerve impulse and by a reduced postsynaptic response to the released quanta.
doi:10.1016/j.nmd.2011.03.002
PMCID: PMC3100385  PMID: 21482111
Centronuclear myopathy; neuromuscular junction; myasthenic syndrome; synaptic vesicle cycle
7.  Congenital myasthenia–related AChR δ subunit mutation interferes with intersubunit communication essential for channel gating 
The Journal of Clinical Investigation  2008;118(5):1867-1876.
Congenital myasthenias (CMs) arise from defects in neuromuscular junction–associated proteins. Deciphering the molecular bases of the CMs is required for therapy and illuminates structure-function relationships in these proteins. Here, we analyze the effects of a mutation in 1 of 4 homologous subunits in the AChR from a CM patient, a Leu to Pro mutation at position 42 of the δ subunit. The mutation is located in a region of contact between subunits required for rapid opening of the AChR channel and impedes the rate of channel opening. Substitutions of Gly, Lys, or Asp for δL42, or substitutions of Pro along the local protein chain, also slowed channel opening. Substitution of Pro for Leu in the ε subunit slowed opening, whereas this substitution had no effect in the β subunit and actually sped opening in the α subunit. Analyses of energetic coupling between residues at the subunit interface showed that δL42 is functionally linked to αT127, a key residue in the adjacent α subunit required for rapid channel opening. Thus, δL42 is part of an intersubunit network that enables ACh binding to rapidly open the AChR channel, which may be compromised in patients with CM.
doi:10.1172/JCI34527
PMCID: PMC2289798  PMID: 18398509
8.  THE THERAPY OF CONGENITAL MYASTHENIC SYNDROMES 
Summary
Congenital myasthenic syndromes (CMSs) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more mechanisms. Specific diagnosis of a CMS is important as some medications that benefit one type of CMS can be detrimental in another type. In some CMSs, strong clinical clues point to a specific diagnosis. In other CMSs, morphologic and in vitro electrophysiologic studies of the neuromuscular junction, determination of the number of acetylcholine receptors (AchRs) per junction, and molecular genetic studies may be required for a specific diagnosis. Strategies for therapy are based on whether a given CMS decreases or increases the synaptic response to acetylcholine (ACh). Cholinesterase inhibitors that increase the synaptic response to ACh and 3,4-diaminopyridine, which increases ACh release, are useful when the synaptic response to ACh is attenuated. Long-lived open-channel blockers of the AChR, quinidine and fluoxetine, are useful when the synaptic response is increased by abnormally prolonged opening episodes of the AChR channel. Ephedrine has beneficial effects in some CMSs but its mechanism of action is not understood.
doi:10.1016/j.nurt.2007.01.001
PMCID: PMC1978489  PMID: 17395135
Congenital myasthenic syndromes; acetylcholine receptor; acetylcholinesterase; choline acetyltransferase; rapsyn; Dok-7; MuSK; cholinesterase inhibitors; 3,4-diaminopyridine; quinidine; fluoxetine
9.  Reducing bodies and myofibrillar myopathy features in FHL1 muscular dystrophy 
Neurology  2011;77(22):1951-1959.
Objective:
Some pathologic features of the FHL1 myopathies and the myofibrillar myopathies (MFMs) overlap; we therefore searched for mutations in FHL1 in our cohort of 50 patients with genetically undiagnosed MFM.
Methods:
Mutations in FHL1 were identified by direct sequencing. Polymorphisms were excluded by using allele-specific PCR in 200 control subjects. Structural changes in muscle were analyzed by histochemistry, immunocytochemistry, and electron microscopy.
Results:
We detected 2 novel and 1 previously identified missense mutation in 5 patients. Patients 1–4 presented before age 30, display menadione–nitro blue tetrazolium–positive reducing bodies, and harbor mutations in the FHL1 LIM2 domain. Patient 5 presented at age 75 and has no reducing bodies, and his mutation is not in a LIM domain. The clinical features include progressive muscle weakness, hypertrophied muscles, rigid spine, and joint contractures, and 1 patient also has peripheral neuropathy. High-resolution electron microscopy reveals the reducing bodies composed of 13-nm tubulofilaments initially emanating from Z-disks. At a more advanced stage, abundant reducing bodies appear in the cytoplasm and nuclei with concomitant myofibrillar disintegration, accumulation of cytoplasmic degradation products, and aggregation of endoplasmic reticulum and sarcotubular profiles.
Conclusions:
FHL1 dystrophies can be associated with MFM pathology. Mutations in the LIM2 domain are associated with reducing bodies composed of distinct tubulofilaments. A mutation extraneous to LIM domains resulted in a mild late-onset phenotype with MFM pathology but no reducing bodies.
doi:10.1212/WNL.0b013e31823a0ebe
PMCID: PMC3235356  PMID: 22094483
10.  Functional Consequences and Structural Interpretation of Mutations of Human Choline Acetyltransferase 
Human Mutation  2011;32(11):1259-1267.
Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyzes synthesis of acetylcholine from acetyl-CoA and choline in cholinergic neurons. Mutations in CHAT (MIM # 118490) cause potentially lethal congenital myasthenic syndromes associated with episodic apnea (ChAT-CMS) (MIM # 254210). Here we analyze the functional consequences of 12 missense and 1 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, p.Ser572Trp) reduce enzyme expression to <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, 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.
doi:10.1002/humu.21560
PMCID: PMC3196808  PMID: 21786365
Choline acetyltransferase; congenital myasthenic syndrome; enzyme kinetics; atomic structural model; thermal stability
11.  Recent Structural and Mechanistic Insights into Endplate Acetylcholine Receptors 
Voluntary movement mediated by skeletal muscle relies on endplate acetylcholine receptors (AChR) to detect nerve-released ACh and depolarize themuscle fiber. Recent structural and mechanistic studies of the endplate AChR have catalyzed a leap in our understanding of the molecular steps in this chemical-to-electrical transduction process. Studies of acetylcholine binding protein (AChBP) give insight into ACh recognition, the first step in activation of the AChR. An atomic structural model of the Torpedo AChR at a resolution of 0.4 nm, together with single-ion channel recording methods, allow tracing of the link between the agonist binding event and gating of the ion channel, as well as determination of how the channel moves when it opens to allow flow of cations. Structural models of the human AChR enable precise mapping of disease-causing mutations, while studies of the speed with which single AChR channels open and close cast light on pathogenic mechanisms.
doi:10.1196/annals.1405.041
PMCID: PMC3478106  PMID: 18567853
acetylcholine receptor; acetylcholine binding protein; agonist recognition; binding-gating coupling mechanism; congenital myasthenic syndrome
12.  Inclusion Body Myositis 
The American Journal of Pathology  2011;179(3):1347-1359.
Sporadic inclusion body myositis (IBM) is a muscle disease with two separate pathogenic components, degeneration and inflammation. Typically, nonnecrotic myofibers are focally surrounded and invaded by CD8+ T cells and macrophages. Both attacked and nonattacked myofibers express high levels of human leukocyte antigen class I (HLA-I) molecules, a prerequisite for antigen presentation to CD8+ T cells. However, only a subgroup of HLA-I+ myofibers is attacked by immune cells. By using IHC, we classified myofibers from five patients with sporadic IBM as attacked (AIBM) or nonattacked (NIBM) and isolated the intracellular contents of myofibers separately by laser microdissection. For comparison, we isolated myofibers from control persons (HCTRL). The samples were analyzed by microarray hybridization and quantitative PCR. HLA-I up-regulation was observed in AIBM and NIBM, whereas HCTRL were negative for HLA-I. In contrast, the inducible chain of the interferon (IFN) γ receptor (IFNGR2) and several IFN-γ–induced genes were up-regulated in AIBM compared with NIBM and HCTRL fibers. Confocal microscopy confirmed segmental IFNGR2 up-regulation on the membranes of AIBM, which positively correlated with the number of adjacent CD8+ T cells. Thus, the differential up-regulation of the IFN-γ signaling cascade observed in the attacked fibers is related to local inflammation, whereas the ubiquitous HLA-I expression on IBM muscle fibers does not require IFNGR expression.
doi:10.1016/j.ajpath.2011.05.055
PMCID: PMC3157228  PMID: 21855683
13.  BENEFICIAL EFFECT OF ALBUTEROL IN CONGENTIAL MYASTHENIC SYNDROME WITH EPSILON SUBUINT MUTATIONS 
Muscle & nerve  2011;44(2):289-291.
Mutations in the epsilon subunit of the acetylcholine receptor (AChR) are a common cause for congenital myasthenic syndrome (CMS). Patients are usually treated with acetylcholine esterase inhibitors and 3,4-diaminopyridine with modest clinical benefit. We report two patients with CMS due to mutations in the AChR epsilon subunit. The first patient carries two heterozygous frameshift mutations, ε127ins5 and ε1293insG. The second patient is homozygous for εC142Y mutation that curtails AChR expression to 22% of wild type in HEK cells. Treatment with pyridostigmine and 3,4 diaminopyridine had a limited beneficial effect in the first patient, and the second patient became wheelchair bound during therapy. The additional use of albuterol produced dramatic improvement in strength and in activities of daily living in both patients. The efficacy and safety of albuterol in patients who harbor identified low-expressor or null mutations in the epsilon or other subunits of AChR merits a well designed clinical trial.
doi:10.1002/mus.22153
PMCID: PMC3136566  PMID: 21721016
14.  Mutation causing severe myasthenia reveals functional asymmetry of AChR signature cystine loops in agonist binding and gating 
Journal of Clinical Investigation  2003;111(4):497-505.
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 α 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 αV132L mutation. The analysis reveals that αV132L 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 δ subunit impairs channel gating approximately fourfold with little effect on ACh binding, while corresponding mutations in the β and ε subunits are without effect. The unique functional contribution of the α subunit cys-loop likely owes to its direct connection via a β strand to αW149 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.
doi:10.1172/JCI200316997
PMCID: PMC151927  PMID: 12588888
15.  Myasthenic syndrome AChRα C-loop mutant disrupts initiation of channel gating 
The Journal of Clinical Investigation  2012;122(7):2613-2621.
Congenital myasthenic syndromes (CMSs) are neuromuscular disorders that can be caused by defects in ace­tylcholine 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 β-strand 10, which connects to the M1 transmembrane domain. Furthermore, V188M weakens inter-residue coupling of K145 in β-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.
doi:10.1172/JCI63415
PMCID: PMC3386830  PMID: 22728938
16.  Mutation causing congenital myasthenia reveals acetylcholine receptor β/δ subunit interaction essential for assembly 
Journal of Clinical Investigation  1999;104(10):1403-1410.
We describe a severe postsynaptic congenital myasthenic syndrome with marked endplate acetylcholine receptor (AChR) deficiency caused by 2 heteroallelic mutations in the β subunit gene. One mutation causes skipping of exon 8, truncating the β subunit before its M1 transmembrane domain, and abolishing surface expression of pentameric AChR. The other mutation, a 3-codon deletion (β426delEQE) in the long cytoplasmic loop between the M3 and M4 domains, curtails but does not abolish expression. By coexpressing β426delEQE with combinations of wild-type subunits in 293 HEK cells, we demonstrate that β426delEQE impairs AChR assembly by disrupting a specific interaction between β and δ subunits. Studies with related deletion and missense mutants indicate that secondary structure in this region of the β subunit is crucial for interaction with the δ subunit. The findings imply that the mutated residues are positioned at the interface between β and δ subunits and demonstrate contribution of this local region of the long cytoplasmic loop to AChR assembly.
J. Clin. Invest. 104:1403–1410 (1999).
PMCID: PMC409847  PMID: 10562302
17.  Sporadic Centronuclear Myopathy with Muscle Pseudohypertrophy, Neutropenia, and Necklace Fibers due to a DNM2 mutation 
Neuromuscular disorders : NMD  2010;20(12):801-804.
Dynamin 2 gene (DNM2) mutations result in an autosomal dominant centronuclear myopathy (CNM) and a Charcot-Marie-Tooth (CMT) neuropathy. DNM2-CMT but not DNM2-CNM patients were noted to have neutropenia. We here report a man with paravertebral muscles hypertrophy and mild neutropenia. His muscle biopsy was typical for CNM with additional “necklace” fibers. Sequencing of DNM2 revealed a known heterozygous c.1269C>T (p.Arg369Trp) mutation. Necklace fibers were considered as a pathological hallmark of late onset X-linked CNM due to mutations in MTM1 but have not been observed in DNM2-CNM. The findings broaden the features of DNM2-myopathy.
doi:10.1016/j.nmd.2010.07.273
PMCID: PMC2991611  PMID: 20817456
Centronuclear myopathy; DNM2; dynamin 2; muscle hypertrophy; muscle pseudohypertrophy, necklace fibers; neutropenia
18.  Inclusion Body Myositis 
The American Journal of Pathology  2011;179(3):1347-1359.
Sporadic inclusion body myositis (IBM) is a muscle disease with two separate pathogenic components, degeneration and inflammation. Typically, nonnecrotic myofibers are focally surrounded and invaded by CD8+ T cells and macrophages. Both attacked and nonattacked myofibers express high levels of human leukocyte antigen class I (HLA-I) molecules, a prerequisite for antigen presentation to CD8+ T cells. However, only a subgroup of HLA-I+ myofibers is attacked by immune cells. By using IHC, we classified myofibers from five patients with sporadic IBM as attacked (AIBM) or nonattacked (NIBM) and isolated the intracellular contents of myofibers separately by laser microdissection. For comparison, we isolated myofibers from control persons (HCTRL). The samples were analyzed by microarray hybridization and quantitative PCR. HLA-I up-regulation was observed in AIBM and NIBM, whereas HCTRL were negative for HLA-I. In contrast, the inducible chain of the interferon (IFN) γ receptor (IFNGR2) and several IFN-γ–induced genes were up-regulated in AIBM compared with NIBM and HCTRL fibers. Confocal microscopy confirmed segmental IFNGR2 up-regulation on the membranes of AIBM, which positively correlated with the number of adjacent CD8+ T cells. Thus, the differential up-regulation of the IFN-γ signaling cascade observed in the attacked fibers is related to local inflammation, whereas the ubiquitous HLA-I expression on IBM muscle fibers does not require IFNGR expression.
doi:10.1016/j.ajpath.2011.05.055
PMCID: PMC3157228  PMID: 21855683
19.  What Have We Learned from the Congenital Myasthenic Syndromes 
The congenital myasthenic syndromes have now been traced to an array of molecular targets at the neuromuscular junction encoded by no fewer than 11 disease genes. The disease genes were identified by the candidate gene approach, using clues derived from clinical, electrophysiological, cytochemical, and ultrastructural features. For example, electrophysiologic studies in patients suffering from sudden episodes of apnea pointed to a defect in acetylcholine resynthesis and CHAT as the candidate gene (Ohno et al., Proc Natl Acad Sci USA 98:2017–2022–2001); refractoriness to anticholinesterase medications and partial or complete absence of acetylcholinesterase (AChE) from the endplates (EPs) has pointed to one of the two genes (COLQ and ACHET) encoding AChE, though mutations were observed only in COLQ. After a series of patients carrying mutations in a disease gene have been identified, the emerging genotype–phenotype correlations provided clues for targeted mutation analysis in other patients. Mutations in EP-specific proteins also prompted expression studies that proved pathogenicity, highlighted important functional domains of the abnormal proteins, and pointed to rational therapy.
doi:10.1007/s12031-009-9229-0
PMCID: PMC3050586  PMID: 19688192
Congenital myasthenic syndromes; Acetylcholine esterase; Choline acetyltransferase; Acetylcholine receptor; Dok-7
20.  Tannic acid facilitates expression of the polypyrimidine tract binding protein and alleviates deleterious inclusion of CHRNA1 exon P3A due to an hnRNP H-disrupting mutation in congenital myasthenic syndrome 
Human Molecular Genetics  2009;18(7):1229-1237.
We recently reported that the intronic splice-site mutation IVS3-8G>A of CHRNA1 that encodes the muscle nicotinic acetylcholine receptor α subunit disrupts binding of a splicing repressor, hnRNP H. This, in turn, results in exclusive inclusion of the downstream exon P3A. The P3A(+) transcript encodes a non-functional α subunit that comprises 50% of the transcripts in normal human skeletal muscle, but its functional significance remains undetermined. In an effort to search for a potential therapy, we screened off-label effects of 960 bioactive chemical compounds and found that tannic acid ameliorates the aberrant splicing due to IVS3-8G>A but without altering the expression of hnRNP H. Therefore, we searched for another splicing trans-factor. We found that the polypyrimidine tract binding protein (PTB) binds close to the 3′ end of CHRNA1 intron 3, that PTB induces skipping of exon P3A and that tannic acid increases the expression of PTB in a dose-dependent manner. Deletion assays of the PTB promoter region revealed that the tannic acid-responsive element is between positions −232 and −74 from the translation initiation site. These observations open the door to the discovery of novel therapies based on PTB overexpression and to detecting possible untoward effects of the overexpression.
doi:10.1093/hmg/ddp023
PMCID: PMC2655771  PMID: 19147685
21.  Mutation in BAG3 Causes Severe Dominant Childhood Muscular Dystrophy 
Annals of neurology  2009;65(1):83-89.
Objective
Myofibrillar myopathies (MFM) are morphologically distinct but genetically heterogeneous muscular dystrophies in which disintegration of Z disks and then of myofibrils is followed by ectopic accumulation of multiple proteins. Cardiomyopathy, neuropathy, and dominant inheritance are frequent associated features. Mutations in αB-crystallin, desmin, myotilin, Zasp, or filamin-C can cause MFM, and were detected in 32/85 patients of the Mayo MFM cohort. Bag3, another Z-disk associated protein, has antiapoptotic properties and its targeted deletion in mice causes fulminant myopathy with early lethality. We therefore searched for mutations in BAG3 in 53 unrelated MFM patients.
Methods
We searched for mutations in BAG3 by direct sequencing and excluded polymorphism using allele-specific PCR in relatives and 200 control subjects. We analyzed structural changes in muscle by histochemistry, immunocytochemistry and electron microscopy, examined mobility of the mutant Bag3 by nondenaturing electrophoresis, and searched for abnormal aggregation of the mutant protein in COS-7 cells.
Results
We identified a heterozygous p.Pro209Leu mutation in three patients. All presented in childhood, had progressive limb and axial muscle weakness, and developed cardiomyopathy and severe respiratory insufficiency in their teens; two had rigid spines and one a peripheral neuropathy. Electron microscopy showed disintegration of Z disks, extensive accumulation of granular debris and larger inclusions, and apoptosis of 8% of the nuclei. On nondenaturing electrophoresis of muscle extracts, the Bag3 complex migrated faster in patient than control extracts, and expression of FLAG-labeled mutant and wild-type Bag3 in COS cells revealed abnormal aggregation of the mutant protein.
Interpretation
We conclude mutation in Bag3 defines a novel severe autosomal dominant childhood muscular dystrophy.
doi:10.1002/ana.21553
PMCID: PMC2639628  PMID: 19085932
22.  hnRNP H enhances skipping of a nonfunctional exon P3A in CHRNA1 and a mutation disrupting its binding causes congenital myasthenic syndrome 
Human Molecular Genetics  2008;17(24):4022-4035.
In humans and great apes, CHRNA1 encoding the muscle nicotinic acetylcholine receptor α subunit carries an inframe exon P3A, the inclusion of which yields a nonfunctional α subunit. In muscle, the P3A(−) and P3A(+) transcripts are generated in a 1:1 ratio but the functional significance and regulation of the alternative splicing remain elusive. An intronic mutation (IVS3-8G>A), identified in a patient with congenital myasthenic syndrome, disrupts an intronic splicing silencer (ISS) and results in exclusive inclusion of the downstream P3A exon. We found that the ISS-binding splicing trans-factor was heterogeneous nuclear ribonucleoprotein (hnRNP) H and the mutation attenuated the affinity of hnRNP for the ISS ∼100-fold. We next showed that direct placement of hnRNP H to the 3′ end of intron 3 silences, and siRNA-mediated downregulation of hnRNP H enhances recognition of exon P3A. Analysis of the human genome suggested that the hnRNPH-binding UGGG motif is overrepresented close to the 3′ ends of introns. Pursuing this clue, we showed that alternative exons of GRIP1, FAS, VPS13C and NRCAM are downregulated by hnRNP H. Our findings imply that the presence of the hnRNP H-binding motif close to the 3′ end of an intron is an essential but underestimated splicing regulator of the downstream exon.
doi:10.1093/hmg/ddn305
PMCID: PMC2638575  PMID: 18806275
23.  Dok-7 Myasthenia: Phenotypic and Molecular Genetic Studies in 16 Patients 
Annals of neurology  2008;64(1):71-87.
Objective
Detailed analysis of phenotypic and molecular genetic aspects of Dok-7 myasthenia in 16 patients.
Methods
We assessed our patients by clinical and electromyographic studies, by intercostal muscle biopsies for in vitro microelectrode analysis of neuromuscular transmission and quantitative electron microscopy EM of 409 end plates (EPs), and by mutation analysis, and expression studies of the mutants.
Results
The clinical spectrum varied from mild static limb-girdle weakness to severe generalized progressive disease. The synaptic contacts were single or multiple, and some, but not all, were small. In vitro microelectrode studies indicated variable decreases of the number of released quanta and of the synaptic response to acetylcholine; acetylcholine receptor (AChR) channel kinetics were normal. EM analysis demonstrated widespread and previously unrecognized destruction and remodeling of the EPs. Each patient carries 2 or more heteroallelic mutations: 11 in genomic DNA, 7 of which are novel; and 6 identifiable only in complementary DNA or cloned complementary DNA, 3 of which are novel. The pathogenicity of the mutations was confirmed by expression studies. Although the functions of Dok-7 include AChR β-subunit phosphorylation and maintaining AChR site density, patient EPs showed normal AChR β-subunit phosphorylation, and the AChR density on the remaining junctional folds appeared normal.
Interpretation
First, the clinical features of Dok-7 myasthenia are highly variable. Second, some mutations are complex and identifiable only in cloned complementary DNA. Third, Dok-7 is essential for maintaining not only the size but also the structural integrity of the EP. Fourth, the profound structural alterations at the EPs likely contribute importantly to the reduced safety margin of neuromuscular transmission.
doi:10.1002/ana.21408
PMCID: PMC2570015  PMID: 18626973
24.  Naturally Occurring Mutations at the Acetylcholine Receptor Binding Site Independently Alter ACh Binding and Channel Gating 
The Journal of General Physiology  2002;120(4):483-496.
By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, ɛN182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant αδ site. Studies of the analogous mutation in the δ subunit, δN187Y, disclose rate constants for ACh occupancy of the nonmutant αɛ site. The second CMS mutation, ɛD175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. ɛD175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, ɛN182Y localizes to the interface with the α subunit, and ɛD175 to the entrance of the ACh binding cavity. Both ɛN182Y and ɛD175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring ɛN182 and ɛD175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.
doi:10.1085/jgp.20028568
PMCID: PMC2229537  PMID: 12356851
congenital myasthenic syndrome; single channel kinetics; agonist binding; channel gating; mutation analysis
25.  Fundamental Gating Mechanism of Nicotinic Receptor Channel Revealed by Mutation Causing a Congenital Myasthenic Syndrome 
The Journal of General Physiology  2000;116(3):449-462.
We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation εA411P in the amphipathic helix of the acetylcholine receptor (AChR) ε subunit. Myasthenic patients from three unrelated families are either homozygous for εA411P or are heterozygous and harbor a null mutation in the second ε allele, indicating that εA411P is recessive. We expressed human AChRs containing wild-type or A411P ε 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 εA411P. Prolines engineered into positions flanking residue 411 of the ε subunit greatly increase the range of activation kinetics similar to εA411P, whereas prolines engineered into positions equivalent to εA411 in β and δ subunits are without effect. Thus, the amphipathic helix of the ε 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.
PMCID: PMC2233692  PMID: 10962020
congenital myasthenic syndrome; single channel kinetics; hidden Markov modeling; channel gating; energy landscape

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