Congenital Myasthenic Syndromes PDF

HHS Public Access Author manuscript Lancet Neurol. Author manuscript; available in PMC 2016 April 01. Author Manuscript Published in final edited form as: Lancet Neurol. 2015 April ; 14(4): 42...

HHS Public Access Author manuscript Lancet Neurol. Author manuscript; available in PMC 2016 April 01. Author Manuscript Published in final edited form as: Lancet Neurol. 2015 April ; 14(4): 420–434. doi:10.1016/S1474-4422(14)70201-7. Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment Andrew G. Engel, Xin-Ming Shen, Duygu Selcen, and Steven M. Sine Department of Neurology (AG Engel MD, X-M Shen PhD, D Selcen MD) and Department of Physiology and Biomedical Engineering (SM Sine PhD), Mayo Clinic, Rochester, MN 55905 USA Author Manuscript Abstract The congenital myasthenic syndromes are diverse disorders linked by abnormal signal transmission at the motor endplate that stem from defects in single or multiple proteins. Multiple endplate proteins are affected by mutations of single enzymes required for protein glycosylation, and deletion of PREPL exerts its effect by activating adaptor protein 1. Finally, neuromuscular transmission is also impaired in some congenital myopathies. The specific diagnosis of some syndromes is facilitated by clinical clues pointing to a disease gene. In absence of such clues, exome sequencing is a useful tool for finding the disease gene. Deeper understanding of disease mechanisms come from structural and in vitro electrophysiologic studies of the patient endplate, and from engineering the mutant and wild-type gene into a suitable expression system that can be interrogated by appropriate electrophysiologic and biochemical studies. Most CMS are treatable. Importantly, however, some medication beneficial in one syndrome can be detrimental in another. Author Manuscript Introduction The congenital myasthenic syndromes (CMS) are inherited disorders in which the safety margin of neuromuscular transmission is impaired by one or more specific mechanisms. The CMS have been recognized as distinct clinical entities since the 1970s, after the autoimmune origin of myasthenia gravis and of the Lambert-Eaton myasthenic syndromes had been established. Initially, the CMS were delineated by combined clinical, in vitro electrophysiologic, and structural studies. The study of the CMS gained further impetus when sequences of genes coding for EP-associated proteins were determined and with the advent of Sanger sequencing. In the past three years, whole exome sequencing facilitated discovery of novel CMS at an accelerated pace and by now no fewer than 20 CMS disease Author Manuscript gene have been identified. Figure 1 shows the distribution of identified CMS disease proteins at the EP. In this review we consider the factors that affect the safety margin of neuromuscular transmission, classify the CMS identified to date, describe their distinguishing features and pathogenesis, and consider available therapies. Correspondence to Dr. Andrew G Engel, Mayo Clinic, Rochester, MN 55905, USA. Contributors All authors contributed equally to the literature search, figures, study design, data analysis, data interpretation, and manuscript preparation. Conflicts of Interest None of the authors reports a conflict of interest.  Engel et al. Page 2 The safety margin of neuromuscular transmission Author Manuscript The safety margin of neuromuscular transmission (NMT) is a function of the difference between the depolarization caused by the EP potential (EPP) and the depolarization required to activate the voltage gated Nav1.4 channels deployed on the postsynaptic membrane.1 The amplitude of the EPP is a function of the number of acetylcholine (ACh) molecules per synaptic vesicle, the number of synaptic vesicles released by nerve impulse, and the efficacy of the released quanta. Quantal efficacy is determined by the EP geometry, the density and functional state of AChE in the synaptic space, the density, affinity for ACh, and kinetic properties of AChR, and the density and kinetic properties of Nav1.4.1,2 Classification of the CMS Table 1 shows a classification of the currently recognized CMS based on 356 index patients Author Manuscript investigated at the Mayo Clinic. Initially, the CMS were classified according to the location of the mutant protein as presynaptic, synaptic basal lamina-associated, and postsynaptic. The current classification takes into account the CMS caused by defects in protein glycosylation where the abnormal proteins are located at any EP site, and assigns a separate group to the CMS caused by defects of EP development and maintenance. Of interest, in another series of 680 CMS patients, frequencies of mutations in ChAT, ColQ, AChR subunits, rapsyn, and Dok7 were identical to those investigated at the Mayo Clinic.3 Diagnosis Generic diagnosis A generic diagnosis of a CMS can be made on the basis of onset at birth to early childhood, fatigable weakness affecting especially the ocular and other cranial muscles, a positive Author Manuscript family history, and a decremental EMG response or an abnormal single-fiber EMG. However, some CMS present later in life, the weakness can affect proximal and torso rather than cranial muscles, and the decremental EMG response may be detected only after prolonged subtetanic stimulation. Tests for anti-AChR and anti-MuSK antibodies are indicated in sporadic patients after the age of 1 year and in arthrogrypotic infants even if the mother has no myasthenic symptoms. Panel 1 lists the differential diagnosis of the CMS. Genetic diagnosis The genetic diagnosis of a specific CMS is greatly facilitated when clinical, and EMG studies point to a candidate gene (Panel 2). If a sufficient number of affected and unaffected relatives is available, linkage analysis can point to a candidate chromosomal locus. This approach works best in inbred populations or multiplex families.4 Author Manuscript Testing for CMS mutations in previously identified CMS genes is now commercially available but is best used in a targeted manner based on specific clinical clues. In recent years, whole exome sequencing has been used to identify CMS mutations. This approach presently captures ~97% of the entire exome but reads only 75% of the exome with more than 20× coverage. Analysis is facilitated if DNA from both parents and more than one affected family member is tested. Exome sequencing with the bioinformatics analysis is still Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 3 expensive and the putative mutations must be confirmed by Sanger sequencing. The analysis Author Manuscript can miss pathogenic noncoding variants and large deletions or duplications but the latter can be identified by array based comparative genomic hybridization.5 Finally, synonymous variants that can cause exon skipping are often filtered out. If a novel CMS disease gene is discovered, then expression studies with the genetically engineered mutant protein can be used to confirm its pathogenicity.6 Deeper insights into disease mechanisms can be obtained by in vitro analysis of neuromuscular transmission and structural studies of the neuromuscular junction (Panel 3). Although available at only few medical centers, they are important for identifying direct effects of the mutations on neuromuscular transmission, characterizing novel CMS, and providing clues for therapy. Currently available therapies Current therapies for the CMS include cholinergic agonists, namely pyridostigmine and 3,4- Author Manuscript diaminopyridine (3,4-DAP), long-lived open-channel blockers of the AChR ion channel, and adrenergic agonists. Pyridostigmine acts by inhibiting AChE in the synaptic basal lamina which increases the number of AChRs activated by a single quantum. 3,4-Diaminopyridine (3,4-DAP) increases the number of ACh quanta released by nerve impulse. Alone, and especially in combination, they increase the amplitude of the EP potential and thereby the safety margin of neuromuscular transmission. Thus they are beneficial in patients with EP AChR deficiency and also in the fast-channel syndromes in which the safety margin of transmission is compromised by a decreased synaptic response to ACh and the abnormally fast decay of the synaptic current. Fluoxetine and quinidine are long-lived open-channel blockers of AChR used in the Author Manuscript treatment of the slow-channel syndrome. By curtailing the duration of the prolonged synaptic currents, they prevent a depolarization block and desensitization of AChR at physiologic rates of stimulation and mitigate the cationic overloading of the postsynaptic region that causes degeneration of the junctional folds and alters the EP geometry. The adrenergic agonists albuterol and ephedrine were empirically found to be effective in the CMS caused by mutations in the ColQ component of AChE, Dok-7, and laminin-β2 as well as in some patients harboring low-expressor mutations of the AChR. The mechanisms by which these agents improve neuromuscular transmission is not understood. Finally, it is important to note that agents that benefit one type of CMS can be ineffective or harmful in another type. For example, patients harboring low-expressor or fast-channel Author Manuscript mutation in AChR are improved by cholinergic agonists whereas patients with slow-channel mutations in AChR are worsened by these medications. Patients harboring mutations in Dok-7 are rapidly worsened by cholinergic agonists but are improved by adrenergic agonists. Therefore it is essential that a molecular diagnosis should inform the choice of therapy. Finally, the cholinergic agonists pyridostigmine and 3,4-DAP exert their effect as soon as the medication is absorbed whereas the adrenergic agonists and the AChR channel blockers act more slowly over days, weeks, or months. Table 2 summarizes the pharmacotherapy of the currently recognized CMS. Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 4 Presynaptic syndromes Author Manuscript Choline acetyltransferase (ChAT) deficiency ChAT catalyzes the synthesis of ACh from acetyl-CoA and choline in cholinergic neurons. The diagnosis is suggested by sudden episodes of apnea provoked by stress or without apparent cause occurring in patients with few or no myasthenic symptoms. Some patients are apneic and hypotonic at birth; others are normal at birth and develop apneic attacks during infancy or childhood.7–11 However, apneic episodes also can occur in patients with Na-channel myasthenia2 or with mutations in rapsyn.12,13 In some children an apneic attack is followed by ventilatory failure for weeks.14 Few patients never breathe spontaneously, and some develop cerebral atrophy from hypoxemia.11 A clue to the identity of the disease gene came from the observation that subtetanic stimulation reduced the amplitude of the compound muscle action potential (CMAP) and of EPP to 50% below the baseline (normal Author Manuscript G) have a mild phenotype with ptosis, prognathism, severe masticatory and facial muscle weakness, and hypernasal speech.77 Congenital defects of glycosylation Author Manuscript Glycosylation increases solubility, folding, stability, assembly, and intracellular transport of nascent peptides. O-glycosylation occurs in the Golgi apparatus with addition of sugar residues to hydroxyl groups of serine and threonine; N-glycosylation occurs in the endoplasmic reticulum in sequential reactions that decorate the amino group of asparagine with a core glycan composed of 2 glucose, 9 mannose and 2 N-acetylglucosamine (GlcNAc)78,79 To date, defects in four enzymes subserving glycosylation cause a CMS: GFPT1 (glutamine fructose-6-phosphate transaminase),4,80 DPAGT1 (dolichyl-phosphate [UDP-N-acetylglucosamine] N-acetylglucosaminephosphotransferase 1),81,82 ALG2 (alpha-1,3-mannosyl transferase), and ALG14 (UDP-N-acetylglucosaminyltransferase subunit).83 Tubular aggregates of the sarcoplasmic reticulum (SR) in muscle fibers are a phenotypic clue to the diagnosis but are not present in all patients. Because glycosylated proteins are present at all EP sites, NMT is compromised by a combination of pre- and Author Manuscript postsynaptic abnormalities. Defects in GFPT1 GFPT1 controls glucose entry into the hexosamine pathway, and hence formation of precursors for N- and O-linked protein glycosylation. A CMS caused by defects in GFPT1 causing limb-girdle muscle weakness responsive to pyridostigmine was reported in 16 patients in 2011.4 A subsequent study of 11 patients revealed slowly progressive weakness in 10, but one patient with mutations disrupting the muscle-specific exon of GFPT1 never moved in utero, was arthrogrypotic at birth, and remains bedfast and tube-fed at age 8 years. She has a severe myopathy with numerous dilated and degenerating vesicular profiles, autophagic vacuoles, and bizarre apoptotic nuclei.80 Muscle specimens in 9 less affected patients revealed tubular aggregates in 6. Electron microscopy demonstrated abnormally Author Manuscript small EP regions and poorly developed junctional folds. In vitro electrophysiology studies revealed a reduced synaptic response to ACh and decreased quantal release in the most severely affected patient. That many EPs in this CMS are underdeveloped likely stems from hypoglycosylation and altered function of EP-associated glycoproteins, such as MuSK, agrin, and dystroglycans. Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 11 Defects in DPAGT1 Author Manuscript DPAGT1 catalyzes the first committed step of N-linked protein glycosylation. DPAGT1 deficiency predicts impaired asparagine glycosylation of multiple proteins distributed throughout the organism, but in the first 5 patients harboring DPAGT1 mutations only neuromuscular transmission was adversely affected; this was attributed to reduced AChR expression at the EP but the patient EPs were not examined.81 A subsequent study of two siblings and of a third patient with DPAGT1 deficiency extended the phenotypic spectrum of the disease.82 These patients have moderately severe to severe weakness and are intellectually disabled. The siblings respond poorly to pyridostigmine and 3,4-DAP; the third patient responds partially to pyridostigmine and albuterol. Whole exome sequencing revealed that the siblings harbored an M1L mutation that reduces protein expression, and H375Y which decreases enzyme activity. The third patient carries V264M that abolishes enzyme activity and a synonymous L120L mutation that markedly augments exon skipping Author Manuscript resulting in many skipped and few nonskipped alleles. Intercostal muscle studies showed fiber type disproportion, small tubular aggregates, and an autophagic vacuolar myopathy. Electron microscopy revealed few degenerating EPs and small pre- and postsynaptic regions. Evoked quantal release, postsynaptic response to ACh, and the EP AChR content were all reduced to ~50% of normal. Immunoblots of muscle extracts with two different antibodies indicated decreased to absent glycosylation of different proteins, including that of STIM1, an SR-associated calcium sensor that operates in concert with Orai1 on the plasma membrane to homeostatically regulate the SR calcium content.84 Because mutations in STIM1 cause a tubular aggregate myopathy,85 STIM1 hypoglycosylation is a likely cause of the tubular aggregates in muscle in N-glycosylation disorders. Author Manuscript Defects in ALG2 and ALG14 ALG2 catalyzes the second and third committed steps of N-glycosylation. In one kinship four affected siblings were homozygous for an insertion/deletion mutation, and another patient was homozygous for a low-expressor V68G mutation. ALG14 forms a multiglycosyltransferase complex with ALG13 and DPAGT1 and thus contributes to the first committed step of N-glycosylation. In one family two affected siblings carried heteroallelic P65L and V68G mutations. EP ultrastructure and parameters of neuromuscular transmission were not investigated.83 Other myasthenic syndromes PREPL deletion syndrome Author Manuscript The hypotonia-cystinuria syndrome is caused by recessive deletions involving the SLC3A1 and PREPL genes at chromosome 2p21. The major clinical features are type A cystinuria, growth hormone deficiency, muscle weakness, ptosis, and feeding problems. A patient with isolated PREPL deficiency had myasthenic symptoms since birth, a positive edrophonium test and growth hormone deficiency but no cystinuria, and responded transiently to pyridostigmine during infancy.86 She harbors a paternally inherited nonsense mutation in PREPL and a maternally inherited deletion involving both PREPL and SLC3A1; therefore Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 12 the PREPL deficiency determines the phenotype. PREPL expression was absent from the Author Manuscript patient’s muscle and EPs. EP studies revealed decreased evoked quantal release and small MEPPs despite robust EP AChR expression.86 Because PREPL is an essential activator of the clathrin associated adaptor protein 1 (AP1),87 and AP1 is required by the vesicular ACh transporter to fill the synaptic vesicles with ACh,88 the small MEPP is attributed to a decreased vesicular content of ACh. Plectin deficiency Organelle and tissue specific isoforms of plectin, encoded by PLEC, crosslink intermediate filaments to their targets in different tissues and thereby assure their cytoskeletal support. Defects in plectin can cause epidermolysis bullosa simplex (EBS),89 muscular dystrophy,90,91 and a myasthenic syndrome.92,93 The muscular dystrophy is caused by loss of cytoskeletal support of the muscle fiber organelles, which become dislocated, and of the Author Manuscript sarcolemma which displays multiple small defects allowing calcium ingress into the fibers.93 The two patients investigated by us respectively harbor Q2057X and R2319X, and a shared c.12043dupG mutation92,93 Both patients had EBS since infancy and later developed a progressive myopathy and a CMS refractory to pyridostigmine. Both had a decremental EMG response on repetitive nerve stimulation, and half-normal MEPPs attributed to degeneration of the junctional folds with loss of AChR and altered EP geometry.93 Defect in Nav1.4 This CMS was detected in a single patient with brief abrupt attacks of muscle weakness and respiratory arrest that caused an anoxic encephalopathy. The synaptic response to ACh and quantal release by nerve impulse were normal, but normal amplitude EPPs failed to generate Author Manuscript muscle action potentials, pointing to a defect in action potential generation. Mutation analysis of SCN4A, the gene encoding Nav1.4, revealed two mutations (S246L in the S4/S5 linker in domain I and V1442E in S4/S5 linker in domain IV). Nav1.4 expression at the EPs was normal. Expression studies in HEK cells of the V1442E-sodium channels revealed marked enhancement of fast inactivation near the resting membrane potential and enhanced use-dependent inactivation on high frequency stimulation. S246L had only minor kinetic effects and is likely a benign mutation. The safety margin of NMT is compromised because most Nav1.4 channels are inexcitable in the resting state.2 The patient responded partially to therapy with pyridostigmine and acetazolamide. Myasthenias associated with congenital myopathies Eyelid ptosis, ophthalmoparesis, weakness of facial muscles, exercise intolerance, a Author Manuscript decremental EMG study, and response to pyridostigmine have been documented in patients with centronuclear myopathies (CNM) caused by mutations in amphiphysin (BIN1),94 myotubularin (MTM1),95 and dynamin 2 (DNM2)96 as well as in other CNM patients with no identified mutations.97 Importantly, knockdown of MTM1 or DNM2 in zebrafish causes reduced spontaneous and touch-evoked movements that respond dramatically to edrophonium.95,96 Detailed investigation of neuromuscular transmission in an adult CNM patient with myasthenic symptoms and no identified mutations revealed EP remodeling, Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 13 mild EP AChR deficiency, simplified postsynaptic regions, 60% reduction of the MEPP Author Manuscript amplitude, and a 40% decrease of evoked quantal release attributed to a decreased number of synaptic vesicles available for release.97 Interestingly, two patients with a congenital myopathy caused by tropomyosin 3 deficiency had signs and symptoms mimicking a CMS.98 Conclusions and future directions Initially, molecular bases for the CMS were approached with in-depth clinical evaluation combined with electrophysiologic and morphometric analyses of patient endplates. With the introduction of gene cloning and sequencing of complementary DNAs, the candidate gene approach provided a powerful complement to these analyses. After a variant in a candidate gene was identified, the goal was to demonstrate its pathogenicity, which could be achieved by incorporating the wild type and mutant genes into a heterologous expression system and Author Manuscript then assessing the level of expression of the mutant protein and its functional properties by appropriate methods that included the patch-clamp studies to dissect the kinetic effects of AChR mutants, and enzyme assays of the wild-type and mutant species of ChAT, DPAGT1 and AChE. The net result was demonstration that the CMS were caused by a diversity of disease targets and molecular mechanisms, which together guided an individualized therapy. Despite the power of the candidate gene approach, in some CMS the disease gene has remained elusive. For these CMS, whole exome sequencing, available in the past 3 years, proved to be a lodestone to discovery of unsuspected defects in genes subserving protein glycosylation, and more are surely in the pipeline. The power of this approach is enhanced if multiple family members, and especially trios are analyzed, and interpretation of the results is greatly facilitated by phenotypic clues. Although whole exome sequencing fails to identify Author Manuscript large scale duplications or deletions, these can now be detected by microarray based comparative gene hybridization. Another future approach will be the use of custom made microarrays designed to detect mutations in heretofore identified CMS disease genes. A drawback of this method will be that it cannot identify newly emerging CMS disease genes. However, in many patients it will obviate the need for more expensive whole exome sequencing. It is highly likely that more CMS disease genes will be discovered but demonstration of pathogenicity associated with individual mutations will remain a necessity. This will take advantage of tried and true methods for gene expression and functional comparison of the wild type and mutant gene products. Clinicians armed with knowledge of the molecular mechanism behind the aberrant gene product will be in an advantageous position to promote rational and individualized therapy of each form of CMS. Author Manuscript Supplementary Material Refer to Web version on PubMed Central for supplementary material. Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 14 Acknowledgments Author Manuscript Sources of support: Drs. Andrew G Engel and Xin-Ming Shen were supported by NIH Grant NS06277. SMS was supported by NIH grant NS031744. References 1. Wood SJ, Slater CP. Safety factor at the neuromuscular junction. Prog Neurobiol. 2001; 64:393– 429. [PubMed: 11275359] 2. Tsujino A, Maertens C, Ohno K, et al. Myasthenic syndrome caused by mutation of the SCN4A sodium channel. Proc Natl Acad Sci USA. 2003; 100:7377–7382. [PubMed: 12766226] 3. Abicht A, Dusl M, Guergueltcheva V, et al. Congenital myasthenic syndromes: achievements and limitations of phenotype-guided gene-after-gene sequencing in diagnostic practice: a study of 680 patients. Hum Mutat. 2014; 33:1474–1484. [PubMed: 22678886] 4. Senderek J, Muller JS, Dusl M, et al. Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect. Am J Hum Genet. 2011; 88:162–172. [PubMed: 21310273] Author Manuscript 5. Shinawi M, Cheung SW. The array CGH and its clinical applications. Drug Discov Today. 2008; 13:760–770. [PubMed: 18617013] 6. Engel AG. The investigation of congenital myasthenic syndromes. Ann N Y Acad Sci. 1993; 681:425–434. [PubMed: 7689310] 7. Byring RF, Pihko H, Shen X-M, et al. Congenital myasthenic syndrome associated with episodic apnea and sudden infant death. Neuromuscul Disord. 2002; 12:548–553. [PubMed: 12117478] 8. Maselli RA, Chen D, Mo D, et al. Choline acetyltransferase mutations in myasthenic syndrome due to deficient acetylcholine resynthesis. Muscle Nerve. 2003; 27:180–187. [PubMed: 12548525] 9. Mallory LA, Shaw JG, Burgess SL, et al. Congenital myasthenic syndrome with episodic apnea. Pediatr Neurol. 2009; 41:42–45. [PubMed: 19520274] 10. Schara U, Christen H-J, Durmus H, et al. Long-term follow-up in patients with congenital myasthenic syndrome due to CHAT mutations. Eur J Paediatr Neurol. 2010; 14:326–333. [PubMed: 19900826] 11. Shen X-M, Crawford TO, Brengman J, et al. Functional consequences and structural interpretation Author Manuscript of mutations in human choline acetyltransferase. Hum Mutat. 2011; 32:1259–1267. [PubMed: 21786365] 12. Ohno K, Engel AG, Shen X-M, et al. Rapsyn mutations in humans cause endplate acetylcholine receptor deficiency and myasthenic syndrome. Am J Hum Genet. 2002; 70:875–885. [PubMed: 11791205] 13. Burke G, Cossins J, Maxwell S, et al. Rapsyn mutations in hereditary myasthenia. Distinct early- and late-onset phenotypes. Neurology. 2003; 61:826–828. [PubMed: 14504330] 14. Kraner S, Lufenberg I, Strassburg HM, Sieb JP, Steinlein OK. Congenital myasthenic syndrome with episodic apnea in patients homozygous for a CHAT missense mutation. Arch Neurol. 2003; 60:761–763. [PubMed: 12756141] 15. Ohno K, Tsujino A, Shen XM, et al. Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans. Proc Natl Acad Sci USA. 2001; 98:2017– 2022. [PubMed: 11172068] 16. Massoulié J, Pezzementi L, Bon S, Krejci E, Valette F-M. Molecular and cellular biology of cholinesterases. Prog Neurobiol. 1993; 41:31–91. [PubMed: 8321908] Author Manuscript 17. Cartaud A, Strochlic L, Guerra M, et al. MuSK is required for anchoring acetylcholinesterase at the neuromuscular junction. J Cell Biol. 2004; 165:505–515. [PubMed: 15159418] 18. Engel AG, Lambert EH, Gomez MR. A new myasthenic syndrome with end-plate acetylcholinesterase deficiency, small nerve terminals, and reduced acetylcholine release. Ann Neurol. 1977; 1:315–330. [PubMed: 214017] 19. Hutchinson DO, Walls TJ, Nakano S, et al. Congenital endplate acetylcholinesterase deficiency. Brain. 1993; 116:633–653. [PubMed: 8390325] Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 15 20. Ohno K, Brengman JM, Tsujino A, Engel AG. Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzyme. Proc Natl Author Manuscript Acad Sci USA. 1998; 95:9654–9659. [PubMed: 9689136] 21. Bestue-Cardiel M, de-Cabazon-Alvarez AS, Capablo-Liesa JL, et al. Congenital endplate acetylcholinesterase deficiency responsive to ephedrine. Neurology. 2005; 65:144–146. [PubMed: 16009904] 22. Mihaylova V, Muller JS, Vilchez JJ, Salih MA, et al. Clinical and molecular genetic findings in COLQ-mutant congenital myasthenic syndromes. Brain. 2008; 131:747–759. [PubMed: 18180250] 23. Liewluck T, Selcen D, Engel AG. Beneficial effects of albuterol in congenital endplate acetylcholinesterase deficiency and DOK-7 myasthenia. Muscle Nerve. 2011; 44:789–794. [PubMed: 21952943] 24. Chan SH, Wong VC, Engel AG. Congenital myasthenia syndrome with a novel COLQ mutation responsive to albuterol. Pediatr Neurol. 2012; 47:137–140. [PubMed: 22759693] 25. Maselli RA, Ng JJ, Andreson JA, et al. Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome. J Med Genet. 2009; 46:203–208. [PubMed: 19251977] Author Manuscript 26. Unwin N. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J Mol Biol. 2005; 346:967–989. [PubMed: 15701510] 27. Milone M, Wang H-L, Ohno K, et al. Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor ɛ subunit. Neuron. 1998; 20:575–588. [PubMed: 9539130] 28. Wang H-L, Ohno K, Milone M, et al. Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome. J Gen Physiol. 2000; 116:449– 460. [PubMed: 10962020] 29. Brejc K, van Dijk WV, Schuurmans M, et al. Crystal structure of ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. NAT. 2001; 411:269–276. 30. Sine SM, Claudio T, Sigworth FJ. Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts: single-channel current kinetics reveal distinct agonist binding affinities. J Gen Physiol. 1990; 96:395–437. [PubMed: 1698917] 31. Sine SM. The nicotinic receptor ligand binding domain. J Neurobiol. 2002; 53(4):431–446. Author Manuscript [PubMed: 12436411] 32. Engel AG, Ohno K, Sine SM. Sleuthing molecular targets for neurological diseases at the neuromuscular junction. Nature Rev Neurosci. 2003; 4:339–352. [PubMed: 12728262] 33. Engel AG, Ohno K, Bouzat C, Sine SM, Griggs RG. End-plate acetylcholine receptor deficiency due to nonsense mutations in the ɛ subunit. Ann Neurol. 1996; 40:810–817. [PubMed: 8957026] 34. Ohno K, Quiram P, Milone M, et al. Congenital myasthenic syndromes due to heteroallelic nonsense/missense mutations in the acetylcholine receptor ɛ subunit gene: identification and functional characterization of six new mutations. Hum Mol Genet. 1997; 6:753–766. [PubMed: 9158150] 35. Plomp JJ, van Kempen GThH, De Baets MB, et al. Acetylcholine release in myasthenia gravis: Regulation at single end-plate level. Ann Neurol. 1995; 37:627–636. [PubMed: 7755358] 36. Engel AG. The therapy of congenital myasthenic syndromes. Neurotherapeutics. 2007; 4:252–257. [PubMed: 17395135] 37. Sadeh M, Shen X-M, Engel AG. Beneficial effect of albuterol in congenital myasthenic syndrome with ɛ subunit mutations. Muscle Nerve. 2011; 44:289–291. [PubMed: 21721016] Author Manuscript 38. Ohno K, Hutchinson DO, Milone M, et al. Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the ɛ subunit. Proc Natl Acad Sci USA. 1995; 92:758–762. [PubMed: 7531341] 39. Harper CM, Engel AG. Quinidine sulfate therapy for the slow-channel congenital myasthenic syndrome. Ann Neurol. 1998; 43:480–484. [PubMed: 9546329] 40. Harper CM, Fukudome T, Engel AG. Treatment of slow channel congenital myasthenic syndrome with fluoxetine. Neurology. 2003; 60:170–173. 41. Colomer J, Muller JS, Vernet A, et al. Long-term improvement of slow-channel myasthenic syndrome with fluoxetine. Neuromuscul Disord. 2006; 16:329–333. [PubMed: 16621558] Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 16 42. Chaouch A, Muller JS, Guergueltcheva V, et al. A retrospective clinical study of the slow-channel congenital myasthenic syndrome. J Neurol. 2012; 259:474–481. [PubMed: 21822932] Author Manuscript 43. Ohno K, Wang H-L, Milone M, et al. Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor ɛ subunit. Neuron. 1996; 17:157–170. [PubMed: 8755487] 44. Shen XM, Brengman J, Edvardson S, Sine SM, Engel AG. Highly fatal fast-channel congenital syndrome caused by AChR ɛ subunit mutation at the agonist binding site. Neurology. 2012; 79:449–454. [PubMed: 22592360] 45. Shen X-M, Ohno K, Tsujino A, et al. Mutation causing severe myasthenia reveals functional asymmetry of AChR signature Cys-loops in agonist binding and gating. J Clin Invest. 2003; 111:497–505. [PubMed: 12588888] 46. Shen X-M, Brengman J, Sine SM, Engel AG. Myasthenic syndrome AChRα C-loop mutant disrupts initiation of channel gating. J Clin Invest. 2012; 122:2613–2621. [PubMed: 22728938] 47. Shen X-M, Fukuda T, Ohno K, Sine SM, Engel AG. Congenital myasthenia-related AChR δ subunit mutation interferes with intersubunit communication essential for channel gating. J Clin Invest. 2008; 118:1867–1876. [PubMed: 18398509] Author Manuscript 48. Wang H-L, Milone M, Ohno K, et al. Acetylcholine receptor M3 domain: Stereochemical and volume contributions to channel gating. Nature Neurosci. 1999; 2:226–233. [PubMed: 10195214] 49. Shen X-M, Ohno K, Sine SM, Engel AG. Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle AChR M3-M4 linker. Brain. 2005; 128:345–355. [PubMed: 15615813] 50. Sine SM, Shen X-M, Wang H-L, et al. Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating. J Gen Physiol. 2002; 120:483– 496. [PubMed: 12356851] 51. Burden SJ, Yumoto N, Zhang W. The role of MuSK in synapse formation and neuromuscular disease. Cold Spring Harb Perspect Biol. 2013; 5:a009167. [PubMed: 23637281] 52. Huze C, Bauche S, Richard P, et al. Identification of an agrin mutation that causes congenital myasthenia and affects synapse function. Am J Hum Genet. 2009; 85:155–167. [PubMed: 19631309] 53. Maselli RA, Fernandez JM, Arredondo J, et al. LG2 agrin mutation causing severe congenital Author Manuscript myasthenic syndrome mimics functional characteristics of non-neural agrin (z-) agrin. Hum Genet. 2012; 131:1123–1135. [PubMed: 22205389] 54. Ohkawara B, Cabrera-Serrano M, Nakat T, et al. LRP4 third β-propeller domain mutations cause novel congenital myasthenic syndrome by compromising agrin-mediated MuSK signalling in a position-specific manner. Hum Mol Genet. 2014; 23:1856–1868. [PubMed: 24234652] 55. Chevessier F, Faraut B, Ravel-Chapuis A, et al. MUSK, a new target for mutations causing congenital myasthenic syndrome. Hum Mol Genet. 2004; 13:3229–3240. [PubMed: 15496425] 56. Mihaylova V, Salih MA, Mukhtar MM, et al. Refinement of the clinical phenotype in MUSK- related congenital myasthenic syndromes. Neurology. 2009; 73:1926–1928. [PubMed: 19949040] 57. Maselli R, Arredondo J, Cagney O, et al. Mutations in MUSK causing congenital myasthenic syndrome impair MuSK-Dok-7 interaction. Hum Mol Genet. 2010; 19:2370–2379. [PubMed: 20371544] 58. Ben Ammar A, Soltanzadeh P, Bauchê S, et al. A mutation causes MuSK reduced sensitivity to agrin and congenital myasthenia. PLoS One. 2013 Jan 9.8:e53826. [PubMed: 23326516] 59. Chevessier F, Girard E, Molgo J, et al. A mouse model for congenital myasthenic syndrome due to Author Manuscript MuSK mutations reveals defects in structure and function of neuromuscular junctions. Hum Mol Genet. 2008; 17:3577–3595. [PubMed: 18718936] 60. Gallenmuller C, Muller-Felber W, Dusl M, et al. Salbutamol-responsive limb-girdle congenital myasthenic syndrome due to a novel missese mutaion and heteroallelic deletion in MUSK. Neuromuscul Disord. 2014; 24:31–35. 2014. [PubMed: 24183479] 61. Okada K, Inoue A, Okada M, et al. The muscle protein Dok-7 is essential for neuromuscular synaptogenesis. Science. 2006; 312:1802–1805. [PubMed: 16794080] 62. Beeson D, Higuchi O, Palace J, et al. Dok-7 mutations underlie a neuromuscular junction synaptopathy. Science. 2006; 313:1975–1978. [PubMed: 16917026] Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 17 63. Muller JS, Herczegfalvi A, Vilchez JJ, et al. Phenotypical spectrum of DOK7 mutations in congenital myasthenic syndromes. Brain. 2007; 130:1497–1506. [PubMed: 17439981] Author Manuscript 64. Selcen D, Milone M, Shen X-M, et al. Dok-7 myasthenia: phenotypic and molecular genetic studies in 16 patients. Ann Neurol. 2008; 64:71–87. [PubMed: 18626973] 65. Anderson JA, Ng JJ, Bowe C, et al. Variable phenotypes associated with mutations in DOK7. Muscle Nerve. 2008; 37:448–456. [PubMed: 18161030] 66. Ammar AB, Petit F, Alexandri K, et al. Phenotype-genotype analysis in 15 patients presenting a congenital myasthenic syndrome due to mutations in DOK7. J Neurol. 2010; 257:754–766. [PubMed: 20012313] 67. Slater CR, Fawcett PRW, Walls TJ, et al. Pre- and postsynaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with ‘limb-girdle myasthenia’. Brain. 2006; 127:2061–2076. [PubMed: 16870884] 68. Schara U, Barisic N, Deschauer M, et al. Ephedrine therapy in eight patients with congenital myasthenic syndrome due to DOK7 mutations. Neuromuscul Disord. 2010; 19:828–832. [PubMed: 19837590] 69. Ramarao MK, Cohen JB. Mechanism of nicotinic acetylcholine receptor cluster formation by Author Manuscript rapsyn. Proc Natl Acad Sci USA. 1998; 95:4007–4012. [PubMed: 9520483] 70. Ramarao MK, Bianchetta MJ, Lanken J, Cohen JB. Role of rapsyn tetratricopeptide repeat and coiled-coil domains in self-association and nicotinic acetylcholine receptor clustering. J Biol Chem. 2001; 276:7475–7483. [PubMed: 11087759] 71. Zuber B, Unwin N. Structure and superorganization of the acetylcholine receptor-rapsyn complex. Proc Natl Acad Sci U S A. 2013; 110:10622–10627. [PubMed: 23754381] 72. Milone M, Shen XM, Selcen D, et al. Myasthenic syndrome due to defects in rapsyn: Clinical and molecular findings in 39 patients. Neurology. 2009; 73:228–235. [PubMed: 19620612] 73. Banwell BL, Ohno K, Sieb JP, Engel AG. Novel truncating RAPSN mutation causing congenital myasthenic syndrome responsive to 3,4-diaminopyridine. Neuromuscul Disord. 2004; 14:202–207. [PubMed: 15036330] 74. Skeie GO, Aurlien H, Müller JS, Norgard G, Bindoff LA. Unusual features in a boy with rapsyn N88K mutation. Neurology. 2006; 67:2262–2263. [PubMed: 17190963] 75. Müller JS, Mildner G, Müller-Felber W, et al. Rapsyn N88K is a frequent cause of CMS in Author Manuscript European patients. Neurology. 2003; 60:1805–1811. [PubMed: 12796535] 76. Cossins J, Burke G, Maxwell S, et al. Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations. Brain. 2006; 129:2773–2783. [PubMed: 16945936] 77. Ohno K, Sadeh M, Blatt I, Brengman JM, Engel AG. E-box mutations in RAPSN promoter region in eight cases with congenital myasthenic syndrome. Hum Mol Genet. 2003; 12:739–748. [PubMed: 12651869] 78. Haeuptle MA, Hennet T. Congenital disorders of glycosylation: An update on defects affecting the biosynthesis of dolichol-linked oligosaccharides. Hum Mutat. 2009; 30:1628–1641. [PubMed: 19862844] 79. Freeze HH, Chong JX, Bamshad MJ, Ng BG. Solving glycosylation disorders: Fundamental approaches reveal complicated pathways. Am J Hum Genet. 2014; 94:161–165. [PubMed: 24507773] 80. Selcen D, Shen X-M, Milone M, et al. GFPT1-myasthenia: Clinical, structural, and electrophysiologic heterogeneity. Neurology. 2013; 23:370–378. [PubMed: 23794683] 81. Belaya K, Finlayson S, Slater C, et al. Mutations in DPAGT1 cause a limb-girdle congenital Author Manuscript myasthenic syndrome with tubular aggregates. Am J Hum Genet. 2012; 91:1–9. 82. Selcen D, Shen X-M, Li Y, et al. DPAGT1 myasthenia and myopathy. Genetic, phenotypic, and expression studies. Neurology. 2014; 82:1822–1830. [PubMed: 24759841] 83. Cossins J, Belaya K, Hicks D, et al. Congenital myasthenic syndromes due to mutations in ALG2 and ALG14. Brain. 2013; 136:944–956. [PubMed: 23404334] 84. Soboloff J, Rothberg BS, Madesh M, Gill DL. STIM proteins: dynamic calcium signal transducers. Nat Rev Mol Cell Biol. 2012; 13:549–565. [PubMed: 22914293] Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 18 85. Bohm J, Chevessier F, De Paula AM, et al. Constitutive activation of STIM1 causes tubular aggregate myopathy. Am J Hum Genet. 2013; 92:271–278. [PubMed: 23332920] Author Manuscript 86. Regal L, Shen XM, Selcen D, et al. PREPL deficiency with or without cystinuria causes a novel myasthenic syndrome. Neurology. 2014; 82:1254–1260. [PubMed: 24610330] 87. Radhakrishnan K, Baltes J, Creemers JWM, Schu P. Trans-Golgi network morphology and sorting is regulated by prolyl-oligopeptidase-like protein PREPL and AP-1 complex subunit μ1A. J Cell Sci. 2013; 126:1155–1163. [PubMed: 23321636] 88. Kim M-H, Hersh LB. The vesicular acetylcholine transporter interacts with clathrin-associated adaptor complexes AP-1 and AP-2. J Biol Chem. 2004; 279:12580–12587. [PubMed: 14724281] 89. Rezniczeck GA, Walko G, Wiche G. Plectin defects lead to various forms of epidermolysis bullosa simplex. Dermatol Clin. 2009; 28:33–41. 90. Smith FJD, Eady RAJ, Leigh IM, et al. Plectin deficiency results in muscular dystrophy with epidermolysis bullosa. Nat Genet. 1996; 13:450–457. [PubMed: 8696340] 91. McMillan JR, Akiyama M, Rouan F, et al. Plectin defects in epidermolysis bullosa simplex with muscular dystrophy. Muscle Nerve. 2007; 35:24–35. [PubMed: 16967486] 92. Banwell BL, Russel J, Fukudome T, et al. Myopathy, myasthenic syndrome, and epidermolysis Author Manuscript bullosa simplex due to plectin deficiency. J Neuropathol Exp Neurol. 1999; 58:832–846. [PubMed: 10446808] 93. Selcen D, Juel VC, Hobson-Webb LD, et al. Myasthenic syndrome caused by plectinopathy. Neurology. 2011; 76:327–336. [PubMed: 21263134] 94. Claeys KG, Maisonobe T, Bohm J, et al. Phenotype of a patient with recessive centronuclear myopathy and a novel BIN1 mutation. Neurology. 2010; 74:519–521. [PubMed: 20142620] 95. Robb SA, Sewry CA, Dowling JJ, et al. Impaired neuromuscular transmission and response to aceylcholinesterase inhibitors in centronuclear myopathy. Neuromuscul Disord. 2011; 21:379– 386. [PubMed: 21440438] 96. Gibbs EM, Clarke NF, Rose K, et al. Neuromuscular junction abnrormalities in DNM2-related centronuclear myopathy. J Mol Med (Berl). 2013; 91:727–737. [PubMed: 23338057] 97. Liewluck T, Shen X-M, Milone M, Engel AG. Endplate structure and parameters of neuromuscular transmission in sporadic centronuclear myopathy associated with myasthenia. Neuromuscul Disord. 2011; 21:387–395. [PubMed: 21482111] Author Manuscript 98. Munot P, Lashley D, Jungbluth H, et al. Congenital fibre type disproportion associated with mutations in the tropomyosin 3 (TPM3) gene mimicking congenital myasthenia. Neuromuscul Disord. 2010; 20:796–800. [PubMed: 20951040] Author Manuscript Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 19 Search strategy for the article Author Manuscript PubMed was searched for articles published on congenital myasthenic syndromes between January 2004 and April 2014. The primary search term was: Congenital myasthenic syndrome. The secondary search terms were: acetylcholine receptor; choline acetyltransferase; ColQ; laminin beta-2; agrin; LRP4; MuSK; Dok-7; rapsyn; GFPT1; DPAGT1; ALG2, ALG14; Prepl; plectin; centronuclear myopathies; Nav1.4 channel. The authors also searched their own reprint files, clinical histories of their patients, and data files of their own research studies. Author Manuscript Author Manuscript Author Manuscript Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 20 Author Manuscript Author Manuscript Figure 1. Schematic diagram of an EP with locations of pre-synaptic, synaptic and postsynaptic CMS Author Manuscript disease proteins. Green line, synaptic basal lamina; red line, AChR on crests of the junctional folds; blue line, LRP4, MuSK, Dok-7, and rapsyn closely associated with AChR. SC, Schwann cell; NT, nerve terminal. (Modified from figure 1, Neuromuscul Disord 2012;22:99–111). Author Manuscript Lancet Neurol. Author manuscript; available in PMC 2016 April 01.  Engel et al. Page 21 Author Manuscript Author Manuscript Author Manuscript Figure 2. ChAT deficiency. (A) Subtetanic stimulation rapidly decreases the endplate potential (EPP) which returns to the baseline slowly over more than 10 min. 3,4-diaminopyridine (3,4-DAP) which increases quantal release accelerates the decline of the EPP, whereas a low Ca2+/high Mg2+ solution which reduces quantal release prevents the abnormal decline of the EPP. (B) Positions of mutated residues in active site tunnel of ChAT, kinetic landscapes of wild-type and mutant enzymes obtained from reaction velocities over a range and AcCoA Author Manuscript concentrations, and normalized kinetic parameters of wild-type and mutant ChAT. The catalytic efficiencies of the enzymes with AcCoA (kcat/Kma), choline (kcat/Kmb), and both substrates (kcat/KiaKmb), were calculated as described in Reference 11. The catalytic efficiencies of the mutant enzymes are