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.

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.

1) The circulating anti-muscle-specific tyrosine kinase antibodies caused neither muscle-specific tyrosine kinase nor acetylcholine receptor deficiency at the endplates; 2) the reduced intercostal miniature endplate potential and current amplitudes were not accounted for by acetylcholine receptor deficiency; 3) the faint immunoglobulin G deposits at the endplates may or may not represent anti-muscle-specific tyrosine kinase antibodies; and 4) the anti-muscle-specific tyrosine kinase antibodies may not be the primary cause of myasthenic symptoms in this patient.

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.Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Between 1995 and 2005, defects in seven end-plate (EP)-associated proteins encoded by 10 different genes have been identified as molecular targets of the CMS. 1 In 2006, Okada and coworkers identified Dok-7 as a muscle-intrinsic activator of MuSK required for synaptogenesis. 2 Dok-7 harbors N-terminal pleckstrin homology (PH) and phosphotyrosineAddress correspondence to Dr Engel, Department of Neurology, Mayo Clinic, Rochester, MN 55905. E-mail: age@mayo.edu. Note Added in Proof Monoallele mutation analysis by the Conversion technology 39 revealed absence of exons 3-6 in one allele in genomic DNA of DOK7 in Patient 9. NIH Public Access Patients and Methods PatientsSixteen patients, 8 men and 8 women, presently 5 to 50 years of age, were investigated. Each patient was initially examined, and four were reexamined 5 to 17 years later after their initial visit (AGE); additional follow-up information came from follow-up letters from referring physicians or patients regarding disease management. All human studies were in accord with guideli...

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 21 -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 a-helical coiled-coil held together by hydrophobic interactions. Pathogenicity of the Ile67Asn mutation was confirmed by 2 measures. First, the Ca 21 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 wildtype 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 a-helical coiled-coil structure of the SNARE complex by replacement of a highly hydrophobic isoleucine by a strongly hydrophilic asparagine. Neurology ® 2014;83:2247-2255 GLOSSARY AChR 5 acetylcholine receptor; cDNA 5 complementary DNA; CMS 5 congenital myasthenic syndrome; EP 5 endplate; MEPP 5 miniature endplate potential; SNAP25B 5 synaptosomal-associated protein, 25B; SNARE 5 soluble N-ethylmaleimide-sensitive factor attachment protein receptor; t-SNAREs 5 target membrane-attached SNAP25B and syntaxin; v-SNARE 5 synaptic vesicle-attached synaptobrevin.The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are core components of the synaptic vesicle fusion machinery. Synaptobrevin attached to the synaptic vesicles is referred to as a v-SNARE, and SNAP25 (synaptosomal-associated protein of 25 kD) and syntaxin attached to the target plasma membrane are designated as t-SNAREs.1 The assembled complex is a coiled-coil in which a-helices are held together by strong hydrophobic interactions. The complex is stabilized by complexin, a small soluble neuronal protein.2 In the resting state, Munc18 binds to a closed form of syntaxin and blocks formation of the SNARE complex but assists the SNARE complex to effect exocytosis in the active state. 3 SNAP25 also participates in endocytosis at hippocampal synapses 4 and negatively modulates the neuronal voltage-gated calcium channel during intense activity.5 Also, ...

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

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.

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.