Engel, Andrew G.

We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) in 3 patients with severe congenital myasthenia: εR218W in the pre-M1 region in 2 patients and εE184K in the β8-β9 linker in 1 patient. Arg218 is conserved in all eukaryotic members of the Cys-loop receptor superfamily, while Glu184 is conserved in the α-, δ-, and ε-subunits of AChRs from all species. εR218W reduces channel gating efficiency 338-fold and AChR expression on the cell surface 5-fold, whereas εE184K reduces channel gating efficiency 11-fold but does not alter AChR cell surface expression. Determinations of the effective channel gating rate constants, combined with mutant cycle analyses, demonstrate strong energetic coupling between εR218 and εE184, and between εR218 and εE45 from the β1-β2 linker, as also observed for equivalent residues in the principal coupling pathway of the α-subunit. Thus, efficient and rapid gating of the AChR channel is achieved not only by coupling between conserved residues within the principal coupling pathway of the α-subunit, but also between corresponding residues in the ε-subunit.

AbstractCongenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is impaired by one or more specific mechanisms. Since the advent of next‐generation sequencing methods, the discovery of novel CMS targets and phenotypes has proceeded at an accelerated rate. Here, we review the current classification of CMS and describe our findings in five of these targets identified and investigated in our laboratory in the past 5 years. Defects in LRP4 hinder synaptic development and maintenance; the defects in PREPL are predicted to diminish filling of the synaptic vesicle with acetylcholine; and defects in SNAP25, Munc13‐1, and synaptotbrevin‐1 impede synaptic vesicle exocytosis.

Congenital myasthenic syndromes are clinically and genetically heterogeneous disorders of neuromuscular transmission. Most are treatable, but certain subtypes worsen with cholinesterase inhibitors. This underlines the importance of genetic diagnosis. Here, the authors report on cases with genetically proven congenital myasthenic syndromes from Turkey. The authors retrospectively reviewed their experience of all patients with congenital myasthenic syndromes, referred over a 5-year period (2011-2016) to the Child Neurology Department of Dokuz Eylül University, Izmir, Turkey. In addition, PubMed was searched for published cases of genetically proven congenital myasthenic syndromes originating from Turkey. In total, the authors identified 43 (8 new patients, 35 recently published patients) cases. Defects in the acetylcholine receptor (n = 15; 35%) were the most common type, followed by synaptic basal-lamina associated (n = 14; 33%) and presynaptic syndromes (n = 10; 23%). The authors had only 3 cases (7%) who had defects in endplate development. One patient had mutation GFPT1 gene (n = 1; 2%). Knowledge on congenital myasthenic syndromes and related genes in Turkey will lead to prompt diagnosis and treatment of these rare neuromuscular disorders.

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

The inhibition of carnitine palmitoyltransferase (CPT, EC 2.3.1.21) by malonyl-CoA, acetyl-CoA and free CoA was studied in sonicated skeletal-muscle homogenates from normal human subjects and from five patients with a mutant CPT [Zierz & Engel (1985) Eur. J. Biochem. 149, 207-214]. (1) Malonyl-CoA, acetyl-CoA and CoA were competitive inhibitors of CPT with palmitoyl-CoA. (2) Acetyl-CoA and CoA inhibited normal and mutant CPT to the same degree, whereas malonyl-CoA inhibited mutant CPT more than normal CPT. (3) Triton X-100 abolished the inhibition of normal CPT by malonyl-CoA, but not by acetyl-CoA or CoA. Triton X-100 by itself caused loss of activity of the mutant CPT. (4) In the concentration range 0.1-0.4 mM, the inhibitory effects of any two of the three inhibitors were synergistic. (5) The inhibitory constants (Ki) for acetyl-CoA and CoA were close to 45 microM. The Ki for malonyl-CoA was 200-fold lower, or 0.22 microM. Addition of 40 microM-acetyl-CoA or CoA resulted in a 3-fold increase in the Ki for acetyl-CoA. Addition of 20 microM-CoA resulted in a 3-fold increase in the Ki for acetyl-CoA. (6) The findings indicate that acetyl-CoA and CoA can inhibit CPT at the catalytic site or a nearby site which is different from that at which malonyl-CoA inhibits CPT. (7) The fact that small changes in the concentration of acetyl-CoA and CoA can antagonize the inhibitory effect of malonyl-CoA suggests that these compounds could modulate the inhibition of CPT by malonyl-CoA.