Choline acetyltransferase (ChAT; EC2.3.1.6) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist inCaenorhabditis elegansandDrosophila, but noCHATmutations have been observed in humans to date. Here we report that mutations inCHATcause a congenital myasthenic syndrome associated with frequently fatal episodes of apnea (CMS-EA). Studies of the neuromuscular junction in this disease show a stimulation-dependent decrease of the amplitude of the miniature endplate potential and no deficiency of the ACh receptor. These findings point to a defect in ACh resynthesis or vesicular filling and toCHATas one of the candidate genes. Direct sequencing ofCHATreveals 10 recessive mutations in five patients with CMS-EA. One mutation (523insCC) is a frameshifting null mutation. Three mutations (I305T, R420C, and E441K) markedly reduce ChAT expression in COS cells. Kinetic studies of nine bacterially expressed ChAT mutants demonstrate that one mutant (E441K) lacks catalytic activity, and eight mutants (L210P, P211A, I305T, R420C, R482G, S498L, V506L, and R560H) have significantly impaired catalytic efficiencies.

In skeletal muscle, acetylcholinesterase (AChE) exists in homomeric globular forms of type T catalytic subunits (ACHE _T ) and heteromeric asymmetric forms composed of 1, 2, or 3 tetrameric ACHE _T attached to a collagenic tail (ColQ). Asymmetric AChE is concentrated at the endplate (EP), where its collagenic tail anchors it into the basal lamina. The ACHE _T gene has been cloned in humans; COLQ cDNA has been cloned in Torpedo and rodents but not in humans. In a disabling congenital myasthenic syndrome, EP AChE deficiency (EAD), the normal asymmetric species of AChE are absent from muscle. EAD could stem from a defect that prevents binding of ColQ to ACHE _T or the insertion of ColQ into the basal lamina. In six EAD patients, we found no mutations in ACHE _T . We therefore cloned human COLQ cDNA, determined the genomic structure and chromosomal localization of COLQ , and then searched for mutations in this gene. We identified six recessive truncation mutations of COLQ in six patients. Coexpression of each COLQ mutant with wild-type ACHE _T in SV40-transformed monkey kidney fibroblast (COS) cells reveals that a mutation proximal to the ColQ attachment domain for ACHE _T prevents association of ColQ with ACHE _T ; mutations distal to the attachment domain generate a mutant ≈10.5S species of AChE composed of one ACHE _T tetramer and a truncated ColQ strand. The ≈10.5S species lack part of the collagen domain and the entire C-terminal domain of ColQ, or they lack only the C-terminal domain, which is required for formation of the triple collagen helix, and this likely prevents their insertion into the basal lamina.

We describe and functionally characterize six mutations of the acetylcholine receptor (AChR) ε subunit gene in three congenital myasthenic syndrome patients. Endplate studies demonstrated severe endplate AChR deficiency, dispersed endplate regions and well preserved junctional folds in all three patients. Electro-physiologic studies were consistent with expression of the fetal γ-AChR at the endplates in one patient, prolongation of some channel events in another and γ-AChR expression as well as some shorter than normal channel events in still another. Genetic analysis revealed two recessive and heteroallelic e subunit gene mutations in each patient. One mutation in each (εC190T [εR64X], ε127ins5 and ε553del7) generates a nonsense codon that predicts truncation of the ε subunit in its N-terminal, extracellular domain; and one mutation in each generates a missense codon (εR147L, εP245L and εR311W). None of the mutations was detected in 100 controls. Expression studies in HEK cells indicate that the three nonsense mutations are null mutations and that surface expression of AChRs harboring the missense mutations is significantly reduced. Kinetic analysis of AChRs harboring the missense mutations show that εR147L is kinetically benign, εP245L prolongs burst open duration 2-fold by slowing the rate of channel closing and εR311W shortens burst duration 2-fold by slowing the rate of channel opening and speeding the rate of ACh dissociation. The modest changes in activation kinetics are probably overshadowed by reduced expression of the missense mutations. The consequences of the endplate AChR deficiency are mitigated by persistent expression of γ-AChR, changes in the release of transmitter quanta and appearance of multiple endplate regions on the muscle fiber.