Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans

Abstract

Choline acetyltransferase (ChAT; EC ) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist in Caenorhabditis elegans and Drosophila, but no CHAT mutations have been observed in humans to date. Here we report that mutations in CHAT cause 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 to CHAT as one of the candidate genes. Direct sequencing of CHAT reveals 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.

Figure 1

Genomic structure and alternative transcripts of human CHAT and 10 identified mutations. CHAT and VACHT are encoded in the cholinergic gene locus (26). Four alternative CHAT transcripts (R, N1, N2, and M) have been reported (15, 17, 41). The S transcript is a moiety identified in the spinal cord. All transcripts encode a 70-kDa ChAT. M transcript encodes an additional 83-kDa ChAT and S transcript codes for an additional 74-kDa ChAT. Vertical shading indicates untranslated regions of VACHT. Diagonal shading show untranslated regions of CHAT. For M and S transcripts, diagonal shading indicates untranslated regions for yielding 83- and 74-kDa ChATs, respectively. Arrows indicate translational start sites. Exons and introns are drawn to indicated scale.

Figure 2

Alignment of the amino acid sequences of human 83-kDa ChAT and of single isoforms identified in pig, mouse, and rat ChAT. Divergent N-terminal ends of the human 74- and 70-kDa ChATs are shown also. Dashes indicate residues identical to the human 83-kDa ChAT. Gaps are inserted for alignment. The right column indicates codon numbers. Arrowheads indicate the 10 recessive mutations identified in five patients. The arrowhead under 523insCC points to the first residue substituted by the frameshift.

Figure 3

Immunoblot identifying translational start sites. (Left) Wild type. A Met-to-Ala substitution at the putative translational start site of 74-kDa ChAT prevents expression of the 74-kDa ChAT (Center). A similar Met-to-Ala substitution of 70-kDa ChAT prevents expression of the 70-kDa ChAT (Right).

Figure 4

Immunoblot demonstrating expression of wild-type and mutant ChATs in COS cells. Expression levels are normalized for cotransfected β-galactosidase activity and compared with that of wild-type. Bars indicate means and standard errors of three to four transfections. * indicates significant difference from wild type (P < 0.01). The 74- to 70-kDa ChAT ratios for the mutants were not significantly different from that of wild type.

Figure 5

SDS/PAGE of purified wild-type and mutant recombinant ChATs expressed in E. coli stained with Coomassie G-250.

Figure 6

Individually scaled kinetic landscapes of wild-type and mutant ChATs. Dots represent observed values. Meshes show curves fitted to Eq. 1, except for panels B and I in which the curves are fitted to Eqs. 2 and 3, respectively (see Materials and Methods).