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Case Reports
. 2014 Dec 9;83(24):2247-55.
doi: 10.1212/WNL.0000000000001079. Epub 2014 Nov 7.

Mutant SNAP25B causes myasthenia, cortical hyperexcitability, ataxia, and intellectual disability

Xin-Ming Shen  1 , Duygu Selcen  2 , Joan Brengman  2 , Andrew G Engel  1
Affiliations
Case Reports

Mutant SNAP25B causes myasthenia, cortical hyperexcitability, ataxia, and intellectual disability

Xin-Ming Shen et al. Neurology. .

Abstract

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(2+)-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 α-helical coiled-coil held together by hydrophobic interactions. Pathogenicity of the Ile67Asn mutation was confirmed by 2 measures. First, the Ca(2+) 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 wild-type 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 α-helical coiled-coil structure of the SNARE complex by replacement of a highly hydrophobic isoleucine by a strongly hydrophilic asparagine.

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Figures

Figure 1
Figure 1. Patient at age 10 years and EP studies
(A) The patient cannot rise from the floor or walk without assistance. (B) Electron micrograph of patient EP shows the nerve terminal harbors abundant synaptic vesicles. Asterisks indicate vesicles focused on the active zones. The postsynaptic region is well developed. Bar = 0.5 μm. (C) Histograms of the quantal content of the EPP (m). The patient values are not normally distributed, with some values much lower and some normal or high normal. (D) Vertical scatterplot of readily releasable quanta (n) at patient and control EPs. Some values at patient EPs are much lower and some as high or higher than at control EPs. EP = endplate; EPP = EP potential.
Figure 2
Figure 2. Analysis of the identified mutation
(A) Scheme of SNAP25B complementary DNA indicating protein domains and position of the mutation. (B) The mutated residue is conserved across vertebrates and Drosophila. (C) Sanger chromatograms of patient and parental genomic DNA shows T>A change in patient but not in parents. (D) Stereo view of the crystal structure of the rat SNARE complex normal to its long axis at the level of the mutated Ile67 based on Protein Data Bank 1SFC. The mutated isoleucine points to center of the complex. Its replacement by a hydrophilic asparagine predicts disruption of hydrophobic interactions between α-helixes. F = father; M = mother; Pt = patient; Sb = synaptobrevin; SNAP25B = synaptosomal-associated protein, 25B; SNARE = soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Sn1 = α-helix near N-terminal end of SNAP25B; Sn2 = α-helix near C-terminal end of SNAP25B; Sx = syntaxin.
Figure 3
Figure 3. Expression studies
(A) Mixing liposomes incorporating wild-type v- and t-SNAREs causes significant right shift in particle size distribution. (B) Mixing liposomes incorporating wild-type v-SNARE with t-SNARE liposomes harboring mutant Snap25B causes no significant shift in particle size distribution. (C) Representative amperometric traces from depolarized chromaffin cells. Each spike represents a single exocytotic event. Mutant SNAP25B and mutant SNAP25B plus wild-type transfected cells generate fewer and lower-amplitude spikes than wild-type SNAP25B-transfected cells. (D) Cumulative exocytotic events during the first minute after depolarization from 15 nontransfected, 11 wild-type SNAP25B-transfected, 14 mutant SNAP25B-transfected, and 17 mutant SNAP25B plus wild-type transfected cells. For each group of cells, each point indicates the mean number of spikes over 5 seconds. Vertical lines indicate 1 SE. Compared with nontransfected or wild-type transfected cells, mutant transfected, or mutant plus wild-type transfected, cells exocytose vesicles at similar markedly reduced rates. SNAP25B = synaptosomal-associated protein, 25B; t-SNAREs = target membrane-attached SNAP25B and syntaxin; v-SNARE = synaptic vesicle-attached synaptobrevin.
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