Synaptotagmin 2 mutations cause an autosomal-dominant form of lambert-eaton myasthenic syndrome and nonprogressive motor neuropathy

Abstract

Synaptotagmin 2 is a synaptic vesicle protein that functions as a calcium sensor for neurotransmission but has not been previously associated with human disease. Via whole-exome sequencing, we identified heterozygous missense mutations in the C2B calcium-binding domain of the gene encoding Synaptotagmin 2 in two multigenerational families presenting with peripheral motor neuron syndromes. An essential calcium-binding aspartate residue, Asp307Ala, was disrupted by a c.920A>C change in one family that presented with an autosomal-dominant presynaptic neuromuscular junction disorder resembling Lambert-Eaton myasthenic syndrome. A c.923C>T variant affecting an adjacent residue (p.Pro308Leu) produced a presynaptic neuromuscular junction defect and a dominant hereditary motor neuropathy in a second family. Characterization of the mutation homologous to the human c.920A>C variant in Drosophila Synaptotagmin revealed a dominant disruption of synaptic vesicle exocytosis using this transgenic model. These findings indicate that Synaptotagmin 2 regulates neurotransmitter release at human peripheral motor nerve terminals. In addition, mutations in the Synaptotagmin 2 C2B domain represent an important cause of presynaptic congenital myasthenic syndromes and link them with hereditary motor axonopathies.

Figure 1

Pedigrees of Affected Families and Lower Limb Deformities (A and B) Family pedigrees for USA1 (A) and UK1 (B). Filled symbols are clinically affected individuals, arrows denote the probands. (C) Foot and toe deformities in three generations in the USA1 family. (D) Asymmetric distal lower limb muscle atrophy (individual II.2 shown here) was noted in affected UK1 females. Splayed toes (family member III.7) were notable in the UK1 kindred.

Figure 2

Postexercise Compound Muscle Action Potential Amplitude Facilitation in USA1 and UK1 and Family Genetic Studies (A) Median nerve distal CMAP before and immediately after 10 s of maximal voluntary contraction for USA1 proband II.2 and her affected offspring III.1 and III.2. Each of these individuals demonstrated a greater than 100% increment in median nerve distal compound muscle action potential amplitude following brief exercise. Individual II.2 also demonstrated a 139% increment in the peroneal motor distal compound muscle action potential amplitude recording from the tibialis anterior muscle. Individuals III.1 and III.2 additionally demonstrated 110% and 200% distal ulnar compound muscle action potential amplitude increments, respectively, after 10 s of exercise. See Table S1 for full electrophysiological data for USA1. (B) Amplitude and area increment of the peroneal CMAP (recording from tibialis anterior) in the UK1 proband (III.2) after 10 s of maximal voluntary contraction (MVC), which persisted beyond 5 min. See Table S2 for electrophysiologic data for UK1. (C) Sanger traces demonstrating the c.920A>C missense mutation in SYT2 in USA1. This produces an amino acid change from aspartate to alanine at the second aspartate residue (p.Asp307Ala) in the cytoplasmic C2B domain of SYT2. A c.923C>T missense mutation in SYT2 in affected UK1 individuals results in a proline-to-leucine amino acid change at the adjacent residue (p.Pro308Leu). Note that the SYT2 gene is encoded at the opposite strand. (D) Position of the identified mutations in each family on the gene structure of SYT2 and the conservation of the affected residues across species.

Figure 3

p.Asp362Ala DSYT1 Fails to Support Neurotransmitter Release in a synaptotagmin-Null Mutant (A) Stereoview of the Asp307 (yellow) and Pro308 (magenta) residues modeled on the rat SYT1 C2B crystal structure. The five essential Ca²⁺-binding residues are highlighted in green and Ca²⁺ ions in red. (B) Immunoblot with anti-SYT (top) or anti-Syntaxin (SYX; control) antisera from head extracts of control or transgenic Drosophila following wild-type (WT) or p.Asp362Ala DSYT induction by elav^C155-GAL4. (C) Representative larval NMJs stained with anti-Myc antisera (green) for animals with Myc-tagged WT or p.Asp362Ala SYT1. The axon is stained with the neuronal marker anti-HRP (magenta). The boxed area is magnified in the bottom panel. Mutant SYT1 targets normally to presynaptic terminals. Scale bars represent 20 μM (top) or 5 μM (bottom). (D) Representative EPSCs recorded in 2 mM external Ca²⁺ in Dsyt1^−/− null larvae (red) and null mutants rescued with WT DSYT1 (black) or p.Asp362Ala DSYT1 (blue). (E) Quantification of mean excitatory junctional current (eEJC) amplitudes in the indicated genotypes (Dsyt1^−/− null, 2.1 ± 0.3 nA, n = 10; elav^C155-GAL4; Dsyt1^−/−; UAS-SYT1, 108.4 ± 13.9 nA, n = 18; elav^C155-GAL4; Dsyt1^−/−; UAS-SYT1 p.Asp362Ala, 2.0 ± 0.5 nA, n = 13). (F) Cumulative vesicle release defined by charge transfer normalized for the maximum in 2.0 mM Ca²⁺ for each genotype (same color code as in D). Each trace was adjusted to a double exponential fit. Both the null and p.Asp362Ala rescued animals display a prominent increase in the slow asynchronous phase of release. (G) Postsynaptic current recordings of spontaneous release at muscle 6 synapses in Dsyt1^−/− null larvae (red) and null mutants rescued with WT DSYT1 (black) or p.Asp362Ala DSYT1 (blue). (H) Quantification of mini frequency in the indicated genotypes (Dsyt1^−/− null, 5.2 ± 0.6 Hz, n = 8; elav^C155-GAL4; Dsyt1^−/−; UAS-SYT1, 1.7 ± 0.2 Hz, n = 16; elav^C155-GAL4; Dsyt1^−/−; UAS-SYT1 p.Asp362Ala, 5.2 ± 0.4 Hz, n = 12). Student’s t test: ^∗∗∗∗p < 0.0001. Error bars represent SEM.

Figure 4

Mutant Synaptotagmin Disrupts Neurotransmitter Release in the Presence of Endogenous Synaptotagmin (A) Representative EPSCs recorded in 0.2 mM extracellular Ca²⁺ at third instar larval muscle 6 synapses for the indicated genotypes (control, induction of WT or p.Asp362Ala DSYT1 by elav^C155-GAL4). (B) Quantification of mean eEJC amplitudes in the indicated genotypes (w^−/−, 120.5 ± 5.3 nA, n = 16; elav^C155-GAL4; UAS-SYT1, 117.5 ± 11 nA, n = 15; elav^C155-GAL4; UAS-SYT1 p.Asp362Ala#1, 26.6 ± 4.7 nA, n = 16; elav^C155-GAL4; UAS-SYT1 p.Asp362Ala#2, 17.3 ± 3.2 nA, n = 16). (C) Postsynaptic current recordings of spontaneous release for the indicated genotypes. (D) Quantification of average mini frequency for the indicated genotypes (w^−/−, 1.6 ± 0.1 Hz, n = 10; elav^C155-GAL4; UAS-SYT1, 2.1 ± 0.2 Hz, n = 10; elav^C155-GAL4; UAS-SYT1 p.Asp362Ala#1, 6.3 ± 0.5 Hz, n = 16; elav^C155-GAL4; UAS-SYT1 p.Asp362Ala#2, 8.2 ± 0.7 Hz, n = 13). Student’s t test: ^∗p < 0.05; ^∗∗∗∗p < 0.0001. Error bars represent SEM. (E) Representative EPSCs during a short 10 Hz tetanic stimulation of the nerve in 0.2 mM external Ca²⁺ for the indicated genotypes. (F) The average for the first ten responses normalized to the first response during the tetanus is shown following induction of WT DSYT 1 (blue), DSYT1 p.Asp362Ala #1 (green), and DSYT1 p.Asp362Ala #2 (red). The average fast tetanic facilitation after ten responses for each genotype is: WT DSYT1 = 0.94 ± 0.02; DSYT1 p.Asp362Ala#1 = 1.29 ± 0.03; DSYT1 p.Asp362Ala#2 = 1.49 ± 0.04. Student’s t test revealed significant differences between WT and p.Asp362Ala (p < 0.0001).