Genetic and Functional Analyses of SHANK2 Mutations Suggest a Multiple Hit Model of Autism Spectrum Disorders
Figure 4
Characterization of the functional impact of SHANK2 mutations in cultured neuronal cells.
A. The colocalization of ProSAP1A/SHANK2-EGFP (postsynaptic marker) and Bassoon (presynaptic marker) indicated that the mutations did not disturb the formation of SHANK2 clusters at excitatory synapses along the dendrites. B. The quantification of synapse density was performed on 20 transfected hippocampal neurons per construct from at least three independent experiments. The majority of the ProSAP1A variants affecting a conserved amino acid among SHANK proteins reduced significantly the synaptic density compared with the variants that affect amino acid non conserved among SHANK proteins (Mann-Whitney U-test: nWT = 20, nmut = 20; US557N = 82.5, pS557N = 0.001; UR569H = 124, pR569H = 0.04; UL629P = 149, pL629P = 0.17; UV717F = 114, pV717F = 0.02; UA729T = 73, pA729T = 0.000; UK780Q = 154, pK780Q = 0.221; UR818H = 108, pR818H = 0.012; UA822T = 154.5, pA822T = 0.224; UV823M = 129, pV823M = 0.056; UY967C = 134, pY967C = 0.076; UG1170R = 78, pG1170R = 0.001; UR1290W = 142, pR1290W = 0.121; UQ1308R = 162, pQ1308R = 0.314; UD1535N = 97, pD1535N = 0.005; UP1586L = 137, pP1586L = 0.910; UL1722P = 79, pL1722P = 0.001, *p<0.05, **p<0.01, ***p<0.001). C. Effect of the variants on synaptic density. The y-axis represents −log P compared to WT (P obtained with Mann-Whitney test). After Bonferroni correction for 16 tests, only P values<0.003 were considered as significant. Variants represented in red were specific to ASD, in orange were shared by ASD and controls, and in green were specific to the controls. Open circles and filled circles represent non conserved and conserved amino acids, respectively. Prim, primary; second, secondary.
