De novo mutations in the gene encoding the synaptic scaffolding protein SHANK3 in patients ascertained for schizophrenia

Abstract

Schizophrenia likely results from poorly understood genetic and environmental factors. We studied the gene encoding the synaptic protein SHANK3 in 285 controls and 185 schizophrenia patients with unaffected parents. Two de novo mutations (R1117X and R536W) were identified in two families, one being found in three affected brothers, suggesting germline mosaicism. Zebrafish and rat hippocampal neuron assays revealed behavior and differentiation defects resulting from the R1117X mutant. As mutations in SHANK3 were previously reported in autism, the occurrence of SHANK3 mutations in subjects with a schizophrenia phenotype suggests a molecular genetic link between these two neurodevelopmental disorders.

Fig. 1.

Families with de novo mutations in the SHANK3 gene. (A) Segregation of the R1117X nonsense mutation in three affected brothers of family PED 419. The proband is indicated by the arrow. (B) Segregation of the R536W missense mutation in the proband but not her unaffected brother in PED 56. (C) Localization on the linear protein structure of SHANK3 of the de novo mutations found in families with schizophrenia. ANK, ankyrin repeats; SH3, Src homology 3 domain; PDZ, postsynaptic density protein (PSD95), Drosophila disk large tumor suppressor (DlgA), and zonula occludens-1 protein (z o-1) domain; SAM, sterile α motif domain. (D) Western blot analysis using HA antibody of HEK293T cell lysate transfected with empty vector (control), HA-Shank3 (WT), R1117X, and R536W. Shank3 WT and R536W have a similar size (200 kDa), whereas the nonsense R1117X results in a truncated protein (123 kDa). (E) Alignment of SHANK3 orthologous peptide sequences near the R536W missense (indicated by the asterisk showing amino acid conservation of the R536 residues in 10 species). GenBank accession numbers: chimp, AC145340; Rhesus monkey, AANU01101358; dog, XP_848271; rat, P_067708; opossum, XP_001366729; lizard, AAWZ01039630; Xenopus, CX494866; zebrafish: zs3.1, CAI20675 and zs3.2, XR_028926.

Fig. 2.

Validation of Shank3 mutations in zebrafish. Knockdown (KD) of either of the zebrafish Shank3 genes (zs3.1, zs3.2) using selective AMOs resulted in severe morphological (A) and behavioral deficits (B) compared with wild type (WT), as illustrated using representative images taken from high-speed video films. Partial rescue was observed with coinjection of AMO and rat WT Shank3 mRNA. The pie charts depict the proportion (% of totals) of normal (control-like; yellow), severely affected (no swimming; red), and mildly affected (slow swimming; blue) embryos in each group. The results with coinjection of AMO and rat WT Shank3 or mutated (R1117X or R536W) mRNA are summarized in bar graphs (C). The asterisk denotes significant (P < 0.001) differences from the KD group.

Fig. 3.

Effect of Shank3 mutants on differentiation of hippocampal neurons. Transfected hippocampal neurons were identified by GFP expression (A). Overexpression of WT Shank3 in neurons leads to an increase in primary neurite outgrowth from somata (B). Overexpression of R536W (C) similarly stimulated neurite outgrowth. In contrast, expression of the R1117X truncating mutation (D) failed to do so. In E, the data are quantified in a bar histogram along with standard deviations for each bar. Neurite outgrowth significantly different from control levels (P < 0.001) is indicated with an asterisk.