Phenotypic and functional analysis of SHANK3 stop mutations identified in individuals with ASD and/or ID

Abstract

BackgroundSHANK proteins are crucial for the formation and plasticity of excitatory synapses. Although mutations in all three SHANK genes are associated with autism spectrum disorder (ASD), SHANK3 appears to be the major ASD gene with a prevalence of approximately 0.5% for SHANK3 mutations in ASD, with higher rates in individuals with ASD and intellectual disability (ID). Interestingly, the most relevant mutations are typically de novo and often are frameshift or nonsense mutations resulting in a premature stop and a truncation of SHANK3 protein.MethodsWe analyzed three different SHANK3 stop mutations that we identified in individuals with ASD and/or ID, one novel (c.5008A > T) and two that we recently described (c.1527G > A, c.2497delG). The mutations were inserted into the human SHANK3a sequence and analyzed for effects on subcellular localization and neuronal morphology when overexpressed in rat primary hippocampal neurons.ResultsClinically, all three individuals harboring these mutations had global developmental delays and ID. In our in vitro assay, c.1527G > A and c.2497delG both result in proteins that lack most of the SHANK3a C-terminus and accumulate in the nucleus of transfected cells. Cells expressing these mutants exhibit converging morphological phenotypes including reduced complexity of the dendritic tree, less spines, and less excitatory, but not inhibitory synapses. In contrast, the truncated protein based on c.5008A > T, which lacks only a short part of the sterile alpha motif (SAM) domain in the very SHANK3a C-terminus, does not accumulate in the nucleus and has minor effects on neuronal morphology.ConclusionsIn spite of the prevalence of SHANK3 disruptions in ASD and ID, only a few human mutations have been functionally characterized; here we characterize three additional mutations. Considering the transcriptional and functional complexity of SHANK3 in healthy neurons, we propose that any heterozygous stop mutation in SHANK3 will lead to a dysequilibrium of SHANK3 isoform expression and alterations in the stoichiometry of SHANK3 protein complexes, resulting in a distinct perturbation of neuronal morphology. This could explain why the clinical phenotype in all three individuals included in this study remains quite severe - regardless of whether there are disruptions in one or more SHANK3 interaction domains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-015-0020-5) contains supplementary material, which is available to authorized users.

Zinc deficiency and low enterocyte zinc transporter expression in human patients with autism related mutations in SHANK3

“…At the moment it still needs to be elucidated how SHANK3 can mediate a regulatory role on transcriptional events, however, the ability of the protein to localize to the nucleus4862 and data presented here might indicate that SHANK3 can not only function as a scaffolding molecule but also as a component of transcriptional complexes. In turn, this would imply that mutations of the SHANK3 gene can directly cause alterations of transcriptional activity in defined tissues and cell types.…”

“…One feature of this complex seems to be the regulation of two Zn importers, ZIP2 and ZIP4 by interacting protein networks and possibly nuclear signaling of SHANK34862. Future research will have to investigate whether the deregulation of Zn homeostasis is a common feature of mutations in “synaptic” proteins that are also found in the GI system.…”

A Subset of Autism-Associated Genes Regulate the Structural Stability of Neurons

“…These multiple putative protein interaction domains enable shank proteins to function as a bridge linking inotropic glutamate receptors, PSD-95, SAPAPS (Naisbitt et al, 1999), Homers (Tu et al, 1999; Hayashi et al, 2009) and the cytoskeleton (Böckers et al, 2001; Qualmann et al, 2004). Altered function of all three SHANK genes have been implicated in autism, with SHANK3 showing the highest prevalence (Moessner et al, 2007; Gauthier et al, 2009; Awadalla et al, 2010; Berkel et al, 2010, 2012; Pinto et al, 2010; Schaaf et al, 2011; Waga et al, 2011; Leblond et al, 2012, 2014; Prasad et al, 2012; Sanders et al, 2012; Sato et al, 2012; Boccuto et al, 2013; Koshimizu et al, 2013; Coe et al, 2014; De Rubeis et al, 2014; Guilmatre et al, 2014; Li et al, 2014a; Cochoy et al, 2015; Krumm et al, 2015; Nemirovsky et al, 2015; Yuen et al, 2015). In general, SHANK2 and 3 promote dendritic spine formation, whereas SHANK1 promotes dendritic spine head size enlargement (Sala et al, 2001; Roussignol et al, 2005; Hung et al, 2008; Verpelli et al, 2011).…”

Characterizing regression in Phelan McDermid Syndrome (22q13 deletion syndrome)

“…Three case reports suggest that regression occurs, and affects motor and language skills (Cochoy et al, 2015; Figura et al, 2014; Macedoni-Lukšic, Krgovic, Zagradišnik, & Kokalj-Vokac, 2013). Three additional investigations have reported on loss of developmental skills.…”