Multi-parametric analysis of 57 SYNGAP1 variants reveal impacts on GTPase signaling, localization, and protein stability

Abstract

SYNGAP1 is a Ras and Rap GTPase with important roles in regulating excitatory synaptic plasticity. While many SYNGAP1 missense and nonsense mutations have been associated with intellectual disability, epilepsy, schizophrenia and autism spectrum disorder (ASD), there are many variants of unknown significance (VUS). In this report, we characterize 58 variants in nine assays that examine multiple aspects of SYNGAP1 function. Specifically, we used multiplex phospho-flow cytometry to measure the impact of variants on pERK, pGSK3β and pCREB and high-content imaging to examine their subcellular localization. We find variants ranging from complete loss-of-function (LoF) to wildtype (WT)-like in their ability to regulate pERK and pGSK3β, while all variants retain at least partial ability to regulate pCREB. Interestingly, our assays reveal that a high percentage of variants located within the disordered domain of unknown function that makes up the C-terminal half of SYNGAP1 exhibited LoF, compared to the more well studied catalytic domain. Moreover, we find protein instability to be a major contributor to dysfunction only for two missense variants both located within the catalytic domain. Using highcontent imaging, we find variants with nuclear enrichment/exclusion and aberrant nuclear speckle localization. These variants are primarily located within the C2 domain known to mediate membrane lipid interactions. We find that mislocalization is distinct from altered catalytic activity, highlighting multiple independent molecular mechanisms underlying variant dysfunction. Our multidimensional dataset allows clustering of variants based on functional phenotypes and provides high-confidence pathogenicity classification..

Mnk1/2 kinases regulate memory and autism-related behaviours via Syngap1

“…5c, bottom). Recent analysis of several human Syngap1 variants 36 and previous work 35 showed that CDK5 phosphorylates Syngap1 at S788.…”

Neuron-specific protein network mapping of autism risk genes identifies shared biological mechanisms and disease relevant pathologies

“…Our BioID2 screen provides functional evidence of the impact ASD-associated de novo missense variants have on the PPI network of three ASD-risk genes, as examples. Time- and resource- intensive studies have also investigated multiple variants in single genes in various animal models 114, 115 . Additional bioinformatic approaches have been used to determine the pathogenicity of rare missense variants; however, the impact on biological pathways remains to be determined 116, 117 .…”

Phenotypes in Children With SYNGAP1 Encephalopathy in China

“…SynGAP sequence contained six various predicted functional domains: pleckstrin homology (PH) domain (27-253AA), C2 domain (263-362AA), RasGAP domain (392-729AA), SH3 domain (785-815AA), coiled-coil (CC) domain (1189-1262AA), and other C-terminal domains of unknown function ( Hamdan et al, 2009 ; Berryer et al, 2013 ; Gamache et al, 2020 ). The SynGAP protein has multiple biological functions and interacts with numerous proteins ( Meili et al, 2021 ). The loss of function of the SYNGAP1 gene has been linked to a variety of neurodevelopmental disorders (NDD), including autism spectrum disorder (ASD), intellectual disability (ID), and epilepsy.…”