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

TitleMulti-parametric analysis of 57 SYNGAP1 variants reveal impacts on GTPase signaling, localization, and protein stability.
Publication TypeJournal Article
Year of Publication2021
AuthorsMeili F, Wei WJ, Sin W-C, Meyers WM, Dascalu I, Callaghan DB, Rogic S, Pavlidis P, Haas K
JournalAm J Hum Genet
Volume108
Issue1
Pagination148-162
Date Published2021 01 07
ISSN1537-6605
KeywordsAutism Spectrum Disorder, Cell Line, Epilepsy, GTP Phosphohydrolases, HEK293 Cells, Humans, Intellectual Disability, Mutation, Neurodevelopmental Disorders, Phenotype, Protein Stability, ras GTPase-Activating Proteins, Signal Transduction
Abstract

SYNGAP1 is a neuronal Ras and Rap GTPase-activating protein 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), whether and how they contribute to individual disease phenotypes is often unknown. Here, we characterize 57 variants in seven assays that examine multiple aspects of SYNGAP1 function. Specifically, we used multiplex phospho-flow cytometry to measure variant impact on protein stability, pERK, pGSK3β, pp38, pCREB, and high-content imaging to examine subcellular localization. We find variants ranging from complete loss-of-function (LoF) to wild-type (WT)-like in their regulation of pERK and pGSK3β, while all variants retain at least partial ability to dephosphorylate pCREB. Interestingly, our assays reveal that a larger proportion of variants located within the disordered domain of unknown function (DUF) comprising the C-terminal half of SYNGAP1 exhibited higher LoF, compared to variants within the better studied catalytic domain. Moreover, we find protein instability to be a major contributor to dysfunction for only two missense variants, both located within the catalytic domain. Using high-content imaging, we find variants located within the C2 domain known to mediate membrane lipid interactions exhibit significantly larger cytoplasmic speckles than WT SYNGAP1. Moreover, this subcellular phenotype shows both correlation with altered catalytic activity and unique deviation from signaling assay results, highlighting multiple independent molecular mechanisms underlying variant dysfunction. Our multidimensional dataset allows clustering of variants based on functional phenotypes and provides high-confidence, multi-functional measures for making pathogenicity predictions.

DOI10.1016/j.ajhg.2020.11.011
Alternate JournalAm J Hum Genet
PubMed ID33308442
PubMed Central IDPMC7820741
Grant List / / CIHR / Canada