Application of Optical Methods for Identification of Missense-Induced Deficits in SynGAP1

Abstract

Disruptions in SynGAP1 function caused by mutations are linked to neurodevelopmental disorders. Most known pathogenic SynGAP1 variants act via truncation and frame shift mutations while the minority of diagnosed cases involve missense mutations. Most of the missense mutations are unique to individual patients. If similar features can be identified among multiple variants, it may help with further development of therapies. The study aimed to investigate the functional consequences of missense variants of SynGAP1, using High Throughput Microscope based neuronal imaging and a protein interaction assay. Neuronal imaging was implemented for measuring SynGAP1 expression and punctate characterization in each missense variant to presumed synaptic puncta in neurons. The methodology included the generation of viruses encoding missense constructs, infection of hippocampal neurons and subsequent neuronal imaging. The ability of variants to oligomerize was measured by in-cell Bioluminescence Energy Transfer Assay. SynGAP1 expression and characterization assay indicated that the overall distribution of WT and missense variants visually look similar, even though the expression, puncta number and size distributions of puncta were different. Development of analysis algorithms for the classification of SynGAP1 missense variants according to their locations is still required. In the BRET assay, characteristics of the substituted amino acid of missense variants influenced the oligomerization. BRET assay can be implemented to detect both increased and decreased interactions of oligomers in SynGAP1. Missense variants unaffected in one assay may still exhibit an effect on another assay. Therefore, a diversity of assays is required to conclude the clinical relevance and overall functional consequences of each missense variant.