DEVELOPMENT OF AN OPTICAL ACTUATOR-REPORTER TOOLSET AND MOLECULAR-COMPUTATIONAL WORKFLOW TO PHENOTYPE PATHOGENICITY OF MISSENSE VARIANTS OF THE RARE MONOGENIC DISEASE GENE SYNGAP1

Rare diseases include ~10000 distinct pathological conditions affecting 5-10% of the population. Missense mutations in monogenic rare disease genes confer considerable additional diversity. Clinical categorisation of most missense mutations as “variants of uncertain significance”, VUS, leaves patients without clear diagnosis and curtails investment in therapeutic development. This multilevel diversity and uncertainty among rare diseases are obstacles to traditional research and drug discovery approaches. To address this challenge, we combine reporter systems and high-throughput perturbation microscopy (HTPM) with deep-learning based image processing and multivariate data analysis pipelines to detect the phenotypic impacts of rare disease gene variants. This has potential to reveal biologically meaningful aberrations caused by known pathogenic variants, which can then be used to identify the possible pathogenicity of specific VUS panels. It may address the burden of VUS diversity by identifying representative variants suitable for reverse screens to reveal compounds ameliorating disease phenotypes. We developed an AAV toolset for long-term non-toxic expression of multiplexed optogenetic actuators with reporters of neuronal activity, structure and the distribution and activity-dependent dispersion of SynGAP1, the second most abundant post-synaptic density protein. This allows longitudinal imaging of neuron cultures in 384 well plates, revealing phenotypic stability, repeatability of optogenetic responses and internal control of compound treatments. After live-cell HTPM, end-point immunocytochemistry detects endogenous markers to investigate possible dominant negative actions. Applied to missense variants of the C2 domain, the workflow revealed feature-based co-clustering of a VUS with pathogenic missense variants, and was used to select a representative variant for reverse chemical screen.