Fig1.: Overview of collaborative workflow of the EpiMVP. Synergy of Project 1 with EpiMVP projects, GVCC and HECT
Milestone 1: Assess in silico and in vitro model systems for VUS functional analyses
- Computational modeling: with the gene and variant curation core (GVCC), generate and assess In silico tools to improve modeling of VUS pathogenicity
- Protein functions: use 2-dimentional (2-D) cultures of HEK293T cells in biochemical tests of each studied VUS
- Cell morphology/intracellular function: test the impact of a VUS on subcellular localization, protein trafficking and/or post translational processing
Fig 2. Computational modeling. Rationale for evaluating VUS and using functional information from projects with GVCC investigators to build and refine the EpiPred tool for variant pathogenesis prediction.
Milestone 2: Establish assays of cell autonomous effects in vitro
We’ll use human pluripotent stem cells (hPSCs) engineered by the human epilepsy tool core (HETC) to express doxycycline inducible Neurogenin 2 to differentiate excitatory neurons (iNeurons) or ASCL1/DLX2 to differentiate inhibitory neurons (iGN). They will be made to express WT or VUS containing genes on a null background to evaluate 2-D cultures of these progenitors and induced neurons to:
- Progenitor proliferation, cell survival
- Potential for differentiation into iNeurons or iGNs and
- Cell morphology, protein intracellular localization,
- Cell motility and
- Gene expression using RNAseq tools
Fig 3.: Neurons differentiated from hPSC expressing patient VUS. Engineered hPSC, iNeurons and iGN reagents from the HETC are used in Project 1 to create 2-D neuronal cultures. Cultures are used to assess morphology, protein expression, synapse formation and turnover.
Milestone 3: Determine the functional impact of VUS on synapse and network properties
These iNeurons (iN and iGN) expressing wildtype, known pathogenic and VUS mutations will be examined for:
- Synapse formation,
- Synaptic spine and synaptic specialization turnover (plasticity),
- Intracellular protein transport and
- Firing properties on multi-electrode arrays (MEA)
Fig 4.: Multielectrode Array activity of neurons differentiated from hPSC expressing patient VUS