Along with the expanding list of potential epilepsy genes comes the challenge of genetic variants of uncertain significance (VUS).
VUS numbers are rising as more patients undergo NGS, but we lack the tools to determine VUS pathogenicity and disease mechanisms. To address these shortfalls, the EpiMVP will optimize cutting-edge multiplatform assays for epilepsy gene function.
The long-term goal of this project is to deliver an in vitro testing pipeline with defined phenotypes in human neuronal models to assay clinically relevant VUS for non-ion-channel epilepsy genes. Project 2 will test variants for the chosen genes in complex structural and functional assays, using human pluripotent stem cell (hPSC) knockout lines generated by the Human Epilepsy Tools Core (HETC). Human in vitro models will include 1) 2-D hPSC cultures: small molecule differentiation into excitatory or inhibitory cortical neurons, excitatory and inhibitory induced neurons (iNeurons) generated by forced transcription factor expression; and 2) Excitatory, inhibitory and fusion (combined excitatory and inhibitory) brain organoid cultures.
Assays of VUS rescue (or deleterious gain-of-function) effects will include morphology, gene expression and neuronal/network activity. The latter includes multielectrode array [MEA] and patch clamp recordings, calcium imaging and depth electrode local field potential [LFP] recordings (in brain organoids).
Our immediate goals are to optimize assays for 1-2 genes per year, determine VUS pathogenicity in vitro for these genes and, in concert with the Variant and Gene Curation Core (VGCC), refine the VUS list for further in vivo testing in Project 3. Two sets of milestones are proposed:
To determine VUS pathogenicity using 2-D hPSC assays (Milestone 1) and brain organoid models (Milestone 2). The HETC, Parent and Ross labs have experience generating cortical neurons via small molecule and iNeuron differentiation, and with multiple brain organoid culture protocols. We will express non-ion channel epilepsy gene variants chosen by the GVCC and Project 1, and via constructs generated by the HETC, in a knockout hPSC background. Assays will include neuronal morphology, gene expression, calcium imaging and electrophysiology in 2-D and brain organoid cultures.
These studies will provide the following deliverables: 1) multiple optimized, cross-validated (between Parent and Ross labs) hPSC platforms to interrogate epilepsy genes; 2) determination of in vitro human neuronal VUS pathogenicity for at least 5 non-ion channel epilepsy genes; 3) human neuronal models for each epilepsy gene; and 4) optimized platforms for future mechanistic and precision therapeutic studies.