Mosaic human cortical organoids model mTOR-related focal cortical dysplasia through <i>DEPDC5</i> deletion

Focal cortical dysplasia type II (FCDII), a major cause of pediatric drug-resistant focal epilepsy, results from brain somatic variants in mTOR pathway genes, including germline and somatic second-hit loss-of-function variants in the mTOR repressor DEPDC5. Here, we present a proof-of-concept model of DEPDC5 two-hit inactivation mosaicism using patient-derived human cortical organoids (hCOs). Mosaic hCOs displayed increased mTOR activity that was rescued by the mTOR inhibitor rapamycin. Mosaic hCOs also exhibited dysmorphic-like neurons and enhanced neuronal excitability, recapitulating key FCDII pathology hallmarks. Single-cell transcriptomics across three developmental stages revealed aberrant differentiation trajectories leading to premature upper-layer neuron generation, upregulated Notch and Wnt signaling pathways in neural progenitors, and altered expression of synaptic- and epilepsy-associated genes in excitatory neurons. In addition, we identified cell-autonomous alterations in metabolism and translation in mosaic DEPDC5 two-hit hCOs. This study provides novel insights into how DEPDC5 deficiency perturbs human corticogenesis, highlighting that mosaic biallelic inactivation of the gene is necessary for FCDII pathogenesis.