Cell-type-informed genotyping of mosaic focal epilepsies reveals cellautonomous and non-cell-autonomous disease-associated transcriptional programs Bizzotto S, Talukdar M, Stronge EA, Ramirez RB, Yang Y, Huang AY, Hu Q, Hou Y, Hylton NK, Finander B, Tillett A, Zhou Z, Chhouk BH, D'Gama AM, Yang E, Green TE, Reutens DC, Mullen SA, Scheffer IE, Hildebrand MS, Buono RJ, Blumcke I, Poduri AH, Khoshkhoo S and Walsh CA. Proc Natl Acad Sci U S A 2025;122(29):e2509622122. While it is widely accepted that somatic variants that activate the PI3K-mTOR pathway are a major cause of drug-resistant focal epilepsy, typically associated with focal cortical dysplasia (FCD) type 2, understanding the mechanism of epileptogenesis requires identifying genotype-associated changes at the single-cell level, which is technically challenging with existing methods. Here, we performed single-nucleus RNA-sequencing (snRNA-seq) of 18 FCD type 2 samples removed surgically for treatment of drug-resistant epilepsy, and 17 non-FCD control samples, and analyzed additional published data comprising >400,000 single nuclei. We also performed simultaneous single-nucleus genotyping and gene expression analysis using two independent approaches: 1) a method that we called genotyping of transcriptomes enhanced with nanopore sequencing (GO-TEN) that combines targeted cDNA long-read sequencing with snRNA-seq, 2) ResolveOME snRNA-seq and DNA genotyping. snRNA-seq showed similar cell identities and proportions between cases and controls, suggesting that mosaic pathogenic variants in PI3K-mTOR pathway genes in FCD exert their effect by disrupting transcription in conserved cell types. GO-TEN and ResolveOME analyses confirmed that pathogenic variant-carrying cells have well-differentiated neuronal or glial identities, with enrichment of variants in cells of the neuroectodermal lineage, pointing to cortical neural progenitors as possible loci of somatic mutation. Within FCD type 2 lesions, we identified upregulation of PI3K-mTOR signaling and related pathways in variant-carrying neurons, downregulation of these pathways in non-variant-carrying neurons, as well as associated changes in microglial activation, cellular metabolism, synaptic homeostasis, and neuronal connectivity, all potentially contributing to epileptogenesis. These genotype-specific changes in mosaic lesions highlight potential disease mechanisms and therapeutic targets.
Lena H. Nguyen (2026) studied this question.