Cell-type-informed genotyping of mosaic focal epilepsies reveals cell-autonomous and non-cell-autonomous disease-associated transcriptional programs

作者：Sara Bizzotto, Maya Talukdar, Edward Stronge, Rosita B. Ramirez, Yingxi Yang, August Yue Huang, Qiwen Hu, Yingping Hou, Norma K. Hylton, Benjamin Finander, A Tillett, Zinan Zhou, Brian H. Chhouk, Alissa M. D’Gama, Edward Yang, Timothy E. Green, David C. Reutens, Saul A. Mullen, Ingrid E. Scheffer, Michael S. Hildebrand, Russell J. Buono, Ingmar Blümcke, Annapurna Poduri, Sattar Khoshkhoo, Christopher A. Walsh · 发表于：Proceedings of the National Academy of Sciences · 年份：2025 · DOI：10.1073/pnas.2509622122 · 被引用次数：13 · 研究领域：Single-cell and spatial transcriptomics、Cancer Genomics and Diagnostics、RNA Research and Splicing

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 lesi...