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

• 成果类型: Article
• 署名作者: Bizzotto, Sara; Talukdar, Maya; Stronge, Edward A.; Ramirez, Rosita B.; Yang, Yingxi; Huang, August Yue; Hu, Qiwen; Hou, Yingping; Hylton, Norma K.; Finander, Benjamin; Tillett, Ashton; Zhou, Zinan; Chhouk, Brian H.; D'Gama, Alissa M.; Yang, Edward; Green, Timothy E.; Reutens, David C.; Mullen, Saul A.; Scheffer, Ingrid E.; Hildebrand, Michael S.; Buono, Russell J.; Blumcke, Ingmar; Poduri, Annapurna H.; Khoshkhoo, Sattar; Walsh, Christopher A.
• 署名单位: Harvard University; Harvard University Medical Affiliates; Boston Children's Hospital; Harvard Medical School; Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute; Institut National de la Sante et de la Recherche Medicale (Inserm); Universite Paris Cite; Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute; Harvard Medical School; Harvard University; Harvard University Medical Affiliates; Brigham & Women's Hospital; Harvard Medical School; Harvard University; Harvard Medical School; Harvard University; Harvard University Medical Affiliates; Boston Children's Hospital; Harvard Medical School; Harvard University; Harvard University Medical Affiliates; Boston Children's Hospital; Harvard Medical School; Harvard University; Harvard University Medical Affiliates; Boston Children's Hospital; Florey Institute of Neuroscience & Mental Health; Howard Florey Institute Affiliates; University of Melbourne; Austin Research Institute; University of Queensland; Royal Brisbane & Women's Hospital; Murdoch Children's Research Institute; Rowan University; Cooper Medical School of Rowan University; University of Erlangen Nuremberg; Cleveland Clinic Foundation; Harvard University; Harvard Medical School; Harvard University Medical Affiliates; Boston Children's Hospital; Harvard University; Harvard University Medical Affiliates; Boston Children's Hospital; Howard Hughes Medical Institute
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10988
• DOI: 10.1073/pnas.2509622122
• 发表日期: 2025-07-22
• 关键词: cortical dysplasia; somatic mutations; spectrum; malformations; PATHWAY; cancer

摘要

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