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, Blümcke Ingmar, Poduri Annapurna H, Khoshkhoo Sattar, Walsh Christopher A
Division of Genetics and Genomics, Manton Center for Orphan Disease Research, Boston, MA 02115.
Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 2025 Jul 22;122(29):e2509622122. doi: 10.1073/pnas.2509622122. Epub 2025 Jul 17.
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.
虽然人们普遍认为，激活PI3K-mTOR通路的体细胞变异是耐药性局灶性癫痫的主要原因，通常与2型局灶性皮质发育不良（FCD）相关，但要了解癫痫发生的机制，需要在单细胞水平上识别与基因型相关的变化，而现有方法在技术上具有挑战性。在这里，我们对18个因耐药性癫痫而手术切除的2型FCD样本和17个非FCD对照样本进行了单核RNA测序（snRNA-seq），并分析了包含超过40万个单核的其他已发表数据。我们还使用两种独立的方法进行了同时单核基因分型和基因表达分析：1）一种我们称为通过纳米孔测序增强转录组基因分型（GO-TEN）的方法，该方法将靶向cDNA长读测序与snRNA-seq相结合；2）ResolveOME snRNA-seq和DNA基因分型。snRNA-seq显示病例和对照之间的细胞类型和比例相似，这表明FCD中PI3K-mTOR通路基因中的镶嵌致病变异通过破坏保守细胞类型中的转录发挥作用。GO-TEN和ResolveOME分析证实，携带致病变异的细胞具有分化良好的神经元或神经胶质细胞类型，神经外胚层谱系细胞中的变异富集，表明皮质神经祖细胞可能是体细胞突变的位点。在2型FCD病变中，我们发现携带变异的神经元中PI3K-mTOR信号通路和相关通路上调，非携带变异的神经元中这些通路下调，以及小胶质细胞激活、细胞代谢、突触稳态和神经元连接的相关变化，所有这些都可能导致癫痫发生。镶嵌病变中这些基因型特异性变化突出了潜在的疾病机制和治疗靶点。
