Cell-type-informed genotyping of mosaic focal epilepsies reveals cell-autonomous and non-cell-autonomous disease-associated transcriptional programs
Abstract
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.
Article Details
Journal Info
Proceedings of the National Academy of Sciences
National Academy of Sciences
Authors (25)
Sara Bizzotto
Division of Genetics and Genomics, Manton Center for Orphan Disease Research
Maya Talukdar
Division of Genetics and Genomics, Manton Center for Orphan Disease Research
Edward A. Stronge
Division of Genetics and Genomics, Manton Center for Orphan Disease Research
Rosita B. Ramirez
Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School
Yingxi Yang
Division of Genetics and Genomics, Manton Center for Orphan Disease Research
August Yue Huang
Division of Genetics and Genomics, Manton Center for Orphan Disease Research
Qiwen Hu
Department of Biomedical Informatics, Harvard Medical School
Yingping Hou
Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School
Norma K. Hylton
Division of Genetics and Genomics, Manton Center for Orphan Disease Research
Benjamin Finander
Division of Genetics and Genomics, Manton Center for Orphan Disease Research
Ashton Tillett
Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School
Zinan Zhou
Division of Genetics and Genomics, Manton Center for Orphan Disease Research
Brian H. Chhouk
Division of Genetics and Genomics, Manton Center for Orphan Disease Research
Alissa M. D’Gama
Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School
Edward Yang
Department of Radiology, Boston Children’s Hospital
Timothy E. Green
Department of Medicine (Austin Health), University of Melbourne
David C. Reutens
Centre for Advanced Imaging, The University of Queensland and Royal Brisbane and Women’s Hospital
Saul A. Mullen
Department of Medicine (Austin Health), University of Melbourne
Ingrid E. Scheffer
Department of Medicine (Austin Health), University of Melbourne
Michael S. Hildebrand
Department of Medicine (Austin Health), University of Melbourne
Russell J. Buono
Department of Biomedical Sciences, Cooper Medical School of Rowan University
Ingmar Blümcke
Department of Neuropathology, University Hospitals Erlangen
Annapurna H. Poduri
Broad Institute of Massachusetts Institute of Technology and Harvard
Sattar Khoshkhoo
Division of Genetics and Genomics, Manton Center for Orphan Disease Research
Christopher A. Walsh