Authors :
Presenting Author: H. Westley Phillips, MD – Stanford University
Alissa D' Gama, MD, PhD – Boston Children's Hospital; Yilan Wang, BS – Boston Children's Hospital; Yasmine Chahine, BA – Boston Children's Hospital; Amanda Swanson, BA – Boston Children's Hospital; Banu Ahtam, D. Phil – Boston Children's Hospital; Jeffrey Bolton, MD – Boston Children's Hospital; Michelle Chiu, MD – Boston Children's Hospital; Joseph Madsen, MD – Boston Children's Hospital; Eunjung Lee, PhD – Boston Children's Hospital; Sanjay Prabhu, MBBS, FRCR – Boston Children's Hospital; Hart Lidov, MD, PhD – Boston Children's Hospital; August Yue Huang, PhD – Boston Children's Hospital; Annapurna Poduri, MD – Boston Children's Hospital; Scellig Stone, MD, PhD – Boston Children's Hospital; Christopher Walsh, MD, PhD – Boston Children's Hospital
Rationale:
Analysis of the molecular profile of resected brain tissue samples from patients who undergo epilepsy surgery suggests that brain-limited somatic mutations, leading to mosaic variants, contributes to epilepsy pathogenesis. Recently, studies have reported the detection of mosaic variants using microbulk brain tissue adherent to removed stereoelectroencephalography (sEEG) electrodes. We aimed to detect and validate pathogenic variants in sEEG electrode-derived DNA from patients with drug resistant epilepsy (DRE) undergoing surgical evaluation, while correlating sEEG data with variant allele frequencies in and adjacent to seizure foci.
Methods:
We prospectively enrolled pediatric patients undergoing invasive intracranial monitoring via sEEG for DRE at Boston Children’s Hospital between July 2021 and June 2022. Following sEEG evaluation, electrodes were individually collected to retain their anatomic orientation. When possible, patient-matched bulk-brain tissue and peripheral blood samples were also collected. DNA was extracted from these samples, and deep next-generation sequencing (gene panel and/or whole-exome sequencing (WES)) was performed. Electrophysiological sEEG electrode-derived data were independently analyzed in an effort to localize the seizure onset zone (SOZ).
Results:
We collected 228 sEEG electrodes, 32 bulk brain tissue specimens, and 10 peripheral blood samples from 16 patients. In eleven patients, we isolated whole genome amplified and unamplified DNA from electrode-derived samples and extracted unamplified DNA from available bulk brain and peripheral blood samples. Using the electrophysiological data provided by sEEG, we selected clinically relevant samples to perform deep targeted gene panel sequencing and WES. We detected four mosaic variants with potential clinical relevance in electrode-derived amplified and unamplified DNA in three patients. In another patient undergoing open surgical intervention following sEEG, a
PIK3CA variant was discovered in the resected brain tissue and subsequently validated in amplified sEEG electrode-derived DNA. Orthogonal validation confirmed these variants, and in some cases, the variant allele frequency across samples suggested a direct relationship to the SOZ. However, tissue retrieval and yield were inconsistent across samples, and discrepancies in variant detection between brain tissue and electrode-derived samples warrant technique optimization.
Conclusions:
We demonstrate the ability to detect and validate mosaic variants from microbulk brain tissue adherent to removed sEEG electrodes, which allows for minimally invasive molecular genetic diagnoses while providing spatial localization of mosaic variants. Although promising, further optimization of sample collection and processing is required.
Funding: Dr. Phillips was supported by grant R25-NS079198 from the National Institutes of Health. Dr. D' Gama was supported by grant T32 HD098061 from the National Institutes of Child Health and Human Development. Dr. Walsh was supported by grant R01-NS035129 from the National Institutes of Health and by the Allen Frontiers Program. Dr. Walsh is an Investigator of the Howard Hughes Medical Institute.