Analysis of DNA from brain tissue on stereo-EEG electrodes reveals mosaic epilepsy-related variants

Abstract Somatic mosaic variants contribute to focal epilepsy, but genetic analysis has been limited to patients with drug-resistant epilepsy (DRE) who undergo surgical resection, as the variants are mainly brain-limited. Stereoelectroencephalography (sEEG) has become part of the evaluation for many patients with focal DRE, and sEEG electrodes provide a potential source of small amounts of brain-derived DNA. We aimed to identify, validate, and assess the distribution of potentially clinically relevant mosaic variants in DNA extracted from trace brain tissue on individual sEEG electrodes. We enrolled a prospective cohort of eleven pediatric patients with DRE who had sEEG electrodes implanted for invasive monitoring, one of whom was previously reported. We extracted unamplified DNA from the trace brain tissue on each sEEG electrode and also performed whole-genome amplification for each sample. We extracted DNA from resected brain tissue and blood/saliva samples where available. We performed deep panel and exome sequencing on a subset of samples from each case and analysis for potentially clinically relevant candidate germline and mosaic variants. We validated candidate mosaic variants using amplicon sequencing and assessed the variant allele fraction (VAF) in amplified and unamplified electrode-derived DNA and across electrodes. We extracted DNA from >150 individual electrodes from 11 individuals and obtained higher concentrations of whole-genome amplified vs unamplified DNA. Immunohistochemistry confirmed the presence of neurons in the brain tissue on electrodes. Deep sequencing and analysis demonstrated similar depth of coverage between amplified and unamplified samples but significantly more called mosaic variants in amplified samples. In addition to the mosaic PIK3CA variant detected in a previously reported case from our group, we identified and validated four potentially clinically relevant mosaic variants in electrode-derived DNA in three patients who underwent laser ablation and did not have resected brain tissue samples available. The variants were detected in both amplified and unamplified electrode-derived DNA, with higher VAFs observed in DNA from electrodes in closest proximity to the electrical seizure focus in some cases. This study demonstrates that mosaic variants can be identified and validated from DNA extracted from trace brain tissue on individual sEEG electrodes in patients with drug-resistant focal epilepsy and in both amplified and unamplified electrode-derived DNA samples. Our findings support a relationship between the extent of regional genetic abnormality and electrophysiology, and suggest that with further optimization, this minimally invasive diagnostic approach holds promise for advancing precision medicine for patients with DRE as part of the surgical evaluation. Competing Interest Statement The authors have declared no competing interest. Funding Statement AMD was supported by NIH/NICHD T32 HD098061. HWP was supported by NIH/NINDS R25 NS079198 and by Credit Unions Kids at Heart. EAL and YW were supported by Allen Discovery Center program, a Paul G. Allen Frontiers Group advised program of the Paul G. Allen Family Foundation. RSS was supported by R00NS112604. AYH was supported by NIH/NIA R56 AG079857 and Alzheimers Association Research Fellowship AARF-22-972287. CAW was supported by NIH/NINDS 5R01NS035129, the Simons Foundation, and the Templeton Foundation. CAW is an Investigator of the Howard Hughes Medical Institute. This research was supported in part by the BCH Rosamund Stone Zander Translational Neuroscience Center Human Neuron Core Repository for Neurological Disorders, Credit Unions Kids at Heart, and the IDDRC (NIH P30 HD018655). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The IRB of Boston Children's Hospital gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes Abbreviations: BCH = Boston Children’s Hospital; DRE = drug-resistant epilepsy; EPC = epilepsia partialis continua; FCD = focal cortical dysplasia; indel = small insertion and deletion; MCD = malformation of cortical development; mTOR = mammalian target of rapamycin; sEEG = stereotactic-EEG; PBS = phosphate buffered saline; SENSA = standardized electrode nomenclature for stereoelectroencephalography applications; SNV = single-nucleotide variant; SOZ = seizure onset zone; VAF = variant allele fraction; VUS = variant of uncertain significance; ES = exome sequencing Data availability The data that support the findings of this study are available from the corresponding author, upon reasonable request.