Identification of a novel mosaic MTOR variant in purified neuronal DNA from depth electrodes in a patient with focal cortical dysplasia

Background: Recent studies have identified brain somatic variants as a cause of focal epilepsy. These studies relied on resected tissue from epilepsy surgery which is not available in most patients. The use of trace tissue adherent to depth electrodes used for stereo electroencephalography (stereo EEG) has been proposed as an alternative but is hampered by the low cell quality and contamination by non-brain cells. Here, we use our improved depth electrode harvesting technique that purifies neuronal nuclei to achieve molecular diagnosis in a patient with focal cortical dysplasia (FCD). Methods: Depth electrode tips were collected, pooled by brain region and seizure onset zone, nuclei isolated and sorted using fluorescence-activated nuclei sorting (FANS). Somatic DNA was amplified from neuronal and astrocyte nuclei using primary template amplification followed by exome sequencing of neuronal DNA from the affected pool, unaffected pool, and saliva. The identified variant was validated using droplet digital PCR. Results: An 11-year-old male with drug-resistant genetic-structural epilepsy due to left anterior insula FCD had seizures from age 3 years. Stereo EEG confirmed seizure onset in the left anterior insula. The two anterior insula electrodes were combined as the affected pool and three frontal electrodes as the unaffected pool. FANS isolated 140 neuronal nuclei from the affected and 245 neuronal nuclei from the unaffected pool. A novel somatic missense MTOR variant (p.Leu489Met, CADD score 23.7) was identified in the affected neuronal sample. Droplet digital PCR confirmed a mosaic gradient (VAF 0.78% in affected neuronal sample, variant was absent in all other samples). Conclusions: Our finding confirms that harvesting neuronal DNA from depth electrodes followed by molecular analysis to identify brain somatic variants is feasible. Our novel method represents a significant improvement compared to the previous method by focusing the analysis on high quality cells of the cell type of interest. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This project has been made possible by the Canada Brain Research Fund (CBRF), an innovative arrangement between the Government of Canada (through Health Canada) and Brain Canada, and by the Azrieli Foundation. It was also supported by Epilepsy Canada, doing business as Epilepsy Canada. KMK also received support from the Department of Clinical Neurosciences, Hotchkiss Brain Institute and Alberta Children′s Hospital Research Institute, University of Calgary. M.T-G was supported by the Alberta Children′s Hospital Foundation and the CIHR-Bridge grant number OGB-185746. Infrastructure in the laboratory of GP was supported by a Canada Foundation for Innovation Grant (John R. Evans Leaders Fund (CFI-JELF) 36624). The views expressed herein do not necessarily represent the views of the Minister of Health or the Government of Canada. ### 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 Conjoint Health Research Ethics Board (CHREB) at the University of Calgary gave ethical approval for this work (REB18-2099). 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 The de-identified phenotypic and exome sequencing data will be made available upon reasonable request from any qualified investigator after institution of a material transfer agreement.