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

Abstract

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.

Detecting somatic variants in purified brain DNA obtained from surgically implanted depth electrodes in epilepsy

“…Using ddPCR, we validated 4/4 somatic variants chosen based on gene association and pathogenicity scores in SEEG-derived neuronal DNA from the affected brain regions and confirmed their absence in the respective SEEG-derived neuronal DNA from unaffected tissue and germline sample. Among the identified variants, one was in MTOR 21 , which is known to be implicated in FCD 2,4,5,8,10,22,23 . We identified a variant in CSDE1, which is associated with 14/28 epilepsy 24 .…”

“…The validated somatic variants had VAFs between 0.78-12.6%, which corresponds to 1.56-25.2% of neurons in the affected brain regions. Additionally, as outlined previously, our technique provides the exact number of nuclei of each cell type that are being amplified 21 . This allows estimation of the power to identify a certain VAF.…”

“…The variants presented here were identified in neuronal nuclei. However non-neuronal cells have also been implicated in epileptogenesis 21 . With our approach, the implication of somatic variants in other cell types such as astrocytes and microglia can also be studied.…”