Somatic Mosaicism in PIK3CA Variant Correlates With Stereoelectroencephalography-Derived Electrophysiology

Abstract

Objectives: Brain-limited pathogenic somatic variants are associated with focal pediatric epilepsy, but reliance on resected brain tissue samples has limited our ability to correlate epileptiform activity with abnormal molecular pathology. We aimed to identify the pathogenic variant and map variant allele fractions (VAFs) across an abnormal region of epileptogenic brain in a patient who underwent stereoelectroencephalography (sEEG) and subsequent motor-sparing left frontal disconnection.

Methods: We extracted genomic DNA from peripheral blood, brain tissue resected from peri-sEEG electrode regions, and microbulk brain tissue adherent to sEEG electrodes. Samples were mapped based on an anatomic relationship with the presumed seizure onset zone (SOZ). We performed deep panel sequencing of amplified and unamplified DNA to identify pathogenic variants with subsequent orthogonal validation.

Results: We detect a pathogenic somatic PIK3CA variant, c.1624G>A (p.E542K), in the brain tissue samples, with VAF inversely correlated with distance from the SOZ. In addition, we identify this variant in amplified electrode-derived samples, albeit with lower VAFs.

Discussion: We demonstrate regional mosaicism across epileptogenic tissue, suggesting a correlation between variant burden and SOZ. We also validate a pathogenic variant from individual amplified sEEG electrode-derived brain specimens, although further optimization of techniques is required.

Figure 1. Workflow to Detect and Validate the PIK3CA E542K Variant

(A) Experimental design to detect brain-limited pathogenic variants in seizure onset zones based on sEEG recordings. The genomic DNA was extracted from resected bulk-brain tissue samples, trace brain tissue adhered to sEEG electrodes, and peripheral blood; a subset of samples underwent deep-panel sequencing and variant calling. (B) The integrated genome viewer (IGV) representation of the somatic PIK3CA E542 mutation in bulk-brain sample F2b. (C) Droplet digital PCR results for bulk-brain sample F2b. Reference signal 190 copies/µl, mutant signal 68.4 copies/µl. Mutant variant allele fraction (VAF): 26.50%. Blue droplets, mutant allele; green droplets, reference allele; orange droplets, both reference and mutant DNA templates; gray droplets, empty. Of note, the VAF reported in the Table takes into account VAFs from replication and control samples.

Figure 2. Representation of Somatic Mosaicism of Pathogenic PIK3CA E542K Variant in a 9-Year-Old Patient With Drug-Resistant Epilepsy and a Left Frontal Malformation of Cortical Development (MCD)

(A) Sagittal 3D surface reconstruction of brain MRI demonstrating a diffuse left frontal malformation of cortical development on a preoperative T1-weighted image. (B) Post-sEEG electrode implantation with sagittal CT coregistration with preoperative T1-weighted MRI depicting electrode F2aOFa trajectory sampling the left anterior middle frontal gyrus to the left orbital frontal cortex. (C–D) Intraoperative microscope imaging (left is anterior) demonstrating (C) predisconnection left frontal craniotomy and (D) postmotor sparing left frontal disconnection with labeled electrode insertion sites (black arrows) indicating ictally active (red box) and inactive (blue box) regions. Of note, the electrode site corresponding to the F3aOFb was outside of the craniotomy and not shown in the image. (E) 3D reformatting of preoperative MRI with superimposed biopsy sites with color gradient (red shapes) demonstrating variant allele fraction of bulk-brain tissue derived PIK3CA E542K variant determined by deep-panel sequencing with dark red representing higher VAF relative to all samples. (F) Lateral view of sEEG implantation plan using the standard electrode nomenclature for sEEG applications naming system (SENSA) with red circles indicating electrode sites where the PIK3CA E542K VAF was detected in sEEG electrode-derived samples.