Contribution of Somatic Ras/Raf/Mitogen-Activated Protein Kinase Variants in the Hippocampus in Drug-Resistant Mesial Temporal Lobe Epilepsy

Abstract

Importance: Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy subtype and is often refractory to antiseizure medications. While most patients with MTLE do not have pathogenic germline genetic variants, the contribution of postzygotic (ie, somatic) variants in the brain is unknown.

Objective: To test the association between pathogenic somatic variants in the hippocampus and MTLE.

Design, setting, and participants: This case-control genetic association study analyzed the DNA derived from hippocampal tissue of neurosurgically treated patients with MTLE and age-matched and sex-matched neurotypical controls. Participants treated at level 4 epilepsy centers were enrolled from 1988 through 2019, and clinical data were collected retrospectively. Whole-exome and gene-panel sequencing (each genomic region sequenced more than 500 times on average) were used to identify candidate pathogenic somatic variants. A subset of novel variants was functionally evaluated using cellular and molecular assays. Patients with nonlesional and lesional (mesial temporal sclerosis, focal cortical dysplasia, and low-grade epilepsy-associated tumors) drug-resistant MTLE who underwent anterior medial temporal lobectomy were eligible. All patients with available frozen tissue and appropriate consents were included. Control brain tissue was obtained from neurotypical donors at brain banks. Data were analyzed from June 2020 to August 2022.

Exposures: Drug-resistant MTLE.

Main outcomes and measures: Presence and abundance of pathogenic somatic variants in the hippocampus vs the unaffected temporal neocortex.

Results: Of 105 included patients with MTLE, 53 (50.5%) were female, and the median (IQR) age was 32 (26-44) years; of 30 neurotypical controls, 11 (36.7%) were female, and the median (IQR) age was 37 (18-53) years. Eleven pathogenic somatic variants enriched in the hippocampus relative to the unaffected temporal neocortex (median [IQR] variant allele frequency, 1.92 [1.5-2.7] vs 0.3 [0-0.9]; P = .01) were detected in patients with MTLE but not in controls. Ten of these variants were in PTPN11, SOS1, KRAS, BRAF, and NF1, all predicted to constitutively activate Ras/Raf/mitogen-activated protein kinase (MAPK) signaling. Immunohistochemical studies of variant-positive hippocampal tissue demonstrated increased Erk1/2 phosphorylation, indicative of Ras/Raf/MAPK activation, predominantly in glial cells. Molecular assays showed abnormal liquid-liquid phase separation for the PTPN11 variants as a possible dominant gain-of-function mechanism.

Conclusions and relevance: Hippocampal somatic variants, particularly those activating Ras/Raf/MAPK signaling, may contribute to the pathogenesis of sporadic, drug-resistant MTLE. These findings may provide a novel genetic mechanism and highlight new therapeutic targets for this common indication for epilepsy surgery.

Figure 1.. Somatic Variants Activating Ras/Raf/Mitogen-Activated Protein Kinase (MAPK) Pathway Genes in Mesial Temporal Lobe Epilepsy

Simplified diagram of the Ras/Raf/MAPK signaling pathway, the pathogenic variants discovered in the mesial temporal lobe epilepsy cohort, and their corresponding proteins. GDP indicates guanosine diphosphate; GOF, gain of function; GTP, guanosine triphosphate; LOF, loss of function; RTK, receptor tyrosine kinase.

Figure 2.. Enrichment of Ras/Raf/Mitogen-Activated Protein Kinase (MAPK) Pathway Variants in the Temporal Lobe and in Mesial Temporal Lobe Epilepsy (MTLE)

A, Retrospective review of Ras/Raf/MAPK pathway variants and PI3K/Akt/mammalian target of rapamycin (mTOR) pathway variants in the lesional focal epilepsy literature. Circle diameters represent normalized case counts in each brain region and the associated bar plots on the right depict the absolute number of cases. B, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the pathogenicity-enriched variants in the MTLE cohort (P < .001 and adjusted P < .05 after correction for multiple hypotheses testing). The dashed orange line represents the adjusted P value of .05.

Figure 3.. Mechanisms of Ras/Raf/Mitogen-Activated Protein Kinase (MAPK) Overactivity in Mesial Temporal Lobe Epilepsy–Associated Variant Shp2 Proteins

A, Immunoblot of the indicated proteins in HEK293T cells transiently expressing wild-type (WT) and variant Shp2. B, Representative pictures from live imaging microscopy of monomeric enhanced green fluorescent protein (mEGFP)–labeled WT and variant Shp2 proteins, transiently expressed in KYSE520 cells. C, The fluorescent puncta (marked by arrowheads in panel B) are quantified. D, Representative images of fluorescence recovery after photobleaching (FRAP) using the transiently expressed mEGFP-labeled G503R Shp2 protein in KYSE520 cells. E, The rate of fluorescence recovery in panel D is quantified. F, Representative images of 8-μM WT and variant Shp2 protein droplet formation in the presence of 10% (w/v) PEG3350 in vitro. G, Droplet formation is quantified by solution turbidity of OD600.

Figure 4.. Evidence of Erk1/2 Overactivation in Mesial Temporal Lobe Epilepsy Tissue Harboring Ras/Raf/Mitogen-Activated Protein Kinase Variants

Each column shows histopathologic images from 1 sample. A and F, A low-grade epilepsy–associated tumor. Original magnification ×40 for image and ×400 for inset. A, Hematoxylin-eosin staining with areas of hypercellularity corresponding to the tumor (inset 1) intermixed with normal brain tissue (inset 2). F, pErk1/2 staining of the corresponding regions is seen in insets 1 and 2. B to E and G, Representative mesial temporal sclerosis–only samples with neuronal dropout in the cornu ammonis region of the hippocampus (panels B to E) but uniform distribution of glial cells across the same region (panel G). The black arrowheads demarcate cornu ammonis neuronal loss. Original magnification ×200. H, Original magnification ×40 for image and ×200 for inset. I, Original magnification ×200. H and I, Erk1/2 phosphorylation (pErk1/2) colocalizes to regions with highest degree of neuronal loss and sclerosis. The arrowheads point to cells with increased pErk1/2 staining, mostly represented by apparent glia (panel I, inset). The black arrowheads point to neurons that have relatively low pErk1/2 staining (panel H, inset).