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

Abstract

ImportanceMesial 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.ObjectiveTo test the association between pathogenic somatic variants in the hippocampus and MTLE.Design, Setting, and ParticipantsThis 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.ExposuresDrug-resistant MTLE.Main Outcomes and MeasuresPresence and abundance of pathogenic somatic variants in the hippocampus vs the unaffected temporal neocortex.ResultsOf 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 RelevanceHippocampal 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.

Oncogenic Pathways Provide Clue to the Etiology of Human Mesial Temporal Lobe Epilepsy

“…10 The work by Khoshkhoo et al 8 builds on previously published research and provides evidence that combined histopathological and genetic analyses will likely guide precision therapies of the future. Additionally, all individuals who were variant positive in the study by Khoshkhoo et al 8 were seizure free more than 2 years following anterior temporal lobectomy, which is in harmony with previously published research showing that complete removal of epileptogenic lesion is the main pre-dictor of optimal postsurgical outcome. 14 One can envision that over time, a combined neuropathological and genetic clinical investigation of resected tissue margins will guide more accurate prognostication and further management.…”

“…Work of Khoshkhoo et al 8 is an important investigation of postzygotic (ie, somatic) variants in mTLE. In this study, the investigators compared results from high-depth (>500×) nextgeneration sequencing of resected hippocampal specimens from 105 individuals affected by a drug-resistant mTLE to data from age-and sex-matched controls.…”

“…At the same time, there is an urgent need to expand clinical genetic testing in epilepsy beyond screening of DNA from peripheral sources (blood, saliva) and in the domain of resected brain specimens, to aid in precision diagnostics and to facilitate rational, mechanistically driven therapies. Work by Khoshkhoo et al 8 provides an exciting impetus, and it is evident that we are just getting started.…”

How can cancer research be illuminated by brain research (and vice versa)?

“…Brain (mal)function has been linked to transcription‐related nucleotide damage (compared to the DNA replication‐related one in cancer), which in turns could promote the molecular diversity of human brain cells even during postmitotic stages 6 . Nonetheless, somatic mutations in genes implicated in the Ras‐Raf‐MAPK signaling cassette, such as protein tyrosine phosphatase nonreceptor type11 ( PTPN11 ), BRAF , KRAS , SOS Ras/Rac guanine nucleotide exchange factor 1 ( SOS1 ), have been identified in the hippocampal region of patients with temporal lobe epilepsy, opening potential novel therapeutic approaches for drug‐resistant cases 7 . Moreover, somatic mutations have been identified in cerebral cortical malformations (eg, pachygyria, periventricular nodular heterotopia), and they mostly refer to the doublecortin ( DCX ) and the lissencephaly type 1 ( LIS1 ) genes 8 .…”

“…6 Nonetheless, somatic mutations in genes implicated in the Ras-Raf-MAPK signaling cassette, such as protein tyrosine phosphatase nonreceptor type11 (PTPN11), BRAF, KRAS, SOS Ras/Rac guanine nucleotide exchange factor 1 (SOS1), have been identified in the hippocampal region of patients with temporal lobe epilepsy, opening potential novel therapeutic approaches for drug-resistant cases. 7 Moreover, somatic mutations have been identified in cerebral cortical malformations (eg, pachygyria, periventricular nodular heterotopia), and they mostly refer to the doublecortin (DCX) and the lissencephaly type 1 (LIS1) genes. 8 The latter observations imply that somatic mutations are prevalent even in nonreplicating cells such as those of the brain parenchyma, and their patterns can be correlated with mutations in the highly replicating tumor cells to provide comparative insights.…”