Somatic Ras/Raf/MAPK Variants Enriched in the Hippocampus in Drug-Resistant Mesial Temporal Lobe Epilepsy

Importance Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy subtype and is often refractory to anti-seizure medications. While most MTLE patients do not have pathogenic germline genetic variants, the contribution of post-zygotic (i.e., somatic) variants in the brain is unknown. Objective To test the association between pathogenic somatic variants in the hippocampus and MTLE. Design This case-control genetic association study analyzed the DNA derived from hippocampal tissue of neurosurgically-treated patients with MTLE and age- and sex-matched neurotypical controls. Participants were enrolled from 1988 through 2019 and clinical data was collected retrospectively. Whole-exome and gene-panel sequencing (depth>500X) were used to identify candidate pathogenic somatic variants. A subset of novel variants were functionally evaluated using cellular and molecular assays. Setting Level 4 epilepsy centers, multi-center study. Participants Non-lesional and lesional (mesial temporal sclerosis, focal cortical dysplasia, and low-grade epilepsy-associated tumors) drug-resistant MTLE patients who underwent anterior medial temporal lobectomy. All patients with available frozen tissue and appropriate consents were included. Control brain tissue was obtained from neurotypical donors at brain banks. Exposures Drug-resistant MTLE. Main Outcomes and Measures Presence and abundance of pathogenic somatic variants in the hippocampus versus the unaffected temporal neocortex. Results Samples were obtained from 105 MTLE patients (52 male, 53 female; age: MED [IQR], 32 [26-44]) and 30 neurotypical controls (19 male, 11 female; age: MED [IQR], 37 [18-53]). Eleven pathogenic somatic variants, enriched in the hippocampus relative to the unaffected temporal neocortex (MED [IQR], 1.92 [1.5-2.7] vs 0.3 [0-0.9], p<0.05), were detected in MTLE patients but not in the controls. Ten of these variants were in PTPN11, SOS1, KRAS, BRAF , and NF1 , all predicted to constitutively activate Ras/Raf/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. ### Competing Interest Statement SK, CAW, and KTK have a planned patent filing pertaining to the findings reported in this manuscript. ### Funding Statement SK was supported by the NIH grants R25NS065743 and K08NS128272 and Doris Duke Physician Scientist Fellowship. ES was supported by the NIH grant T32GM007753. HWP was supported by the NIH grant R25NS079198. SB was supported by the Manton Center for Orphan Disease Research at Boston Childrens Hospital and is now supported by a European Commissions Horizon 2020 Research and Innovation Programme Marie Sklodowska Curie Actions Individual Fellowship grant agreement no. 101026484 CODICES. IES was supported by a National Health and Medical Research Council Australia Investigator Grant 1172897. SFB was supported by an NHMRC Investigator Grant 1196637. MSH was supported by an NHMRC Ideas Grant 2012287. SFB and MSH were supported by NHMRC Project Grants 1129054 and 1079058. EY was supported by the NIH grants R01NS035129 and R01NS094596. ELH, DL, and some of the sequence data generation was supported by the NIH grant R01NS094596. EAL was supported by the Suh Kyungbae Foundation and the NIH grants DP2AG072437 and R01 AG070921 and Allen Discovery Center program a Paul G. Allen Frontiers Group advised program of the Paul G. Allen Family Foundation. CAW was supported by grants from the NIH R01NS035129 and by the Allen Frontiers Program. CAW is an Investigator of the Howard Hughes Medical Institute. KTK was supported by the Yale Rockefeller Centers for Mendelian Genomics and R01NS109358 and R01NS111029 and R01NS117609 and the Simons Foundation and March of Dimes and Hydrocephalus Association and Rudi Schulte Research Institute. ### 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: IRB of Yale-New Haven Hospital gave ethical approval for this work. IRB of Boston Children's Hospital gave ethical approval for this work. Ethics committee of Austin Hospital gave ethical approval for this work. Ethics committee of Royal Melbourne Hospital gave ethical approval for this work. IRB of Boston Children's Hospital reviewed the ethical approvals from all the recruitment sites and provided ethical approval for all research performed with samples obtained from patients at multiple recruitment sites. 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 and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All the whole-exome and gene-panel sequencing data generated for this study will be deposited in the database of Genotypes and Phenotypes (dbGaP).