Contribution of Somatic Ras/Raf/Mitogen-Activated Protein Kinase Variants 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 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

Dengue NS1 antigen contributes to disease severity by inducing interleukin (IL)-10 by monocytes

Both dengue NS1 antigen and serum IL-10 levels have been shown to associate with severe clinical disease in acute dengue infection and IL-10 has also been shown to suppress dengue specific T cell responses. Therefore, we proceeded to investigate the mechanisms by which dengue NS1 contributes to disease pathogenesis and if it is associated with altered IL-10 production. Serum IL-10 and dengue NS1 antigen levels were assessed serially in 36 adult Sri Lankan individuals with acute dengue infection. We found the serum IL-10 levels positively correlated with dengue NS1 antigen levels (Spearmans R=0.47, p&lt;0.0001), and NS1 also correlated with annexin V expression by T cells in acute dengue (Spearman’s R= 0.63, p=0.001). However, NS1 levels did not associate with the functionality of T cell responses or with expression of costimulatory molecules. Therefore, we further assessed the effect of dengue NS1 on monocytes and T cells by co-culturing primary monocytes and peripheral blood mononuclear cells (PBMC), with varying concentrations of NS1 up to 96 hours. Apoptosis of T cells was determined by annexin V expression. Monocytes co-cultured with NS1 produced high levels of IL-10with the highest levels seen at 24 hours, and then gradually declined,. Recombinant NS1 also induced annexin V expression by both CD4+ and CD8+ T cells in a dose dependent manner although a wide individual variation was seen. Therefore, our data show that dengue NS1 appears to contribute to pathogenesis of dengue infection by inducing IL-10 production by monocytes and possibly inducing apoptosis of T cells.</p

Expansion of highly activated iNKT cells with altered phenotype in acute dengue infection.

Invariant natural killer T cells (iNKTs) are capable of rapid activation and production of cytokines upon recognition of antigenic lipids presented by CD1d molecules. They have been shown to play a significant role in many viral infections and were observed to be highly activated in patients with acute dengue infection. In order to further characterize their role in dengue infection, we investigated the proportion of iNKT cells and their phenotype in adult patients with acute dengue infection. The functionality of iNKT cells in patients was investigated by both IFNγ and IL-4 ex vivo ELISpot assays following stimulation with alpha-galactosyl-ceramide (GalCer). We found that circulating iNKT cells proportions were significantly higher (p=0.03) in patients with acute dengue when compared to healthy individuals and were predominantly of the CD4+ subset. iNKT cells of patients with acute dengue had reduced proportions expressing CD8 and CD161 when compared to healthy individuals. The iNKT cells of patients were highly activated and iNKT activation significantly correlated with dengue virus-specific IgG antibody levels. iNKT cells expressing Bcl-6 (p=0.0003) and both Bcl-6 and ICOS (p=0.006) were significantly increased in patients when compared to healthy individuals. Therefore, our data suggest that in acute dengue infection there is an expansion of highly activated CD4+ iNKT cells, with reduced expression of CD161 markers.</p