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 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

Identification of human progenitors of exhausted CD8+T cells associated with elevated IFN-γ response in early phase of viral infection

T cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. Limited knowledge exists of the initial viral and immune dynamics that characterise exhaustion in humans. We studied longitudinal blood samples from a unique cohort of individuals with primary infection using single-cell multi-omics to identify the functions and phenotypes of HCV-specific CD8+ T cells. Early elevated IFN-γ response against the transmitted virus is associated with the rate of immune escape, larger clonal expansion, and early onset of exhaustion. Irrespective of disease outcome, we find heterogeneous subsets of progenitors of exhaustion, based on the level of PD-1 expression and loss of AP-1 transcription factors. Intra-clonal analysis shows distinct trajectories with multiple fates and evolutionary plasticity of precursor cells. These findings challenge the current paradigm on the contribution of CD8+ T cells to HCV disease outcome and provide data for future studies on T cell differentiation in human infections

Mosaicism in tuberous sclerosis complex: Lowering the threshold for clinical reporting

Tuberous sclerosis complex (TSC) is a multi-system genetic disorder. Most patients have germline mutations in TSC1 or TSC2 but, 10%-15% patients do not have TSC1/TSC2 mutations detected on routine clinical genetic testing. We investigated the contribution of low-level mosaic TSC1/TSC2 mutations in unsolved sporadic patients and families with TSC. Thirty-one sporadic TSC patients negative on routine testing and eight families with suspected parental mosaicism were sequenced using deep panel sequencing followed by droplet digital polymerase chain reaction. Pathogenic variants were found in 22/31 (71%) unsolved sporadic patients, 16 were mosaic (median variant allele fraction [VAF] 6.8% in blood) and 6 had missed germline mutations. Parental mosaicism was detected in 5/8 families (median VAF 1% in blood). Clinical testing laboratories typically only report pathogenic variants with allele fractions above 10%. Our findings highlight the critical need to change laboratory practice by implementing higher sensitivity assays to improve diagnostic yield, inform patient management and guide reproductive counseling

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