Director, Pediatric Epilepsy Program Brown University Providence, Rhode Island
Rationale: Rasmussen encephalitis (RE) is a rare cause of pharmacoresistant epilepsy, characterized by progressive hemispheric inflammation resulting in seizures and, if left untreated, hemiplegia, hemianopia and cognitive decline. Data on gene expression in this condition are lacking. With this preliminary study, we sought to investigate aberrant gene expression pathways involved in the pathophysiology of RE. Methods: We conducted RNA-sequencing of fresh-frozen resected epileptogenic tissue from four children with RE (age at surgery = 11.8 ± 0.5 years). We analyzed temporal lobe tissue in three out of four patients and frontal and temporal lobe tissue in one of four patient. Control brain tissue included then specimens from perilesional resected areas in different patients; one with RE and nine with focal cortical dysplasia. RNA-sequencing was performed using standard protocol on Illumina HiSeq 2x150bp sequencing, single index platform. After extraction of normalized gene hit counts, we utilized the DESeq2 package with R version 4.0.1, and compared gene expression between cases and controls. The Wald test was used to generate p-values and log2 fold changes. Genes with an adjusted p-value < 0.05 were called as differentially expressed genes (DEG) for each comparison. Gene enrichment analysis was then performed with PANTHER Overrepresentation Test and the Reactome platform. Results: We identified 1,385 DEGs in RE samples. Functional gene clustering analysis showed over-representation of several pathways involved in neuroinflammation mediated by T-cell response including interferon-signaling (particularly IFN-gamma – CD44, GBP2, GBP3, CACNA2D2, IRF1, IRF2, IRF4, IRF5, IRF9 genes), signaling by interleukins (IL-6 [CAMK2D, CRLF1, IL6R, IL6ST, ITPR2, LIFR, OSMR, SOCS3, STAT1, STAT3], IL-4/IL-13 [IL4R, ANXA1, COL1A2, FSCN1, IRF4, CCR1, ITGAX, CEBPD], and IL-10 [CCR1, CXCL10, CCR5, CD86, CSF1, FPR1, IL18, IL1R1]), phosphorylation of CD3 and TCR zeta chains (HLA-DPA1, HLA-DRA, HLA-DRB1, HLA-DRB-5, ITGB4, PAG1, PTPN22, PTPRC genes). Microtubule-dependent trafficking and aggrephagy were also upregulated, with several overexpressed genes of the Tubulin family (TUBA1A, TUBA1B, TUBB1, TUBB2A, TUBB3, TUBB4B, TUBB6). Similarly, assembly and cell surface presentation of NMDA receptors pathway was also upregulated (CAMK2D, CASK, GRIN3A, LRRC7, NBEA, PAPLN). Conclusions: With this preliminary study, we contribute to expanding the knowledge of pathophysiological processes involved in Rasmussen’s encephalitis. We found upregulation of several neuroinflammatory pathways, with a central role of IFN-gamma signaling, interleukins and microtubule-dependent trafficking. These data support the findings from pathological studies implicating a pivotal role of immunological mechanisms in RE. These data will require validation by qPCR and further study with a larger cohort. Follow-up study on these samples will utilize other platforms such as metabolomics and single-cell sequencing analysis. Funding: Please list any funding that was received in support of this abstract.: This study was supported by institutional funds (Brown University).
