Brain Molecular Mechanisms in Rasmussen Encephalitis

Objective: Identify molecular mechanisms in brain tissue of Rasmussen encephalitis (RE) when compared to people with non-RE epilepsy (PWE) and control cases using whole exome sequencing (WES), RNAseq, and proteomics. Methods: Frozen brain tissue (ages 2-19 years) was obtained from control autopsy (n=14), surgical PWE (n=10), and surgical RE cases (n=27). We evaluated WES variants in RE associated with epilepsy, seizures, RE, and human leukocyte antigens (HLAs). Differential expression was evaluated by RNAseq (adjusted p<0.05) and label-free quantitative mass spectrometry (false discovery rate<5%) in the three groups. Results: WES revealed no common pathogenic variants in RE, although several rare and likely deleterious variants of unknown significance (VUS; ANGPTL7/MTOR, SCN1A, FCGR3B, MTOR) and more common HLA VUS in >25% RE cases (HLA-DRB1, HLA-DQA2) all with allele frequency <5% in the general population. RNAseq in RE vs. PWE (1516 altered transcripts) revealed significant activation of crosstalk between dendritic and natural killer cells (p=7.94x10-6, z=2.65), in RE vs. control (7466 transcripts) neuroinflammation signaling activation (p=6.31x10-13, z=5.07), and in PWE vs. control (945 transcripts) phagosome formation activation (p=2.00x10-13, z=5.61). Proteomics detected fewer altered targets. Significance: In RE, we identified activated immune signaling pathways and immune cell type annotation enrichment that suggest roles of the innate and adaptive immune responses, as well as HLA variants that may increase vulnerability to RE. Follow up studies could evaluate cell type density and subregional localization associated with top targets, clinical history (neuropathology, disease duration), and whether modulating crosstalk between dendritic and natural killer cells may limit disease progression. Overall design: RNAseq. Comparisons were made from frozen brain tissue for RE, PWE, and control. RNA was isolated from brain (10mg) with miRNeasy Purification of Total RNA (Qiagen), using Qiazol with handheld pestle-equipped device, 22-gauge needle. RNA quality and concentration was determined by Agilent Bioanalyzer. Libraries were created by NYU GTC with Illumina Stranded Total RNA Prep with Ribo-Zero Plus (Cat.20040529), and sequenced on Illumina NovaSeq6000 with S2 100-cycle flow cell, 50bp paired-end.