RNA-seq analysis of hippocampal tissues reveals novel candidate genes for drug refractory epilepsy in patients with MTLE-HS

Abstract

Array-based profiling studies have shown implication of aberrant gene expression patterns in epileptogenesis. We have performed transcriptome analysis of hippocampal tissues resected from patients with MTLE-HS using RNAseq approach. Healthy tissues from tumour margins obtained during tumour surgeries were used as non-epileptic controls. RNA sequencing was performed using standard protocols on Illumina HiSeq 2500 platform. Differential gene expression analysis of the RNAseq data revealed 56 significantly regulated genes in MTLE patients. Gene cluster analysis identified 3 important hubs of genes mostly linked to, neuroinflammation and innate immunity, synaptic transmission and neuronal network modulation which are supportive of intrinsic severity hypothesis of pharmacoresistance. This study identified various genes like FN1 which is central in our analysis, NEUROD6, RELN, TGFβR2, NLRP1, SCRT1, CSNK2B, SCN1B, CABP1, KIF5A and antisense RNAs like AQP4-AS1 and KIRREL3-AS2 providing important insight into the understanding of the pathophysiology or genomic basis of drug refractory epilepsy due to MTS.

Truncating mutation in CSNK2B and myoclonic epilepsy

“…Here, we report a male patient with intellectual disability and myoclonic epilepsy beginning during infancy who was found to have a de novo heterozygous frameshift mutation in CSNK2B . The involvement of CSNK2B in epileptogenicity is supported by the results of a previous RNA‐seq analysis of hippocampal tissues in a cohort of patients with mesial temporal lobe epilepsy‐hippocampal sclerosis, which showed the downregulation of CSNK2B expression (Dixit et al., ). Our present mutation analysis data agreed with this semiquantitative expression analysis data of hippocampal tissues from a cohort of patients with refractory epilepsy.…”

Understanding Tissue-specific Gene Regulation

“…To ensure this result was not due to the threshold we used when identifying tissue-specific TFs, we selected the ten transcription factors with the highest and lowest expression enrichment in this brain-tissue subregion (see Supplemental Material and Methods) and performed a detailed investigation of their GSEA profiles ( Figure 4B). NEUROD2 and SP8 were the top tissue-specific transcription factors with brain-function associated targeting profiles; these TFs play important roles in brain function [31][32][33]. In addition, four of the highly non-tissuespecific transcription factors (based on expression)-GRHL1, KLF15, PAX3, and TET1-have positive enrichment for targeting genes with relevant brain functions.…”

Altered glutamatergic tone reveals two distinct resting state networks at the cellular level in hippocampal sclerosis

“…Using RNA-seq analysis of the hippocampal tissue resected from patients with HS, we have previously identified hubs of genes linked to synaptic transmission and neuronal network modulation 18 . We reported significant alterations in the expression levels of transporters, receptors and molecules involved in calcium signalling 18 . However, in that study we could not detect mRNA level alterations of NR2A levels in the RNAseq analysis, which could be attributed to the small sample size.…”

“…qPCR was performed in an independent set of 10 patients (both hippocampal and ATL) and 10 non-seizure control samples. RNA was extracted, purified and analyzed for purity by calculating the RIN values as previously described 18 . Purified RNA was reverse transcribed using high capacity cDNA reverse transcription kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer protocol.…”

“…Purified RNA was reverse transcribed using high capacity cDNA reverse transcription kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer protocol. Real time PCR amplifications were performed in CFX96 Real Time System (Bio-Rad, USA) with the following cycling parameters: an initial hot start of 95 °C for 3 min followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. To normalize qPCR reactions, HPRT (hypoxanthine phosphoribosyl-transferase) was included as a reference gene 18 . The 2 −ΔΔCq (Livak) Method (Bio-Rad CFX Manager software) was used to quantify the relative normalized expression of studied genes.…”