Abstracts

Rationale: Haploinsufficiency of the CHD2 gene leads to early-onset epileptic encephalopathy (EE), accounting for ~1.2% of cases as one of the more commonly mutated genes for this condition [1]. Patients with CHD2 mutations have a spectrum of symptoms in addition to their epilepsy including developmental delay, intellectual disability, autism spectrum disorders, and behavioral problems. While broadly understood to be a chromatin-remodeler, the exact function of CHD2 in the brain has not been fully characterized. To date, CHD2 is the only EE gene involved in remodeling chromatin; therefore understanding the mechanism of its actions may uncover a unique epilepsy gene network and provide more targets for therapeutic intervention. Recent studies have shown an increased rate of neuronal differentiation of neurons in the mouse cortex after CHD2 knockdown via shRNA [2]. The majority of CHD2 pathogenic variants lead to premature truncation of the protein, and there is no correlation between the position of the variant and severity of the disorder, suggesting all truncated proteins are non-functional. We therefore sought to develop a human cellular knockout model of CHD2-related EE to study how CHD2 mutations affect human neural development.  Methods: We used CRISPR/Cas9 editing of a control human induced pluripotent stem cell (iPSC) line to introduce indels in the fourth exon of CHD2, which is included in both known mRNA isoforms and would lead to loss of all CHD2 protein domains. We differentiated mutant and isogenic control iPSCs into cortical-like neural progenitor cells using a dual-SMAD inhibition protocol. At day 10, CHD2 +/- and CHD2 +/+ RNA samples were isolated, and total RNA-seq was performed. Whole-cell patch-clamp recordings were done at later stages.  Results: Using stringent criteria (q value < 0.05), 47 transcripts were found to be upregulated and 192 downregulated. Gene ontology (GO) analysis for up-regulated genes identified many transcripts involved in mature neuronal processes (axonogenesis, neurotransmitter transport, voltage-gated ion channel activity), while the down-regulated genes comprised pathways involved in cell proliferation and extracellular matrix. Mutant neurons in 2-D cultures recorded by whole-cell patch clamp demonstrated greater maximal evoked firing frequency and spontaneously firing than controls. RNA-seq analysis of the neurons is ongoing.  Conclusions: Taken together, our data suggests premature neuronal differentiation and neuronal hyperexcitability of CHD2 heterozygous loss-of-function human iPSCs. Further investigation is needed to understand how CHD2 haploisufficiency leads to premature differentiation and network hyperexcitability in patient brains.1.            Carvill GL, Heavin SB, Yendle SC, McMahon JM, O'Roak BJ, Cook J, et al. Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1. Nature genetics. 2013;45(7):825-30.2.            Shen T, Ji F, Yuan Z, Jiao J. CHD2 is required for embryonic neurogenesis in the developing cerebral cortex. Stem Cells. 2015;33(6):1794-806.  Funding: NIH/NINDS and CURE