Abstracts

Rationale: Developmental and epileptic encephalopathies (DEEs) are a group of pediatric epilepsies wherein recurrent seizures occur in the context of intellectual disability (ID). In a recent study, our group identified a pathogenic, heterozygous de novo c.1768G >A, p.Glu590Lys missense variant in the transcription factor (TF), CUX2, as a novel cause of DEEs. CUX2 represses gene expression during neurodevelopment and function, regulating dendritogenesis, synaptogenesis, proliferation and differentiation in the cerebral cortex. The variant alters a highly conserved residue in a DNA binding domain, changing it from negatively to positively charged. Given the negatively charged nature of DNA, it is likely that the variant enhances CUX2 binding to DNA, aberrantly decreasing gene expression critical for neurodevelopment.

Methods: To determine the effects of the variant we use an induced pluripotent stem cell (iPSC) model. To study CUX2 in a functionally relevant cell type, we differentiated iPSCs to neuronal progenitor cells (NPCs) and will also differentiate cortical excitatory (glutamatergic) neurons. With CRISPR-Cas9, we will generate an isogenic knockout control. To assess CUX2 genome occupancy, we conducted CUT&RUN in NPCs. We will conduct RNA-seq on the neuronal, isogenic lines of CUX2 function to determine changes in gene expression due to the variant. By integrating the two datasets, we will identify CUX2 target genes and the direction of change in expression.

Results: We obtained fibroblasts from a patient in the aforesaid study. She presented with absence seizures that evolved into myoclonic seizures and non-convulsive status epilepticus. She has severe ID and demonstrates stereotypies and obsessional features. The fibroblasts were reprogrammed into iPSCs, and quality control checks were performed. Using the STEMdiff SMADi Neural Induction Kit, we differentiated the iPSCs into NPCs and confirmed expression of CUX2 using qRT-PCR, Western blotting, and immunofluorescence. We conducted CUT&RUN with control line NPCs, determining that CUX2 bound primarily to promoter regions. Gene Ontology (GO) analysis revealed that the genes CUX2 bound to are involved in RNA splicing and proteasomal protein catabolic processes.

Conclusions: We aim to determine the pathogenic mechanism by which p.Glu590Lys affects CUX2 function. Thus far, our study has revealed that CUX2 is associated with promoter regions and we will integrate this information with our RNA-seq data to determine how the CUX2 p.Glu590Lys variant alters CUX2 binding and gene expression. GO analysis suggests that CUX2 is involved in the regulation of RNA processing and protein degradation. Our study lays the foundation for studying other TFs with missense variants in patients with NDDs. Overall, investigation of TFs provides new opportunities for understanding the biological mechanisms that underpin epilepsy, pushing forward the development of therapeutics against these disorders.

Funding: Please list any funding that was received in support of this abstract.: This research was supported by the AES Predoctoral Research Fellowship Award.