Abstract

Our study investigates the pathogenic role of a novel CELF2 gene mutation (p.Pro520Arg) in epilepsy, particularly in infantile epileptic spasms syndrome (IESS), a severe developmental and epileptic encephalopathy. The mutation was identified through whole-exome sequencing in an 11-month-old male infant who presented with seizure onset at 2 months of age and global developmental delay. Brain MRI revealed corpus callosum dysplasia, and EEG showed intermittent hypsarrhythmia. The CELF2 mutation was confirmed as de novo and classified as likely pathogenic according to ACMG guidelines.

To explore the functional consequences of CELF2 deficiency, we established zebrafish models with celf2 knockout and knockdown using CRISPR/Cas9 and morpholino technologies. These zebrafish exhibited hyperactivity, seizure-like behaviors, and abnormal EEG discharges. Behavioral abnormalities were rescued by injection of wild-type CELF2 mRNA, but not by the mutant form, demonstrating that the mutation results in a loss-of-function. In situ hybridization revealed increased expression of key neurodevelopmental genes, including FOS, BDNF, NPAS4, GABRA1, GABRG2, and PYYA, in the brains of celf2 mutants.

Furthermore, multi-omics analyses provided insights into the molecular mechanisms underlying the phenotype. Proteomic and metabolomic profiling showed dysregulation of lipid metabolism pathways and upregulation of Hsp90b1, implicating impaired neuronal lipid handling in seizure susceptibility. Single-cell RNA sequencing identified altered proportions of neuronal subtypes and changes in gene expression related to nucleosome positioning, arginine and proline metabolism, and several signaling pathways, including GnRH and NOD receptor signaling.

In summary, this study demonstrates that CELF2 is a novel epilepsy-related gene whose deficiency leads to significant neurodevelopmental and molecular abnormalities. The integration of behavioral, electrophysiological, transcriptomic, proteomic, and metabolomic data highlights the multifaceted role of CELF2 in brain function and offers new insights into the pathogenesis of genetic epilepsy and potential therapeutic avenues.