Generation and characterization of a knockout mouse of an enhancer of <i>EBF3</i>

Genomic studies of neurodevelopmental disorders (NDDs) have identified several relevant genomic variants. EBF3 is a gene with an excess of protein-coding de novo variants and underlies Hypotonia, Ataxia, and Delayed Development Syndrome. We previously identified noncoding de novo variants in an enhancer of EBF3 and further found enrichment of deletions of this enhancer in NDDs. In this study, we generated a novel mouse line that deletes the highly conserved, orthologous mouse region within the Rr169617 regulatory region, and characterized the molecular and phenotypic aspects of this mouse model. We found a deviation from Mendelian expectation (P=0.02) with significant depletion of the deletion allele (P=5.8×10-4). Rr169617+/- mice had a reduction of Ebf3 expression by 10% and Rr169617-/- mice had a reduction by 20%. Differential expression analyses in E12.5 forebrain, midbrain, and hindbrain in Rr169617+/+ versus Rr169617-/- mice identified dysregulated genes including histone and brain development related genes. A priori phenotyping analysis (open field, hole board and light/dark transition) identified sex-specific differences in mobility only for Rr169617-/- mice across multiple behavioral assays with Rr169617-/- males less mobile than Rr169617-/- females. Furthermore, both sexes when homozygous for the enhancer deletion displayed body composition differences when compared to wildtype mice. Overall, we show that deletion within Rr169617 reduces expression of Ebf3 and results in phenotypic outcomes consistent with potential sex specific behavioral differences.