Objective: Collier/Olf/EBF (COE) transcription factors have distinct expression patterns in the developing and mature nervous system. To date, a neurological disease association has been conclusively established for only the Early B-cell Factor-3 (EBF3) COE family member through the identification of heterozygous loss-of-function variants in individuals with autism spectrum/neurodevelopmental disorders (NDD). Here, we identify a symptom severity risk association with missense variants primarily disrupting the zinc finger domain (ZNF) in EBF3-related NDD. Methods: A phenotypic assessment of 41 individuals was combined with a literature meta-analysis for a total of 83 individuals diagnosed with EBF3-related NDD. Quantitative diagnostic phenotypic and symptom severity scales were developed to compare EBF3 variant type and location to identify genotype-phenotype correlations. To stratify the effects of EBF3 variants disrupting either the DNA-binding domain (DBD) or the ZNF, we used in vivo fruit fly UAS-GAL4 expression and in vitro luciferase assays. Results: We show that patient symptom severity correlates with EBF3 missense variants perturbing the ZNF, which is a key protein domain required for stabilizing the interaction between EBF3 and the target DNA sequence. We found that ZNFassociated variants failed to restore viability in the fruit fly and impaired transcriptional activation. However, the recurrent variant EBF3 p.Arg209Trp in the DBD is capable of partially rescuing viability in the fly and preserved transcriptional activation. Interpretation: We describe a symptom severity risk association with ZNF perturbations and EBF3 loss-of-function in the largest reported cohort to date of EBF3-related NDD patients. This analysis should have potential predictive clinical value for newly identified patients with EBF3 gene variants.

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Hypotonia, Ataxia, and Delayed Development syndrome is a neurodevelopmental disorder caused by heterozygous Early B-Cell Factor 3 (EBF3) loss-of-function variants. Identified in 2016, the full spectrum of clinical findings and the relationship between the EBF3 genotype and clinical outcomes has not been determined beyond its namesake features. We combined a phenotypic assessment of 33 individuals molecularly diagnosed with EBF3 pathogenic variants with a meta-analysis of 34 previously reported individuals. The combined 62 unique individuals enabled comparative cross-sectional phenotype and genotype analysis in the largest cohort to date of affected individuals. Cardinal distinguishing features were identified that facilitate phenotypic stratification for clinical diagnosis. We developed assessment scales to ascertain individuals at risk for pathogenic EBF3 variants, stratify the clinical severity, and connect variant-specific molecular phenotypes to clinical outcomes. Our findings show that a specific class of EBF3 variants affecting the evolutionarily conserved Zinc Finger (ZNF) motif, which is critical for stabilizing the protein interaction with the DNA target sequence, is associated with an increased risk of persistent motor and language impairments. These findings highlight the impact of combining variant-specific molecular phenotypes with comprehensive clinical data to predict neurodevelopmental outcomes and potentially guide personalized decisions for therapeutic interventions.