Functional Analyses in Patient‐Derived Neurons Establish Pathogenicity for STXBP1 Splice Variant c.429+5G>A

Pathogenic STXBP1 variants cause a broad spectrum of neurodevelopmental disorders. We investigated a patient with developmental delay but no seizures, carrying a heterozygous, predicted splice site variant, c.429+5G>A, initially classified as a variant of uncertain significance. Patient‐derived neurons had normal morphology in vitro, but > 40% reduced MUNC18‐1/ STXBP1 protein and mRNA levels, comparable with two established loss‐of‐function variants (Asp262Val and Arg235*). Nonsense‐mediated decay inhibition increased transcript levels, and RT‐PCR/minigene analysis demonstrated Exon 6 skipping, resulting in a frameshift and premature stop codon. Relative to a large cohort of typically developing children, EEG biomarker analysis revealed elevated long‐range temporal correlations in beta and gamma bands, increased delta power, and reduced excitation/inhibition ratio in the beta band. This multimodal assessment demonstrates that c.429+5G>A is a disease‐causing variant, and the value of combining functional and clinical data for accurate variant interpretation. Based on this, the patient was included in the EU STXBP1 registry ESCO.