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.

IPSC-derived neurons; STXBP1-RD; case report; functional validation; spice-site variant.