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

Area of Science:

• Genetics
• Neuroscience
• Molecular Biology

Background:

• Pathogenic variants in the STXBP1 gene are associated with a range of neurodevelopmental disorders.
• Accurate interpretation of genetic variants, especially those of uncertain significance, is crucial for diagnosis and patient management.

Purpose of the Study:

• To investigate the pathogenicity of a splice site variant (c.429+5G>A) in a patient with developmental delay.
• To determine the functional impact of the c.429+5G>A variant on STXBP1 expression and neuronal function.
• To evaluate the utility of combining functional and clinical data for variant interpretation.

Main Methods:

• Analysis of patient-derived neurons to assess morphology and STXBP1/MUNC18-1 protein and mRNA levels.
• Utilized nonsense-mediated decay inhibition, RT-PCR, and minigene assays to investigate transcript alterations.
• Performed EEG biomarker analysis to assess neuronal network function in the patient compared to controls.

Main Results:

• The c.429+5G>A variant led to >40% reduction in STXBP1/MUNC18-1 levels, similar to known loss-of-function variants.
• Exon skipping due to the splice site variant resulted in a frameshift and premature stop codon.
• EEG analysis revealed distinct biomarkers, including altered temporal correlations and excitation/inhibition ratios, in the patient.

Conclusions:

• The c.429+5G>A variant is pathogenic and contributes to neurodevelopmental disorders by reducing STXBP1 levels.
• Multimodal assessment combining functional and clinical data is valuable for accurate variant classification.
• The patient was subsequently enrolled in the European STXBP1 registry (ESCO).