Functional profiling of STXBP1 missense variants using a novel dual‐readout fluorometric assay

Abstract Objective STXBP1 ‐related disorders ( STXBP1 ‐RD) are among the most common genetic neurodevelopmental disorders, marked by early onset epilepsy, global developmental delay, and motor impairments. Many missense variants remain uncharacterized, limiting accurate variant interpretation and hindering development of precision therapies. Current American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) variant curation frameworks provide limited discriminatory power for STXBP1 missense variants due to limited functional data and frequently incomplete parental testing, leaving a substantial proportion of variants classified as variants of uncertain significance. Methods We developed a fluorometric assay that leverages split fluorescence complementation to simultaneously measure STXBP1 protein stability and its binding affinity to a primary interaction partner, syntaxin‐1A (STX1A), including genetically engineered closed and open STX1A conformations, which are essential for SNARE complex function. Using this platform, we evaluated five benign variants, six pathogenic variants, and four variants that result in an atypically mild clinical presentation. Results Benign population variants showed minimal effects on stability or binding, whereas known pathogenic variants consistently demonstrated reduced protein stability and diminished binding to STX1A, with some exhibiting STX1A conformation‐specific binding deficits. Variants associated with atypically mild phenotypes displayed intermediate, variant‐specific profiles. To quantify discriminatory performance, we developed and trained an exploratory support vector machine classifier using stability and binding metrics. With the exception of a single STXBP1 variant in an individual with a mild clinical presentation, the model correctly classified benign and pathogenic variants using leave‐one‐out cross‐validation. For variants with available clinical data, classifier scores showed a statistically significant correlation with clinical severity. Significance Together, these findings demonstrate that a scalable, quantitative assay capturing STXBP1 stability and syntaxin‐binding behavior can differentiate benign from pathogenic variants and provide functional insights into mechanisms of pathogenicity. This platform offers a path toward improved variant interpretation, supports refinement of ACMG/AMP criteria for synaptic disorders, and may inform future therapeutic targeting strategies for STXBP1 ‐RD.