Clinical Variants in Expressing Human STXBP1 Reveal a Novel Class of Pathogenic Variants and Classify Variants of Uncertain Significance

Purpose: Modeling disease variants in animals is useful for drug discovery, understanding disease pathology, and classifying variants of uncertain significance (VUS) as pathogenic or benign.

Methods: Using Clustered Regularly Interspaced Short Palindromic Repeats, we performed a Whole-gene Humanized Animal Model procedure to replace the coding sequence of the animal model's ortholog with the coding sequence for the human gene. Next, we used Clustered Regularly Interspaced Short Palindromic Repeats to introduce precise point variants in the Whole-gene Humanized Animal Model-humanized locus from 3 clinical categories (benign, pathogenic, and VUS). Twenty-six phenotypic features extracted from video recordings were used to train machine learning classifiers on 25 pathogenic and 32 benign variants.

Results: Using multiple models, we were able to obtain a diagnostic sensitivity near 0.9. Twenty-three VUS were also interrogated and 8 of 23 (34.8%) were observed to be functionally abnormal. Interestingly, unsupervised clustering identified 2 distinct subsets of known pathogenic variants with distinct phenotypic features; both p.Tyr75Cys and p.Arg406Cys cluster away from other variants and show an increase in swim speed compared with hSTXBP1 worms. This leads to the hypothesis that the mechanism of disease for these 2 variants may differ from most STXBP1-mutated patients and may account for some of the clinical heterogeneity observed in the patient population.

Conclusion: We have demonstrated that automated analysis of a small animal system is an effective, scalable, and fast way to understand functional consequences of variants in and identify variant-specific intensities of aberrant activity suggesting a genotype-to-phenotype correlation is likely to occur in human clinical variations of .