Resubmission: Systems for Exploring Pathogenicity in Clinical Variants of a Presynaptic Protein Involved in Epilepsy

Technical Summary Epilepsy is a large health problem affectingof the human populationGenetic alterations in STXBPrepresents one of the most common causes of severe Early Infantile Epileptic Encephalopathy and many patients with pathogenic variations in STXBPare resistant to standard anti epileptic treatmentGenome wide DNA sequencing is now being adopted in clinical practice and an increasing number of variants are identified in epilepsy associated genesyet the clinical interpretation of the new variants is challengingSome of the variants are known to be either pathological or benignyet a majority of the gene variations remain unknown for their functional consequenceA large number of Variant of Uncertain SignificanceVUSare becoming commonplace in genes for human diseasesproviding a significant barrier in making diagnoses and implementing therapiesBioinformatic approaches can provide some insight into pathogenic probability of VUS allelesbut functional studies in animal model systems are often needed to make definitive of pathogenicity assignmentsThe expense and long timelines of mouse models production make the use of alternative small animal models attractiveIn this proposalthe Celegans nematode is used as an alternative model capable of fast high throughput production and screeningHuman genes are installed as gene swap replacements of the native disease gene homologsIn preliminary workgene swap humanization of STXBPin the unclocus rescued severe locomotion and behavior defects present in the gene KO animalsFurtherinstallation of two pathogenic variants into the gene swap locus lead to significant disruption of activityIn this proposala larger set of clinically observed variants are installed as a gene swap humanized STXBPlocusthen function deviations are measured with various behavioral and physiological assaysIn Aimof proposala set of pathogenic variants are installed and measured capacity to exhibit detectable defects of functionIn Aima set of known benign variants are installed and measured for presence of functional defectsIn Aima set of VUS alleles are installed and activity is compared to the known pathogenic and benign variantsAchieving all aims provides a body of data for assessing the predictive capacity of humanized STXBPstrains to support or refute the remaining variants for capacity to be pathogenicIn additional future phase II workonce pathogenic behavior of a variant is detected as deviant activity in the functional assaysthe system can then be used to screen for compounds that lead to reversal of activity back towards wild typeAs a resultsuccess of the phase I workcreate a platform for probing variant biology and drug discoveryApplied to other targetssimilar discovery systems can be developed for many of thedisease associated genes of the human genome Narrative Many cases of Epilepsy are caused by defects in presynaptic proteinsDoctors need to understand which of the defects cause diseaseIn this projectsmall animal models are humanized to express defective genes associated with epilepsyThe resulting humanized animal models and their analysis will be used in understanding of disease mechanisms and discovering new therapeutics