Resubmission: Systems for Exploring Pathogenicity in Clinical Variants of a Presynaptic Protein Involved in Epilepsy - Technical Summary
Epilepsy is a large health problem affecting 1% of the human population. Genetic alterations in STXBP1
represents one of the most common causes of severe Early Infantile Epileptic Encephalopathy and many
patients with pathogenic variations in STXBP1 are resistant to standard antiepileptic treatment. Genome wide
DNA sequencing is now being adopted in clinical practice and an increasing number of variants are identified
in epilepsyassociated genes, yet the clinical interpretation of the new variants is challenging. Some of the
variants are known to be either pathological or benign, yet a majority of the gene variations remain unknown
for their functional consequence. A large number of Variant of Uncertain Significance (VUS) are becoming
commonplace in genes for human diseases, providing a significant barrier in making diagnoses and
implementing therapies. Bioinformatic approaches can provide some insight into pathogenic probability of VUS
alleles, but functional studies in animal model systems are often needed to make definitive of pathogenicity
assignments. The expense and long timelines of mouse models production make the use of alternative small
animal models attractive. In this proposal, the C. elegans nematode is used as an alternative model capable of
fast highthroughput production and screening. Human genes are installed as geneswap replacements of the
native diseasegene homologs. In preliminary work, geneswap humanization of STXBP1 in the unc18 locus
rescued severe locomotion and behavior defects present in the gene KO animals. Further, installation of two
pathogenic variants into the geneswap locus lead to significant disruption of activity. In this proposal, a larger
set of clinicallyobserved variants are installed as a geneswap humanized STXBP1 locus, then function
deviations are measured with various behavioral and physiological assays. In Aim 1 of proposal, a set of
pathogenic variants are installed and measured capacity to exhibit detectable defects of function. In Aim 2, a
set of known benign variants are installed and measured for presence of functional defects. In Aim 3, a set of
VUS alleles are installed and activity is compared to the known pathogenic and benign variants. Achieving all
aims provides a body of data for assessing the predictive capacity of humanized STXBP1 strains to support or
refute the remaining variants for capacity to be pathogenic. In additional future phase II work, once pathogenic
behavior of a variant is detected as deviant activity in the functional assays, the system can then be used to
screen for compounds that lead to reversal of activity back towards wild type. As a result, success of the
phase I work, create a platform for probing variant biology and drug discovery. Applied to other targets, similar
discovery systems can be developed for many of the 6000+ disease associated genes of the human genome.
