Functional classification of VUS based on examining the phenotypic difference between the reference and variant alleles in flies. Both rescue and overexpression-based approaches can utilize various phenotypes such as lethality, fertility, lifespan, morphological defects, or behavioral phenotypes as functional readouts of gene function. (A) Rescue-based approaches to assess patient variants require a scorable phenotype caused by LOF of the fly ortholog that is rescued by expression of the reference allele. If the VUS is a LOF, expression of the variant would be unable to rescue to the same level as the reference. (B) Overexpression strategies can be used to classify the variant of interest based on comparing the phenotypes induced by each transgene. LOF alleles are determined by a phenotype that is less severe than the reference phenotype, indicated by “>”, whereas GOF alleles are determined by a phenotype that is more severe than the reference phenotype, indicated by “<”.
