Functional Studies of Genetic Variants Associated with Human Diseases in Notch Signaling-Related Genes Using...

Rare variants in the many genes related to Notch signaling cause diverse Mendelian diseases that affect myriad organ systems. In addition, genome- and exome-wide association studies have linked common and rare variants in Notch-related genes to common diseases and phenotypic traits. Moreover, somatic mutations in these genes have been observed in many types of cancer, some of which are classified as oncogenic and others as tumor suppressive. While functional characterization of some of these variants has been performed through experimental studies, the number of "variants of unknown significance" identified in patients with diverse conditions keeps increasing as high-throughput sequencing technologies become more commonly used in the clinic. Furthermore, as disease gene discovery efforts identify rare variants in human genes that have yet to be linked to a disease, the demand for functional characterization of variants in these "genes of unknown significance" continues to increase. In this chapter, we describe a workflow to functionally characterize a rare variant in a Notch signaling related gene that was found to be associated with late-onset Alzheimer's disease. This pipeline involves informatic analysis of the variant of interest using diverse human and model organism databases, followed by in vivo experiments in the fruit fly Drosophila melanogaster. The protocol described here can be used to study variants that affect amino acids that are not conserved between human and fly. By "humanizing" the almondex gene in Drosophila with mutant alleles and heterologous genomic rescue constructs, a missense variant in TM2D3 (TM2 Domain Containing 3) was shown to be functionally damaging. This, and similar approaches, greatly facilitate functional interpretations of genetic variants in the human genome and propel personalized medicine.