Abstract We identified the uncharacterized human gene C10orf35 (FAM214B) in two genome-wide screens: a suppressor screen for genes whose overexpression corrects the enlarged lysosomes in FIG4 null cells, and a knock-out screen for genes whose inactivation in wildtype cells produces enlarged lysosomes. C10orf35 was a positive hit in both screens, providing strong evidence for a role in lysosome biology. To define the physiological function of C10orf35, we generated a knock-out allele of the mouse ortholog. In this supplemental project, we will characterize the function of C10orf35 by generating homozygous null mice and examining survival, tissue morphology, and lysosome function in cultured mouse embryonic fiboblasts. We will also characterize a C10orf35 variant of unknown significance that was identified through Genematcher in a patient with spastic paraplegia. We will evaluate the ability of the missense variant of unknown significance by transfection of a C10orf35 null HAP1 cell line. C10orf35 is a member of a two-gene family. We generated a null allele of the other mouse family member, C4orf32. We will characterize the phenotype of this null mouse as per above. We will also generate double homozygotes missing both genes, to evaluate the essentiality of this gene family for mammalian survival. This work will provide novel information about a previously uncharacterized gene family in the human genome that is known to be related to lysosome function. The requisite methods and reagents are available in the laboratory. Quinlan Doctrove is a member of an underrepresented minority. He will work closely with a senior researcher who has fourteen years of experience in related projects. This project will provide an opportunity for training in current methods for characterization of the human genome, including characterization of mouse models and functional analysis of pathogenic variants.