Klein, AndrésLas Heras Parraguez, Macarena2024-05-132024-05-132024https://hdl.handle.net/11447/8737Tesis presentada a la Facultad de Medicina Clínica Alemana de la Universidad del Desarrollo, para optar al grado académico de Doctor en Ciencias e Innovación en Medicina.Niemann-Pick Type C (NPC) disease is a devastating neurovisceral disorder caused by loss-of-function mutations in the NPC1 and NPC2 genes. It is characterized by the buildup of non-esterified cholesterol, sphingomyelin and other lipids in endosomes and lysosomes. The clinical presentations are highly heterogeneous and vary from a rapidly progressing neonatal form to an adult-onset chronic neurodegenerative condition. Currently, no effective treatments exist for NPC disease, prompting an urgent need for new therapeutic options. While some disease-causing variants have been associated to specific clinical outcomes, there exist numerous exceptions that highlight the complex nature of NPC, even among individuals of the same family. Differences in disease presentation and severity have also been observed in NPC mouse models of different genetic backgrounds, suggesting that other genetic factors besides the NPC1 mutation might act as disease modifiers. In this thesis, we aimed identify and validate putative modifier genes of NPC disease severity through studies in human and yeast. Our initial strategy involved using genomic and clinical data from a family affected by the disease, whose members exhibited a broad range of signs and symptoms with varying degrees of severity, to perform linkage analyses (cohort of identification). Typically, such findings are validated using a replication cohort; however, there is no publicly available genomic data of NPC patients that includes clinical information. To overcome this challenge and study the potential modifier role of the genes identified from human linkages, we developed a precision medicine platform using yeast cells. This platform included four already sequenced Saccharomyces cerevisiae strains. We treated these yeast of four different genetic backgrounds with U18, an NPC1 protein inhibitor, to mimic NPC disease severities. The strains exhibited varying responses to the U18 treatment. Then, to map additional genes potentially involved in modifying disease severity, we used segregants derived from parental yeast strains with divergent phenotypes to perform linkage analyses. Subsequently, we obtained lists of putative modifiers from both human and yeast linkages. Finally, we xiv selected several of these candidates for loss-of-function validation experiments in yeast. The results obtained revealed that yeast knockouts for two of the selected genes treated with U18 showed a partial rescue of the evaluated phenotypes (µmax and vacuolar phenotypes) compared to the wild-type strain treated with U18. These findings support the potential of these genes as modifiers of NPC disease severity and position them as potential therapeutic targets or biomarkers of severity. This study represents the first successful human linkage conducted to identify potential modifier genes of NPC severity, as well as the first linkages in a model organism with high resolution. This yeast cell-based platform could help to increase our understanding of some diseases, identify new therapeutic targets, and serve as an invaluable tool for assessing the efficacy of potential treatments. The findings generated from this study could have implications not only for NPC, but also for other LSDs and neurodegenerative diseases, including Alzheimer’s disease.116 p.en090036SEnfermedad de Niemann-Pick Type CGenes modificadores de la severidadVariabilidad fenotípicaS. cerevisiaeThesis