Identifying molecular mechanisms contributing to disease etiology and the neurological symptoms associated with pediatric DPD deficiency - PROJECT SUMMARY Dihydropyrimidine dehydrogenase (DPD) deficiency, caused by mutations within DPYD, has been studied mostly in the context of 5-fluorouracil (5-FU) toxicity and its role as a pharmacogene. However, a rare pediatric disorder with severe early onset symptoms, pediatric DPD deficiency, is also linked to genetic variation within DPYD. The molecular mechanisms that contribute to disease onset and associated symptoms are unknown, nor have any effective therapeutic avenues been identified, likely owing to the lack of mechanistic knowledge. Therefore, in the proposed studies, I will use induced pluripotent stem cell (iPSC)- derived cerebral organoid models of pediatric DPD deficiency to identify the mechanisms linking DPD deficiency with the outward symptoms of the condition. The overall goals of the proposed studies are to define disease etiology, determine molecular mechanisms that lead to disease onset, and to identify potential therapeutic targets. We hypothesize that dysregulation of excitatory and inhibitory signaling within the brain contributes to seizure onset and epilepsy due to excitotoxicity and a decrease in β-alanine inhibitory signaling, along with anatomical structural changes across development and differences in cellular composition. We further hypothesize that the symptoms of this disorder could be relieved by correcting genetic variation within DPYD. To address this hypothesis, we will identify differentially expressed genes and dysregulated pathways at bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) levels to determine molecular mechanisms contributing to the disease. We will investigate the potential for dysregulation of gene expression via epigenetic mechanisms and perform functional studies of organoid signaling. Finally, isogenic cell line models will be established to determine if the molecular and functional phenotype of organoids derived from affected patients is specifically dependent on DPYD genetic variation. Overall, these data will describe the etiology of pediatric DPD deficiency, determine molecular mechanisms and pathways contributing to symptom onset, and aid in therapeutic target identification for disease treatment. These studies will provide broad implications into the role of DPYD in neurological development and developmental mechanisms contributing to pediatric genetic epilepsy onset.
