Genetic And Epigenetic Regulation Of Dihydropyrimidinase And Beta-Ureidopropionase In Individuals With Altered Uracil Catabolism And Normal Dihydro Pyrimidine Dehydrogenase Enzyme Activity

Abstract

There remain a number of patients presenting with 5-fluorouracil (5-FU) toxicity despite normal dihydropyrimidine dehydrogenase (DPD) enzyme activity, suggesting possible deficiencies in the two enzymes downstream of DPD in the uracil catabolic pathway: dihydropyrimidinase (DHP), encoded by the DPYS gene, or β-ureidopropionase (BUP-1), encoded by the UPB1 gene. Unlike DPD, which is expressed in peripheral blood mononuclear cells (PBMCs), DHP and BUP-1 enzyme activity is detected in only the liver and kidney, thus requiring an invasive biopsy for determination of activity. This dissertation, therefore, focuses on the role of the genetic and epigenetic regulation of DPYS and UPB1. The genetic variations present in DPYS and UPB1 were identified in 219 healthy volunteers with known DPD enzyme activity and [2-13C]-uracil breath test (UraBT) profiles and 50 cancer patients who experienced 5-FU toxicity despite normal DPD enzyme activity. In the volunteers with a deficient UraBT and normal DPD activity, three non-conservative DPYS mutations were detected, two of which caused a decrease in DHP activity. In the 50 cancer patients, two inactivating UPB1 non-conservative mutations were detected. Epigenetic regulation, mainly methylation, was examined in 43 liver samples (a tissue where DPYS and UPB1 are highly expressed) and in 20 PBMC samples (where neither gene is expressed) to determine if methylation is a possible mechanism of down-regulation/silencing in these genes. Low levels of methylation were iv detected in the DPYS gene in the liver samples and PBMCs, suggesting that methylation may not be a key element in the regulation of DPYS. However, varying levels of methylation were detected in the CpG islands of the UPB1 gene in the liver samples and almost complete methylation was detected in the UPB1 CpG islands of the PBMCs, suggesting that methylation may be involved in the regulation of the UPB1 gene. This study was designed to expand our understanding of the molecular mechanisms governing the regulation of the uracil catabolic pathway and will highlight the importance of screening for the integrity of the entire pathway including DPD, DHP and BUP-1 in cancer patients prior to the administration of flouropyrimidine drugs to effectively lower the incidence of 5-fluorouracil toxicity