Functional Characterization of 12 Dihydropyrimidinase Allelic Variants in Japanese Individuals for the Prediction of 5-Fluorouracil Treatment-Related Toxicity

The drug 5-fluorouracil (5-FU) is the first-choice chemotherapeutic agent against advanced-stage cancers. However, 10% to 30% of treated patients experience grade 3 to 4 toxicity. The deficiency of dihydropyrimidinase (DHPase), which catalyzes the second step of the 5-FU degradation pathway, is correlated with the risk of de-veloping toxicity. Thus, genetic polymorphisms within DPYS, the DHPase-encoding gene, could potentially serve as predictors of severe 5-FU-related toxicity. We identified 12 novel DPYS variants in 3554 Japanese individuals, but the effects of these mutations on function remain unknown. In the current study, we performed in vitro enzymatic analyses of the 12 newly identified DHPase var-iants. Dihydrouracil or dihydro-5-FU hydrolytic ring-opening kinetic parameters, Km and Vmax, and intrinsic clearance (CL;,,t 5 Vmax/Km) of the wild-type DHPase and eight variants were measured. Five of these variants (R118Q, H295R, T418I, Y448H, and T513A) showed significantly reduced CL;,,t compared with that in the wild-type. The parameters for the remaining four variants (V59F, D81H, T136M, and R490H) could not be determined as dihydrouracil and dihydro-5-FU hydrolytic ring-opening activity was undetectable. We also determined DHPase variant protein stability using cyclohex-imide and bortezomib. The mechanism underlying the observed changes in the kinetic parameters was clarified using blue-native polyacrylamide gel electrophoresis and three-dimensional struc-tural modeling. The results suggested that the decrease or loss of DHPase enzymatic activity was due to reduced stability and olig-omerizati on of DHPase variant proteins. Our findings support the use of DPYS polymorphisms as novel pharmacogenomic markers for predicting severe 5-FU-related toxicity in the Japa-nese population. SIGNIFICANCE STATEMENT DHPase contributes to the degradation of 5-fluorouracil, and genetic polymorphisms that cause decreased activity of DHPase can cause severe toxicity. In this study, we performed functional analysis of 12 DHPase variants in the Japanese population and identified 9 genetic polymorphisms that cause reduced DHPase function. In addition, we found that the ability to oligomerize and the conformation of the ac-tive site are important for the enzymatic activity of DHPase.

Automated summary

This study investigates the effects of genetic polymorphisms within the DPYS gene on the enzymatic activity of DHPase, which contributes to the degradation of 5-fluorouracil, and suggests that these polymorphisms could be used as pharmacogenomic markers for predicting severe 5-FU-related toxicity in the Japanese population.