Functional characterization of rare DPYD variants identified in 8,380 Japanese individuals: implications for 5-fluorouracil metabolism

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the metabolism of 5-fluorouracil, and reduced DPD activity resulting from DPYD genetic variants is a major cause of severe 5-fluorouracil -induced toxicity. Although several clinically relevant variants have been identified in Caucasian populations, the functional consequences of rare DPYD variants identified in Japanese individuals remain poorly characterized. In this study, we comprehensively analyzed 26 DPYD variants identified through whole-genome sequencing of 8380 Japanese individuals. Recombinant DPD proteins were expressed in 293FT cells, and enzymatic activity was evaluated using liquid chromatography-tandem mass spectrometry to determine kinetic parameters (Km, Vmax, CLint). Protein expression levels and quaternary structure were assessed by immunoblotting and blue native PAGE, and structural modeling was performed to explore possible structural consequences of selected variants. Among the 26 DPD variants analyzed, 5 exhibited markedly reduced or abolished enzymatic activity compared with wild-type DPD, including 3 loss of function variants. Loss of detectable homodimer formation was observed in Gly194Asp and Glu386Ter, whereas other functionally impaired variants retained homodimer formation, indicating that altered dimerization represents only one potential mechanism contributing to DPD dysfunction. Structural analyses suggested possible local alterations affecting cofactor interactions or the dimer interface in selected variants, providing hypotheses for future mechanistic investigation. These findings provide a comprehensive functional characterization of rare DPYD variants identified in Japanese individuals and support their prioritization for future clinical validation. However, their clinical significance and potential utility for fluoropyrimidine dose individualization require confirmation in patients receiving fluoropyrimidine-based chemotherapy. SIGNIFICANT STATEMENT: This study functionally characterized 26 rare DPYD variants identified in a Japanese population and identified 5 variants with markedly reduced or abolished DPD activity. These findings expand the functional annotation of DPYD variation in a population in which established fluoropyrimidine-associated risk variants are uncommon and provide evidence to prioritize functionally impaired variants for future clinical validation.