Functional characterization of 21 allelic variants of dihydropyrimidine dehydrogenase identified in 1,070 Japanese individuals

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2), encoded by the DPYD gene, is the rate-limiting enzyme in the degradation pathway of endogenous pyrimidine and fluoropyrimidine drugs such as 5-fluorouracil (5-FU). DPD catalyzes the reduction of uracil, thymine, and 5-FU. In Caucasians, DPYD mutatio...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Drug metabolism and disposition 2018-05
Hauptverfasser: Hishinuma, Eiji, Narita, Yoko, Saito, Sakae, Maekawa, Masamitsu, Akai, Fumika, Nakanishi, Yuya, Yasuda, Jun, Nagasaki, Masao, Yamamoto, Masayuki, Yamaguchi, Hiroaki, Mano, Nariyasu, Hirasawa, Noriyasu, Hiratsuka, Masahiro
Format: Artikel
Sprache:eng
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung:Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2), encoded by the DPYD gene, is the rate-limiting enzyme in the degradation pathway of endogenous pyrimidine and fluoropyrimidine drugs such as 5-fluorouracil (5-FU). DPD catalyzes the reduction of uracil, thymine, and 5-FU. In Caucasians, DPYD mutations, including DPYD*2A, DPYD*13, c.2846A>T, and 1129-5923C>G/hapB3, are known to contribute to interindividual variations in the toxicity of 5-FU. However, none of these DPYD polymorphisms have been identified in the Asian population. Recently, 21 DPYD allelic variants, including some novel-single nucleotide variants (SNVs), were identified in 1,070 healthy Japanese individuals by analyzing their whole-genome sequences (WGS), but the functional alterations caused by these variants remain unknown. In this study, in vitro analysis was performed on 22 DPD allelic variants by transiently expressing wild-type DPD and 21 DPD variants in 293FT cells and characterizing their enzymatic activities, using 5-FU as a substrate. DPD expression levels and dimeric forms were determined using immunoblotting and blue native-PAGE, respectively. Additionally, the values of three kinetic parameters, the Michaelis constant (Km), maximum velocity (Vmax), and intrinsic clearance (CLint = Vmax/Km), were determined for the reduction of 5-FU. We found that 10 variants exhibited significantly decreased intrinsic clearance in comparison to wild-type DPD. Moreover, the band patterns observed in the immunoblots of blue native gels indicated that DPD dimerization is required for enzymatic activity in DPD. Thus, the detection of rare DPYD variants might facilitate severe adverse effect prediction of 5-FU-based chemotherapy in the Japanese population.
ISSN:1521-009X