Functional characterization of 21 allelic variants of dihydropyrimidine dehydrogenase identified in 1070 Japanese individuals

Eiji Hishinuma, Yoko Narita, Sakae Saito, Masamitsu Maekawa, Fumika Akai, Yuya Nakanishi, Jun Yasuda, Masao Nagasaki, Masayuki Yamamoto, Hiroaki Yamaguchi, Nariyasu Mano, Noriyasu Hirasawa, Masahiro Hiratsuka

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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 c.1129-5923C>G/hapB3, are known to contribute to interindividual variations in the toxicity of 5-FU; however, none of these DPYD polymorphisms has been identified in the Asian population. Recently, 21 DPYD allelic variants, including some novel single-nucleotide variants (SNVs), were identified in 1070 healthy Japanese individuals by analyzing their whole-genome sequences (WGSs), 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. Eleven variants exhibited significantly decreased intrinsic clearance compared with 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.

Original languageEnglish
Pages (from-to)1083-1090
Number of pages8
JournalDrug Metabolism and Disposition
Volume46
Issue number8
DOIs
Publication statusPublished - 2018 Aug

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

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