TY - JOUR
T1 - Functional characterization of 21 allelic variants of dihydropyrimidine dehydrogenase identified in 1070 Japanese individuals
AU - Hishinuma, Eiji
AU - Narita, Yoko
AU - Saito, Sakae
AU - Maekawa, Masamitsu
AU - Akai, Fumika
AU - Nakanishi, Yuya
AU - Yasuda, Jun
AU - Nagasaki, Masao
AU - Yamamoto, Masayuki
AU - Yamaguchi, Hiroaki
AU - Mano, Nariyasu
AU - Hirasawa, Noriyasu
AU - Hiratsuka, Masahiro
N1 - Funding Information:
This study was supported in part by the Foundation for Promotion of Cancer Research in Japan [Grants 159 and 100], Tohoku Medical Megabank Project from the Ministry of Education,Culture, Sports, Science and Technology (MEXT) and the Japan Agency for Medical Research and Development (AMED) [Grant JP17km0105002]. https://doi.org/10.1124/dmd.118.081737.
Funding Information:
We thank the Biomedical Research Core of Tohoku University Graduate School of Medicine for technical support and Evelyn Marie Gutierrez Rico, Tohoku University, for proofreading the manuscript.
Publisher Copyright:
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics
PY - 2018/8
Y1 - 2018/8
N2 - 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.
AB - 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.
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U2 - 10.1124/dmd.118.081737
DO - 10.1124/dmd.118.081737
M3 - Article
C2 - 29769267
AN - SCOPUS:85050800997
VL - 46
SP - 1083
EP - 1090
JO - Drug Metabolism and Disposition
JF - Drug Metabolism and Disposition
SN - 0090-9556
IS - 8
ER -