Importance of Rare DPYD Genetic Polymorphisms for 5-Fluorouracil Therapy in the Japanese Population

Abstract

Dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene, is the rate-limiting enzyme in 5-fluorouracil (5-FU) degradation. In Caucasians, four DPYD risk variants are recognized to be responsible for interindividual variations in the development of 5-FU toxicity. However, these risk variants have not been identified in Asian populations. Recently, 41 DPYD allelic variants, including 15 novel single nucleotide variants, were identified in 3,554 Japanese individuals by analyzing their whole-genome sequences; however, the effects of these variants on DPD enzymatic activity remain unknown. In the present study, an in vitro analysis was performed on 41 DPD allelic variants and three DPD risk variants to elucidate the changes in enzymatic activity. Wild-type and 44 DPD-variant proteins were heterologously expressed in 293FT cells. DPD expression levels and dimerization of DPD were determined by immunoblotting after SDS-PAGE and blue native PAGE, respectively. The enzymatic activity of DPD was evaluated by quantification of dihydro-5-FU, a metabolite of 5-FU, using high-performance liquid chromatography-tandem mass spectrometry. Moreover, we used 3D simulation modeling to analyze the effect of amino acid substitutions on the conformation of DPD. Among the 41 DPD variants, seven exhibited drastically decreased intrinsic clearance (CLint) compared to the wild-type protein. Moreover, R353C and G926V exhibited no enzymatic activity, and the band patterns observed in the immunoblots after blue native PAGE indicated that DPD dimerization is required for its enzymatic activity. Our data suggest that these variants may contribute to the significant inter-individual variability observed in the pharmacokinetics and pharmacodynamics of 5-FU. In our study, nine DPD variants exhibited drastically decreased or no enzymatic activity due to dimerization inhibition or conformational changes in each domain. Especially, the rare DPYD variants, although at very low frequencies, may serve as important pharmacogenomic markers associated with the severe 5-FU toxicity in Japanese population.

Comparison of genetic susceptibility to lung adenocarcinoma and squamous cell carcinoma in Japanese patients using a novel panel for cancer-related drug-metabolizing enzyme genes

“…Several studies have shown a link between reduced DPYD enzyme activity and increasing the risk of severe toxicity. A recent study has reported that the functional alterations of enzyme activities caused by DYPD variants were characterized 21 . The rs200562975of DPYD identified in the present study reportedly reduced enzymatic activity to less than 70% of wild-type in vitro 21 .…”

“…A recent study has reported that the functional alterations of enzyme activities caused by DYPD variants were characterized 21 . The rs200562975of DPYD identified in the present study reportedly reduced enzymatic activity to less than 70% of wild-type in vitro 21 . However, none of the previous studies examined whether the DPYD variants contribute to the risk of lung cancer.…”

A computational study of the reaction mechanism and stereospecificity of dihydropyrimidinase

“…To predict the enzyme-substrate specificity, both the substrate binding energy and the activation energies of the chemical steps should be considered. 69 Accordingly, we further estimated the binding affinities of the substrates of other enzymes in this family (Scheme 2) within the SkDHPase active site, using eqn (1). The binding energies of the natural substrates DHU and L-DHT are À10.8 and À15.0 kcal mol À1 , which are 2-3 times more negative than for the non-natural substrates (À5.8, À6.0, À6.8 kcal mol À1 for D-DHT, HYD, L-HYD, respectively).…”

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

“…Informed consent was obtained from all research participants, and the study was approved by the Tohoku Medical Megabank Organization ethics committee (permission numbers 2017-4-26, 2017-4-58, and 2017-4-090) and the Tohoku University Graduate School of Pharmaceutical Sciences ethics committee (permission number 14-08). DPYS sequence alterations were identified using Sanger sequencing according to previously described methods (Akai et al, 2015;Hishinuma et al, 2018;Hishinuma et al, 2022). The primer pairs used to detect single nucleotide variants (SNVs) in Sanger sequencing are listed in Table 2.…”

“…In Caucasians, genetic polymorphisms in DPYD, the gene encoding DPD, contribute to individual differences in 5-FU toxicity, and the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines have already been established for dose adjustment based on four risk variants (DPYD*2A, DPYD*13, c.2846A>T, and c.1129-5923C>G/hapB3) (Amstutz et al, 2018). However, these polymorphisms have not been identified in Asian populations (van Kuilenburg, 2004;Maekawa et al, 2007;Hishinuma et al, 2018;Yokoi et al, 2020;Hishinuma et al, 2022). Toxicity associated with 5-FU has also been observed in cancer patients with regular DPD enzymatic activity, suggesting a potential deficiency of DHPase, the second enzyme in the pyrimidine degradation pathway.…”