Functional Analysis of DPYD Variants Associated With 5-Fluorouracil-Based Anticancer Drug Toxicity in Cancer Patients

Aims: We previously identified a strong association between seven rare DPYD variants (Ser199Asn, Ile245Phe, Thr305Lys, Glu386Ter, Ser556Arg, Ala571Asp, and Trp621Cys) and fluoropyrimidine-related toxicity in Japanese cancer patients. Herein, we reported clinical and molecular functional analyses of these seven rare DPYD variants. Methods: Mutant dihydropyrimidine dehydrogenase (DPD) enzymes were expressed in HEK293 cells. DPD activity was measured by quantifying dihydro-5-FU using high-performance liquid chromatography–tandem mass spectrometry. DPD levels and dimerization were analyzed via western blotting and blue native PAGE. In addition, three-dimensional (3D) structural modeling was performed to evaluate the effects of these mutations on enzyme structure and function. Results: Transient expression of DPD variant enzymes in HEK293 cells showed that the Ser199Asn, Ala571Asp, and Glu386Ter variants caused profound reductions in activity compared to the wild type (27%, 5.0%, and undetectable, respectively), while Thr305Lys, Trp621Cys, Ile245Phe, and Ser556Arg retained partial to normal activity (49%, 58%, 118%, and 113%). The results of 3D structural analysis supported the functional findings from HEK293 cell experiments. Clinical evaluation revealed that carriers with the five variants (Ser199Asn, Ile245Phe, Glu386Ter, Ala571Asp, and Trp621Cys) presented with adverse reactions Graded 3 or higher according to the Common Terminology Criteria for Adverse Events. Retrospective assessment of DPD activity in peripheral blood mononuclear cells revealed that patients carrying Ser199Asn, Ala571Asp, or Glu386Ter exhibited reduced DPD activity, correlating with findings from HEK293 cell experiments. Conclusions: Expression studies demonstrated that the rare DPYD variants Ser199Asn, Ala571Asp, and Glu386Ter were associated with decreased DPD enzyme activity and 5-FU toxicity, even in the heterozygous state.