Functional characterization of 50 CYP2D6 allelic variants by assessing primaquine 5-hydroxylation.

Cytochrome P450 2D6 (CYP2D6) is responsible for the metabolic activation of primaquine, an antimalarial drug. CYP2D6 is genetically polymorphic, and these polymorphisms are associated with interindividual variations observed in the therapeutic efficacy of primaquine. To further understand this association, we performed in vitro enzymatic analyses of the wild-type CYP2D6.1 and 49 CYP2D6 allelic variants, which were expressed in 293FT cells, using primaquine as a substrate. The concentrations of CYP2D6 variant holoenzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild type and 27 variants showed a peak at 450 nm. The kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of primaquine 5-hydroxylation were characterized. The kinetic parameters of the wild type and 16 variants were measured, but the values for the remaining 33 variants could not be determined because of low metabolite concentrations. Among the variants, six (i.e., CYP2D6.17, .18, .35, .39, .53, and .70) showed significantly reduced intrinsic clearance compared with that of CYP2D6.1. Three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2D6 variants. Our findings provide insights into the allele-specific activity of CYP2D6 for primaquine, which could be clinically useful for malaria treatment and eradication efforts.

Functional Characterization of 21 Allelic Variants of Dihydropyrimidine Dehydrogenase Identified in 1070 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 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.

Functional characterization of 21 allelic variants of dihydropyrimidinase.

Dihydropyrimidinase (DHP, EC 3.5.2.2), encoded by the gene DPYS, is the second enzyme in the catabolic pathway of pyrimidine and of fluoropyrimidine drugs such as 5-fluorouracil, which are commonly used in anticancer treatment; DHP catalyzes the hydrolytic ring opening of dihydrouracil and dihydro-5-fluorouracil. DPYS mutations are known to contribute to interindividual variations in the toxicity of fluoropyrimidine drugs, but the functional characterization of DHP allelic variants remains inadequate. In this study, in vitro analysis was performed on 22 allelic variants of DHP by transiently expressing wild-type DHP and 21 DHP variants in 293FT cells and characterizing their enzymatic activities by using dihydrouracil and dihydro-5-fluorouracil as substrates. DHP expression levels and oligomeric forms were determined using immunoblotting and blue native PAGE, respectively, and the stability of the DHP variants was assessed by examining the proteins in variant-transfected cells treated with cycloheximide or bortezomib. Moreover, three kinetic parameters, Km, Vmax, and intrinsic clearance (Vmax/Km), for the hydrolysis of dihydrouracil and dihydro-5-fluorouracil were determined. We found that 5/21 variants showed significantly decreased intrinsic clearance as compared to wild-type DHP, and that 9/21 variants were expressed at low levels and were inactive due to proteasome-mediated degradation. The band patterns observed in the immunoblotting of blue native gels corresponded to DHP activity, and, notably, 18/21 DHP variants exhibited decreased or null enzymatic activity and these variants also showed a drastically reduced ability to form large oligomers. Thus, detection of DPYS genetic polymorphisms might facilitate the prediction severe adverse effects of fluoropyrimidine-based treatments.

Genetic polymorphisms of dihydropyrimidinase in a Japanese patient with capecitabine-induced toxicity.

Dihydropyrimidinase (DHP) is the second enzyme in the catabolic pathway of uracil, thymine, and chemotherapeutic fluoropyrimidine agents such as 5-fluorouracil (5-FU). Thus, DHP deficiency might be associated with 5-FU toxicity during fluoropyrimidine chemotherapy. We performed genetic analyses of the family of a patient with advanced colon cancer who underwent radical colectomy followed by treatment with 5-FU prodrug capecitabine and developed severe toxicity attributable to a lack of DHP. We measured urinary uracil and dihydrouracil, and genotyped DPYS in the patient and her family. We also measured the allele frequency of DPYS polymorphisms in 391 unrelated Japanese subjects. The patient had compound heterozygous missense and nonsense polymorphisms comprising c.1001A>G (p.Gln334Arg) in exon 6 and c.1393C>T (p.Arg465Ter) in exon 8, which are known to result in a DHP enzyme with little or no activity. The urinary dihydrouracil/uracil ratio in the patient was 17.08, while the mean ± SD urinary dihydrouracil/uracil ratio in family members who were heterozygous or homozygous for wild-type DPYS was 0.25 ± 0.06. In unrelated subjects, 8 of 391 individuals were heterozygous for the c.1001A>G mutation, while the c.1393C>T mutation was not identified. This is the first report of a DHP-deficient patient with DPYS compound heterozygous polymorphisms who was treated with a fluoropyrimidine, and our findings suggest that polymorphisms in the DPYS gene are pharmacogenomic markers associated with severe 5-FU toxicity in Japanese patients.

Novel single nucleotide polymorphisms of the dihydropyrimidinase gene (DPYS) in Japanese individuals.

Genetic polymorphisms of the dihydropyrimidinase gene (DPYS) may be associated with the development of severe toxicity to 5-fluorouracil, a drug used to treat solid tumors. In this study, we analyzed the nine coding exons and exon-intron junctions of DPYS in 183 Japanese individuals. We detected two novel single nucleotide polymorphisms (SNPs)-285C > T (Thr95Thr) and 349T > C (Trp117Arg)-in exon 2. The nonsynonymous SNP 349T > C was analyzed in 208 Japanese individuals. Although the allele frequency of the SNP in the Japanese population was found to be extremely low (0.13%), the enzymatic activity of the variant protein might be reduced compared with that of the wild-type protein.