Comparative functional analysis of DPYD variants of potential clinical relevance to dihydropyrimidine dehydrogenase activity.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of the uracil catabolic pathway, being critically important for inactivation of the commonly prescribed anti-cancer drug 5-fluorouracil (5-FU). DPD impairment leads to increased exposure to 5-FU and, in turn, increased anabolism of 5-FU to cytotoxic nucleotides, resulting in more severe clinical adverse effects. Numerous variants within the gene coding for DPD, DPYD, have been described, although only a few have been demonstrated to reduce DPD enzyme activity. To identify DPYD variants that alter enzyme function, we expressed 80 protein-coding variants in an isogenic mammalian system and measured their capacities to convert 5-FU to dihydro-fluorouracil, the product of DPD catabolism. The M166V, E828K, K861R, and P1023T variants exhibited significantly higher enzyme activity than wild-type DPD (120%, P = 0.025; 116%, P = 0.049; 130%, P = 0.0077; 138%, P = 0.048, respectively). Consistent with clinical association studies of 5-FU toxicity, the D949V substitution reduced enzyme activity by 41% (P = 0.0031). Enzyme activity was also significantly reduced for 30 additional variants, 19 of which had <25% activity. None of those 30 variants have been previously reported to associate with 5-FU toxicity in clinical association studies, which have been conducted primarily in populations of European ancestry. Using publicly available genotype databases, we confirmed the rarity of these variants in European populations but showed that they are detected at appreciable frequencies in other populations. These data strongly suggest that testing for the reported deficient DPYD variations could dramatically improve predictive genetic tests for 5-FU sensitivity, especially in individuals of non-European descent.

microRNAs miR-27a and miR-27b directly regulate liver dihydropyrimidine dehydrogenase expression through two conserved binding sites.

Dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) is the rate-limiting enzyme in the uracil catabolic pathway and has a pivotal role in the pharmacokinetics of the commonly prescribed anticancer drug 5-fluorouracil (5-FU). Deficiency of DPD, whether due to inadequate expression or deleterious variants in DPYD, has been linked to severe toxic responses to 5-FU. Little is known about the mechanisms governing DPD expression in the liver. In this report, we show increased accumulation of RNA-induced silencing complex (RISC) proteins on DPYD mRNA in cells overexpressing the highly homologous microRNAs (miRNA) miR-27a and miR-27b. These miRNAs were shown to repress DPD expression through two conserved recognition sites in DPYD. The IC50 of 5-FU for HCT116 cells overexpressing miR-27a or miR-27b was 4.4 µmol/L (both), significantly lower than that for cells expressing a nontargeting (scramble) control miRNA (14.3 µmol/L; P = 3.3 × 10(-5) and P = 1.5 × 10(-7), respectively). Mouse liver DPD enzyme activity was inversely correlated with expression levels of miR-27a (R(2) = 0.49; P = 0.0012) and miR-27b (R(2) = 0.29; P = 0.022). A common variant in the hairpin loop region of hsa-mir-27a (rs895819) was also shown to be associated with elevated expression of the miR-27a in a panel of cell lines (P = 0.029) and in a transgenic overexpression model (P = 0.0011). Furthermore, rs895819 was associated with reduced DPD enzyme activity (P = 0.028) in a cohort of 40 healthy volunteers. Taken together, these results suggest that miR-27a and miR-27b expression may be pharmacologically relevant modulators of DPD enzyme function in the liver. Furthermore, our data suggest that rs895819 may be a potential risk allele for 5-FU sensitivity.

Phenotypic profiling of DPYD variations relevant to 5-fluorouracil sensitivity using real-time cellular analysis and in vitro measurement of enzyme activity.

In the 45 years since its development, the pyrimidine analog 5-fluorouracil (5-FU) has become an integral component of many cancer treatments, most notably for the management of colorectal cancer. An appreciable fraction of patients who receive 5-FU suffer severe adverse toxicities, which in extreme cases may result in death. Dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) rapidly degrades 85% of administered 5-FU, and as such, limits the amount of drug available for conversion into active metabolites. Clinical studies have suggested that genetic variations in DPYD increase the risk for 5-FU toxicity, however, there is not a clear consensus about which variations are relevant predictors. In the present study, DPYD variants were expressed in mammalian cells, and the enzymatic activity of expressed protein was determined relative to wild-type (WT). Relative sensitivity to 5-FU for cells expressing DPYD variations was also measured. The DPYD*2A variant (exon 14 deletion caused by IVS14+1G>A) was confirmed to be catalytically inactive. Compared with WT, two variants, S534N and C29R, showed significantly higher enzymatic activity. Cells expressing S534N were more resistant to 5-FU-mediated toxicity compared with cells expressing WT DPYD. These findings support the hypothesis that selected DPYD alleles are protective against severe 5-FU toxicity, and, as a consequence, may decrease the effectiveness of 5-FU an antitumor drug in carriers. In addition, this study shows a method that may be useful for phenotyping other genetic variations in pharmacologically relevant pathways.