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

Abstract

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.

Figure 1. Predicted impact of missense DPYD variations on DPD protein function

A, the probability of each variation being damaging to protein function or structure was determined using PolyPhen-2. B, for each probability reported in panel A, the estimated FPR was calculated by the software. Each mark represents an individual amino acid substitution. Shading indicates the qualitative classifier predicted by PolyPhen-2 (black, benign; gray, possibly damaging; white, probably damaging).

Figure 2. In vitro enzyme activity of DPD variants

Dot blots were used to calculate relative expression of DPD (A) and alpha tubulin (B). Relative DPD enzyme activity was determined for selected variants predicted to be probably damaging (C), possibly damaging (D), and benign (E). F, enzyme activity was determined for R21X, E386X, P633[FS], F100[FS], and the somatic mutation G252V. For panels C-F, each “x” represents a single biological replicate, which was the average of 3 technical replicates. The mean of biological replicates is presented as a horizontal bar ± standard deviation. The mean relative activity and standard deviation for wildtype DPD are presented as horizontal gray and dashed lines, respectively. Variants with activities that are significantly different from wildtype are color coded according to the scale on the right: red, *2A-like (<12.5% activity); yellow, I560S-like (12.5%-25% activity); green, D949V-like (>25% activity); and blue, significantly greater than wildtype.

Figure 3. Location of variants relative to structural features of DPD

An alignment of vertebrate DPD amino acid sequences was prepared using Clustal Omega. Sequence consensus is denoted below the alignment (asterisk, fully conserved residue; colon, strongly similar properties; period, weakly similar properties; no symbol, dissimilar amino acids). Predicted secondary structural features are indicated by colored bars above the alignment (red, alpha helices; blue, beta turns). Differences between the predicted human structure and the published pig structure are denoted by horizontal (not present in predicted structure) and vertical (not present in published structure) white lines overlaid on the secondary structural prediction. Boxes indicate functional elements (4Fe-4S, iron-sulfur coordinating domains; FAD, FAD binding domain; NADP, NADP binding domain; FMN, FMN binding domain; UBC, uracil binding domain; ASL, active site loop; F, FAD interacting residue; N, NADP interacting residue; F2, FMN interacting residue; PA, pyrimidine associated residue). Amino acid changes that significantly reduced enzyme activity are colored as described for Figure 2. In instances where multiple amino acid variations have been reported for a given residue (i.e. R592W and R592Q), the color indicates the more severe phenotype.

Figure 4. Location of deficient variants relative to major structural domains of DPD

The position of damaging amino acid variants are depicted within the N-terminal Fe-S cluster containing alpha-helical domain I (A), the FAD binding domain II and NADPH binding domain III (B), the pyrimidine binding domain IV (C), and the C terminal Fe-S cluster containing domain V (D). Deleterious amino acids changes are colored as described for Figure 2. The position of I560S, which was not evaluated in this study, is shown in panel C for the benefit of the reader.

Figure 5. Frequencies of deleterious variants in publicly available population data

Allele frequency data is presented for the NHLBI ESP African American (AA) and European American (EU) populations, and for the 1000 Genomes populations of West African (AFR), American (AMR), East Asian (ASN), and European (EUR) ancestry. The allele frequencies for *2A, I560S, and D949V are presented as red, yellow, and green bars containing diagonal black lines. Additional variants that resulted in significant reductions in DPD enzyme activity are clustered by similarity to known deficient variants (as depicted in Fig. 2) and are represented as solid colored bars.