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 dihydrofluorouracil, the product of DPD catabolism. The M166V, E828K, K861R, and P1023T variants exhibited significantly higher enzyme activity than wildtype 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.

Towards a test to predict 5-fluorouracil toxicity: Pharmacokinetic data for thymine and two sequential metabolites following oral thymine administration to healthy adult males

“…A large number of other mutations have a much lower incidence (Offer et al, 2014). The DPYD c.1129-5923C>G/hapB3 deep intronic mutation has been associated with half of severe toxicity and a relatively high carrier rate of 4.6% in a Swiss population (Froehlich et al, 2015) but its applicability to other, non-Swiss populations requires confirmation.…”

Translating DPYD Genotype into DPD Phenotype: Using the DPYD Gene Activity Score

“…With the gene activity score we can facilitate in a more specific dose adjustment in fluoropyrimidine treatment using current knowledge on differences in DPD enzyme activity due to DPYD variants. [13,19,24,25]. DPYD*2A leads to skipping of the entire exon 14 and deletion of 165 base pairs which results in a truncated protein that is catalytically inactive [22,26].…”

“…The c.2846A>T polymorphism leads to a structural change in the DPD enzyme that interferes with cofactor binding or electron transport [16]. Reported allele frequencies of c.2846A>T vary from 0.1 to 1.1% in African-Americans and Caucasians, respectively [13,19,24,46]. In vitro data show that homozygous expression of the c.2846A>T variant results in an activity of 59% compared with wildtype (p = 0.0031) [13].…”

“…In addition to DPYD*2A, other SNPs in DPYD have been described to result in decreased DPD enzyme activity, including DPYD*13 (c.1679T>G; I560S; rs55886062), c.2846A>T (D949V; rs67376798) and c.1236G>A (E412E; rs56038477, in haplotype B3) [13][14][15]. However, not all of these SNPs result in a similar decrease in DPD enzyme activity as DPYD*2A [3,14,16].…”

Genotype–phenotype correlations in 5-fluorouracil metabolism: a candidate DPYD haplotype to improve toxicity prediction

“…6 Efforts are ongoing to identify further functional polymorphism in the DPYD, and indeed several variants have been described so far. 7,8 Recently, the Clinical Pharmacogenetic Implementation Consortium, based on an ample literature survey and on the consistency of results from structure-function analysis, proposed genotype-based recommendation for varying fluorouracil dosing, according to the presence of the allelic variants *2A, *13 and rs67376798. 9 Even so, also the last two SNPs are rare variants.…”