Methylation of the DPYD Promoter: An Alternative Mechanism for Dihydropyrimidine Dehydrogenase Deficiency in Cancer Patients

Abstract

Purpose: Dihydropyrimidine dehydrogenase (DPD) deficiency, a known pharmacogenetic syndrome associated with 5-fluorouracil (5-FU) toxicity, has been detected in 3% to 5% of the population. Genotypic studies have identified >32 sequence variants in the DPYD gene; however, in a number of cases, sequence variants could not explain the molecular basis of DPD deficiency. Recent studies in cell lines indicate that hypermethylation of the DPYD promoter might downegulate DPD expression. The current study investigates the role of methylation in cancer patients with an unexplained molecular basis of DPD deficiency. Experimental Design: DPD deficiency was identified phenotypically by both enzyme assay and uracil breath test, and genotypically by denaturing high-performance liquid chromatography. The methylation status was evaluated in PCR products (209 bp) of bisulfite-modified DPYD promoter, using a novel denaturing high-performance liquid chromatography method that distinguishes between methylated and unmethylated alleles. Clinical samples included five volunteers with normal DPD enzyme activity, five DPD-deficient volunteers, and five DPD-deficient cancer patients with a history of 5-FU toxicity. Results: No evidence of methylation was detected in samples from volunteers with normal DPD. Methylation was detected in five of five DPD-deficient volunteers and in three of five of the DPD-deficient cancer patient samples. Of note, one of the two samples from patients with DPD-deficient cancer with no evidence of methylation had the mutation DPYD*2A, whereas the other had DPYD*13. Discussion: Methylation of the DPYD promoter region is associated with down-regulation of DPD activity in clinical samples and should be considered as a potentially important regulatory mechanism of DPD activity and basis for 5-FU toxicity in cancer patients.Dihydropyrimidine dehydrogenase (DPD) enzyme deficiency, a known pharmacogenetic syndrome detected in 3% to 5% of the population (1), has been associated with toxicity to 5-fluorouracil (5-FU) cancer chemotherapy and death in some cases with profound deficiency of the enzyme (2). DPD is the first enzyme in a three-step catabolic pathway responsible for the degradation of f85% of administered 5-FU. Genotypic studies have identified >32 sequence variants in the DPYD gene (1, 3). Expression analysis of these variants showed that many were polymorphisms with no obvious functional significance (4), with the exception of a few mutations. One example commonly associated with DPD deficiency and severe toxicity to 5-FU is an intronic sequence variation (IVS14 + 1 G > A, DPYD*2A), which results in a truncated protein that lacks 55 amino acids due to the skipping of exon 14 (5). A second example is a less common mutation (1679T > G, I560S, DPYD*13), which is associated with decreased DPD activity and 5-FU toxicity (6) due to a nonconservative amino acid change from isoleucine to serine at codon 560 (I560), which is 100% conserved among human, mouse, rat, bovine, and pig species, suggesting its imp...

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