Methylation of the DPYD promoter: an alternative mechanism for dihydropyrimidine dehydrogenase deficiency in cancer patients.
OBJECTIVE
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 down-regulate DPD expression. The current study investigates the role of methylation in cancer patients with an unexplained molecular basis of DPD deficiency.


METHODS
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.


CONCLUSIONS
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.
Ezzeldin
Hany H
HH
Division of Clinical Pharmacology and Toxicology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-3300, USA.
Lee
Adam M
AM
Mattison
Lori K
LK
Diasio
Robert B
RB
D000328:Adult; D000964:Antimetabolites, Antineoplastic / Q000633:toxicity; D002851:Chromatography, High Pressure Liquid; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D054067:Dihydropyrimidine Dehydrogenase Deficiency; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000235:genetics*; D015536:Down-Regulation; D005260:Female; D005472:Fluorouracil / Q000633:toxicity; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009369:Neoplasms / Q000201:enzymology*; D009691:Nucleic Acid Denaturation; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics*
D000964:Antimetabolites, Antineoplastic; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil