title: Mutational analysis of the human dihydropyrimidine dehydrogenase gene by denaturing high-performance liquid chromatography.
abstract: Mutations in the DPYD gene, which encodes dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in the catabolism of pyrimidines, are responsible for an inborn error of metabolism associated with thymine-uraciluria and neurological symptoms. Because the antimetabolite 5-fluorouracil (5-FU) is metabolized by the same enzyme, deficient DPYD alleles may also constitute a risk factor for severe toxicity following treatment with this anticancer drug. The aim of this study was to develop a comprehensive and rapid method to detect sequence variations within the DPYD gene. Using polymerase chain reaction (PCR) amplification and denaturing high-performance liquid chromatography (DHPLC), we established a protocol that makes it possible to screen all 23 exons of the DPYD gene and their exon-intron boundaries for both known and unknown mutations under identical conditions. A novel one-step PCR mutagenesis procedure was developed to generate heterozygous mutant amplicons as positive controls to optimize DHPLC detection of any sequence variation. DHPLC analysis was shown to result in mutation-specific elution profiles and to be able to distinguish different base changes within the same exon or different heterozygous combinations of mutations within the same exon. By analyzing the DPYD gene in 16 affected individuals, a total of 47 base changes were detected, representing eight known mutations and three novel intronic base changes. Sequence analysis confirmed all base changes detected. This method will be useful in identifying patients at risk for toxicity prior to 5-FU treatment, as well as in the analysis of individual patients with thymine-uraciluria.
authors.affiliation: Margarete Fischer-Bosch Institute of Clinical Pharmacology, D-70376 Stuttgart, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Fischer
authors.forename: Matthias
authors.initials: M
authors.lastname: Schwab
authors.forename: Michel
authors.initials: M
authors.lastname: Eichelbaum
authors.forename: Ulrich M
authors.initials: UM
authors.lastname: Zanger
mesh_terms: D000483:Alleles; D000964:Antimetabolites, Antineoplastic / Q000009:adverse effects / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D054067:Dihydropyrimidine Dehydrogenase Deficiency; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D005472:Fluorouracil / Q000009:adverse effects / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D007438:Introns; D016296:Mutagenesis; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011686:Purine-Pyrimidine Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics
chemical_list: D000964:Antimetabolites, Antineoplastic; D004247:DNA; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil