High-throughput genotyping by DHPLC of the dihydropyrimidine dehydrogenase gene implicated in (fluoro)pyrimidine catabolism.
Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme in the degradation of pyrimidines and pyrimidine base analogs including the anticancer drugs 5-fluorouracil (5-FU) and Xeloda. A decreased DPD enzyme activity has been described in cancer patients experiencing severe and life-threatening toxicity after 5-FU treatment and distinct sequence variants in the DPD gene (DPYD) have been associated with impaired enzyme function. The most prominent mutation in the DPD deficient patient group, a mutation in the splicing donor consensus sequence of intron 14, IVS14+1g>a, resulting in a truncated protein, has been observed in the Caucasian population at frequencies as high as 0.91%-0.94%. This underlines the need for a test system for DPYD mutations in patients undergoing chemotherapy with 5-FU or with Xeloda. To set up a fast and sensitive method to identify variant DPYD alleles, we analyzed 50 healthy individuals by denaturing high performance liquid chromatography (DHPLC). A primer set spanning the whole coding region and the exon-intron boundaries of DPYD was used. In addition, a cDNA-based assay was developed to rapidly identify the 165 base pair deletion in the corresponding RNA of IVS14+1g>a mutation carriers. The optimal mutation detection was elaborated for each of the PCR fragments. DHPLC analysis detected 5 different genetic alterations occurring in the coding region of the gene, as well as 10 intronic sequence variants, respectively. In conclusion, high-throughput screening for DPYD variants by DHPLC may be a reliable tool in the investigation of the molecular basis of DPD deficiency. Furthermore, it will help to identify patients at risk for toxic side effects upon chemotherapy using 5-FU and will facilitate individual treatment of patients.
Gross
Eva
E
Department of Obstetrics and Gynecology, Technical University of Munich, D-81675 Munich, Germany.
Seck
Katharina
K
Neubauer
Steffi
S
Mayr
Jutta
J
Hellebrand
Heide
H
Ratanaphan
Adisorn
A
Lutz
Verena
V
Stockinger
Hubertus
H
Kiechle
Marion
M
D000328:Adult; D000483:Alleles; D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000009:adverse effects / Q000493:pharmacokinetics*; D001711:Biotransformation; D000069287:Capecitabine; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D042943:Dihydrouracil Dehydrogenase (NADP); D005091:Exons / Q000235:genetics; D005260:Female; D005472:Fluorouracil / Q000009:adverse effects / Q000493:pharmacokinetics*; D005796:Genes; D005838:Genotype; D006801:Humans; D008658:Inactivation, Metabolic; D007438:Introns / Q000235:genetics; D008297:Male; D020125:Mutation, Missense; D010088:Oxidoreductases / Q000172:deficiency / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D011743:Pyrimidines / Q000378:metabolism* / Q000493:pharmacokinetics; D022821:RNA Splice Sites / Q000235:genetics; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D013997:Time Factors; D044465:White People / Q000235:genetics
D000970:Antineoplastic Agents; D011743:Pyrimidines; D022821:RNA Splice Sites; D003841:Deoxycytidine; D000069287:Capecitabine; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil