High-throughput genotyping by DHPLC of the dihydropyrimidine dehydrogenase gene implicated in (fluoro)pyrimidine catabolism.

Eva Gross, Katharina Seck, Steffi Neubauer, Jutta Mayr, Heide Hellebrand, Adisorn Ratanaphan, Verena Lutz, Hubertus Stockinger, Marion Kiechle

Department of Obstetrics and Gynecology, Technical University of Munich, D-81675 Munich, Germany.

International journal of oncology 2003 Feb

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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.

Citation

Eva Gross, Katharina Seck, Steffi Neubauer, Jutta Mayr, Heide Hellebrand, Adisorn Ratanaphan, Verena Lutz, Hubertus Stockinger, Marion Kiechle. High-throughput genotyping by DHPLC of the dihydropyrimidine dehydrogenase gene implicated in (fluoro)pyrimidine catabolism. International journal of oncology. 2003 Feb;22(2):325-32