Mutational Analysis of the Human Dihydropyrimidine Dehydrogenase Gene by Denaturing High-Performance Liquid Chromatography
| العنوان: | Mutational Analysis of the Human Dihydropyrimidine Dehydrogenase Gene by Denaturing High-Performance Liquid Chromatography |
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| المؤلفون: | Ulrich M. Zanger, Michel Eichelbaum, Joachim Fischer, Matthias Schwab |
| المصدر: | Genetic Testing. 7:97-105 |
| بيانات النشر: | Mary Ann Liebert Inc, 2003. |
| سنة النشر: | 2003 |
| مصطلحات موضوعية: | Antimetabolites, Antineoplastic, Purine-Pyrimidine Metabolism, Inborn Errors, congenital, hereditary, and neonatal diseases and abnormalities, Dihydropyrimidine Dehydrogenase Deficiency, DNA Mutational Analysis, Biology, Nucleic Acid Denaturation, Polymerase Chain Reaction, Denaturing high performance liquid chromatography, Dihydropyrimidine dehydrogenase, medicine, Humans, Gene, Alleles, Chromatography, High Pressure Liquid, Dihydrouracil Dehydrogenase (NADP), Genetics (clinical), chemistry.chemical_classification, Base Sequence, Catabolism, Genetic Variation, DNA, Exons, medicine.disease, Molecular biology, Introns, Mutational analysis, Enzyme, chemistry, Biochemistry, Mutagenesis, Inborn error of metabolism, DPYD, Fluorouracil |
| الوصف: | 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. |
| تدمد: | 1557-7473 1090-6576 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::554cc29b83d4c3a07e4a814e8a8a0abcTest https://doi.org/10.1089/109065703322146777Test |
| حقوق: | CLOSED |
| رقم الانضمام: | edsair.doi.dedup.....554cc29b83d4c3a07e4a814e8a8a0abc |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15577473 10906576 |
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