RESUMEN
PURPOSE: Complete or partial loss of dihydropyrimidine dehydrogenase (DPD) function has been described in cancer patients with intolerance to fluoropyrimidine drugs like 5-fluorouracil (5-FU) or Xeloda. The intention of this population study is to assess and to evaluate gene variations in the entire coding region of the dihydropyrimidine dehydrogenase gene (DPYD), which could be implicated in DPD malfunction. EXPERIMENTAL DESIGN: A cohort of 157 individuals was genotyped by denaturing high-performance liquid chromatography; 100 of these genotypes were compared with functional studies on DPD activity and mRNA expression. RESULTS: Twenty-three variants in coding and noncoding regions of the DPYD gene were detected, giving rise to 15 common haplotypes with a frequency of >1%. Rare sequence alterations included a frameshift mutation (295-298delTCAT) and three novel point mutations, 1218G>A (Met406Ile), 1236G>A (Glu412Glu), and 3067C>T (Pro1023Ser). DPD enzyme activity showed high variation in the analyzed population and correlated with DPD mRNA expression. In particular, the novel variants were not accompanied with decreased enzyme activity. However, a statistically significant deviation from the median DPD activity of the population was associated with the mutations 1601G>A (Ser534Asn) and 2846A>T (Asp949Val). CONCLUSION: This work presents an analysis of DPYD gene variations in a large cohort of Caucasians. The results reflect the genetic and enzymatic variability of DPD in the population and may contribute to further insight into the pharmacogenetic disorder of DPD deficiency.
Asunto(s)
Dihidrouracilo Deshidrogenasa (NADP)/genética , Fluorouracilo/metabolismo , Mutación , Población Blanca/genética , Alelos , Secuencia de Bases , Cromatografía Líquida de Alta Presión/métodos , Estudios de Cohortes , ADN/sangre , ADN/química , ADN/genética , Análisis Mutacional de ADN , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Regulación Enzimológica de la Expresión Génica , Frecuencia de los Genes , Haplotipos , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estadística como AsuntoRESUMEN
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.