Denaturing high performance liquid chromatography analysis of the DPYD gene in patients with lethal 5-fluorouracil toxicity.
Dihydropyrimidine dehydrogenase (DPD) enzyme deficiency is a pharmacogenetic syndrome with possible fatal outcome following 5-fluorouracil (5-FU) treatment. Several studies examining the molecular basis for DPD deficiency have identified over 30 sequence variations in the DPYD gene (which codes for the DPD enzyme). Our laboratory has recently developed and validated a denaturing high performance liquid chromatography method capable of identifying both known and unknown sequence variations in the DPYD gene. In the present study, we used this denaturing high performance liquid chromatography approach to examine the DPYD genotype of three patients who experienced lethal toxicity after administration of 5-FU. DPD enzyme activity could only be measured in one patient before death and demonstrated that lethal toxicity can occur in a partially DPD-deficient individual. Multiple heterozygous sequence variations (both known and unknown) were detected in all three patients including the novel variants 545T>A, M182K and 2329G>T, A777S. We conclude that (a) lethal toxicity can occur in partially DPD-deficient individuals after administration of 5-FU and is not exclusive to profoundly DPD-deficient individuals as suggested previously, (b) the complicated heterozygote genotype seen in these patients, combined with DPD deficiency being an autosomal codominant inherited syndrome, precludes the use of simple genotyping assays that identify only one or two mutations as a method for identifying DPD-deficient individuals; and (c) these multiple heterozygote genotypes (which are more difficult to accurately characterize) may be responsible for some of the conflicting reports which suggests a lack of correlation between phenotype and genotype. (+info)
New insights in dihydropyrimidine dehydrogenase deficiency: a pivotal role for beta-aminoisobutyric acid?
DPD (dihydropyrimidine dehydrogenase) constitutes the first step of the pyrimidine degradation pathway, in which the pyrimidine bases uracil and thymine are catabolized to beta-alanine and the R-enantiomer of beta-AIB (beta-aminoisobutyric acid) respectively. The S-enantiomer of beta-AIB is predominantly derived from the catabolism of valine. It has been suggested that an altered homoeostasis of beta-alanine underlies some of the clinical abnormalities encountered in patients with a DPD deficiency. In the present study, we demonstrated that only a slightly decreased concentration of beta-alanine was present in the urine and plasma, whereas normal levels of beta-alanine were present in the cerebrospinal fluid of patients with a DPD deficiency. Therefore the metabolism of beta-alanine-containing peptides, such as carnosine, may be an important factor involved in the homoeostasis of beta-alanine in patients with DPD deficiency. The mean concentration of beta-AIB was approx. 2-3-fold lower in cerebrospinal fluid and urine of patients with a DPD deficiency, when compared with controls. In contrast, strongly decreased levels (10-fold) of beta-AIB were present in the plasma of DPD patients. Our results demonstrate that, under pathological conditions, the catabolism of valine can result in the production of significant amounts of beta-AIB. Furthermore, the observation that the R-enantiomer of beta-AIB is abundantly present in the urine of DPD patients suggests that significant cross-over exists between the thymine and valine catabolic pathways. (+info)
Rapid identification of dihydropyrimidine dehydrogenase deficiency by using a novel 2-13C-uracil breath test.
PURPOSE: Dihydropyrimidine dehydrogenase (DPD)-deficient cancer patients have been shown to develop severe toxicity after administration of 5-fluorouracil. Routine determination of DPD activity is limited by time-consuming and labor-intensive methods. The purpose of this study was to develop a simple and rapid 2-(13)C-uracil breath test, which could be applied in most clinical settings to detect DPD-deficient cancer patients. EXPERIMENTAL DESIGN: Fifty-eight individuals (50 "normal," 7 partially, and 1 profoundly DPD-deficient) ingested an aqueous solution of 2-(13)C-uracil (6 mg/kg). (13)CO(2) levels were determined in exhaled breath at various time intervals up to 180 min using IR spectroscopy (UBiT-IR(300)). DPD enzyme activity and DPYD genotype were determined by radioassay and denaturing high-performance liquid chromatography, respectively. RESULTS: The mean (+/-SE) C(max), T(max), delta over baseline values at 50 min (DOB(50)) and cumulative percentage of (13)C dose recovered (PDR) for normal, partially, and profoundly DPD-deficient individuals were 186.4 +/- 3.9, 117.1 +/- 9.8, and 3.6 DOB; 52 +/- 2, 100 +/- 18.4, and 120 min; 174.1 +/- 4.6, 89.6 +/- 11.6, and 0.9 DOB(50); and 53.8 +/- 1.0, 36.9 +/- 2.4, and <1 PDR, respectively. The differences between the normal and DPD-deficient individuals were highly significant (all Ps <0.001). CONCLUSIONS: We demonstrated statistically significant differences in the 2-(13)C-uracil breath test indices (C(max), T(max), DOB(50), and PDR) among healthy and DPD-deficient individuals. These data suggest that a single time-point determination (50 min) could rapidly identify DPD-deficient individuals with a less costly and time-consuming method that is applicable for most hospitals or physicians' offices. (+info)
Dehydropyrimidine dehydrogenase deficiency in a cancer patient undergoing 5-fluorouracil chemotherapy.
We present a case of a Caucasian cancer patient undergoing 5-fluorouracil (5-FU)-containing chemotherapy in our department. The 49-year-old female patient suffered from adverse effects representing WHO grade 3 toxicity. Genotyping revealed that she carried the exon 14-skipping mutation which is known to result in dehydropyrimidine dehydrogenase (DPD) deficiency. DPD is the enzyme that converts 5-FU to inactive metabolites and therefore dictates the amount of 5-FU that is available to be metabolised to cytotoxic nucleotides. Consequently DPD deficiency is the cause of severe adverse and sometimes lethal reactions to 5-FU. In conclusion the identification of cancer patients at increased risk of severe toxicity prior to the administration of 5-FU would be desirable. (+info)
5FU and oxaliplatin-containing chemotherapy in two dihydropyrimidine dehydrogenase-deficient patients.
Patients with a germline mutation leading to a deficiency of the dihydropyrimidine dehydrogenase (DPD) enzyme are at risk from developing severe toxicity on the administration of 5FU-containing chemotherapy. We report on the implications of this inborn genetic error in two patients who received 5FU and oxaliplatin. A possible co-medication effect of oxaliplatin is considered, as are the consequences of screening for DPD deficiency. (+info)
Relationships among plasma [2-(13)C]uracil concentrations, breath (13)CO(2) expiration, and dihydropyrimidine dehydrogenase (DPD) activity in the liver in normal and dpd-deficient dogs.
Dihydropyrimidine dehydrogenase (DPD), the first enzyme in the sequential metabolism of pyrimidine, regulates blood concentrations of 5-fluorouracil and is deeply involved in its toxicity. This study was designed to examine the effects of a DPD inhibitor on blood concentrations of [2-(13)C]uracil ([(13)C]uracil) and (13)CO(2) concentration (Delta(13)C) expired in breath after oral or intravenous administration of [(13)C]uracil to DPD-suppressed dogs prepared by pretreatment with 5-(trans-2-bromovinyl)uracil (BVU), a DPD inhibitor. Area under the curve (AUC(t)) of [(13) C]uracil after oral administration at 20 micromol/kg to dogs pretreated with BVU at 2, 5, and 40 mmol/kg were 37-, 88- and 120-fold higher than those of the control dogs, respectively. In contrast, breath AUC(t) values of Delta(13)C were reduced to 0.88-, 0.47- and 0.13-fold the control values, respectively. Upon intravenous administration of [(13)C]uracil at 20 micromol/kg to dogs pretreated with BVU at 0.5, 2, and 40 micromol/kg, blood AUC(t) values of [(13)C]uracil were 1.4-, 4.2-, and 13-fold higher than those of the control group, respectively, whereas breath AUC(t) values were reduced to 1.0-, 0.83-, and 0.07-fold the respective control values. DPD activities in the liver cytosol of dogs pretreated with BVU at 0.5, 2, 5, and 40 micromol/kg were decreased to 0.71-, 0.12-, 0.06-, and 0.04-fold those of the control dogs, respectively. These findings demonstrate that breath output (Delta(13)C) is a good marker of hepatic DPD activity in vivo. (+info)
Methylation of the DPYD promoter: an alternative mechanism for dihydropyrimidine dehydrogenase deficiency in cancer patients.
PURPOSE: Dihydropyrimidine dehydrogenase (DPD) deficiency, a known pharmacogenetic syndrome associated with 5-fluorouracil (5-FU) toxicity, has been detected in 3% to 5% of the population. Genotypic studies have identified >32 sequence variants in the DPYD gene; however, in a number of cases, sequence variants could not explain the molecular basis of DPD deficiency. Recent studies in cell lines indicate that hypermethylation of the DPYD promoter might down-regulate DPD expression. The current study investigates the role of methylation in cancer patients with an unexplained molecular basis of DPD deficiency. EXPERIMENTAL DESIGN: DPD deficiency was identified phenotypically by both enzyme assay and uracil breath test, and genotypically by denaturing high-performance liquid chromatography. The methylation status was evaluated in PCR products (209 bp) of bisulfite-modified DPYD promoter, using a novel denaturing high-performance liquid chromatography method that distinguishes between methylated and unmethylated alleles. Clinical samples included five volunteers with normal DPD enzyme activity, five DPD-deficient volunteers, and five DPD-deficient cancer patients with a history of 5-FU toxicity. RESULTS: No evidence of methylation was detected in samples from volunteers with normal DPD. Methylation was detected in five of five DPD-deficient volunteers and in three of five of the DPD-deficient cancer patient samples. Of note, one of the two samples from patients with DPD-deficient cancer with no evidence of methylation had the mutation DPYD*2A, whereas the other had DPYD*13. DISCUSSION: Methylation of the DPYD promoter region is associated with down-regulation of DPD activity in clinical samples and should be considered as a potentially important regulatory mechanism of DPD activity and basis for 5-FU toxicity in cancer patients. (+info)
Unpredicted severe toxicity after 5-fluorouracil treatment due to dihydropyrimidine dehydrogenase deficiency.
Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). Thus, patients with a DPD deficiency are at risk of developing severe 5-FU-associated toxicity. A 37-year-old female with gastric cancer underwent a curative operation, followed by adjuvant chemotherapy consisting of 5-FU and epirubicin. After the first cycle of chemotherapy, the patient manifested grade 2 mucositis and febrile neutropenia, and when her treatment was subsequently continued with doxifluridine she developed severe mucositis and febrile neutropenia. A PCR study revealed that her DPD mRNA level was lower than that in a control group. Thus, when considering the routine use of 5-FU for the treatment of cancer patients, an analysis of DPD activity or screening for DPD mutations is warranted in confined patients who experience unpredicted severe toxicity after initial 5-FU administration, even though DPD deficiency is a rare metabolic defect. (+info)