DPYD*2A mutation: the most common mutation associated with DPD deficiency

Background Dihydropyrimidine dehydrogenase (DPD) enzyme is responsible for the elimination of approximately 80% of administered dose of 5-FU. DPD deficiency has been associated with severe 5-FU toxicity. Syndrome of DPD deficiency manifests as diarrhea, stomatitis, mucositis, and neurotoxicity and in some cases death. This is a true pharmacogenetic syndrome, with symptoms being unrecognizable until exposure to the drug. Patients and methods A 75-year-old patient with metastatic pancreatic adenocarcinoma developed grade 4 thrombocytopenia, grade 3 coagulopathy, and grade 3 neurologic toxicity with a fatal outcome following administration of 5-FU. Due to pancytopenia, DPD activity could not be determined in peripheral blood mononuclear cells (PBMC) using a previously described radioassay. Therefore, screening and genotypic analysis of homozygous and heterozygous, known and unknown sequence variants, in the DPYD gene were performed using DHPLC as previously described. All DPYD sequence variants identified by DHPLC were confirmed by DNA sequencing using a dideoxynucleotide chain termination method and capillary electrophoresis on an ABI 310 Automated DNA Sequencer. Results Genotyping analysis of the DPYD gene revealed the presence of the heterozygous mutation, IVS14 + 1 G > A, DPYD*2A. Conclusion Genotypic analysis using DHPLC can be employed to screen DPD deficiency in a patient with severe neutropenia. The mutation IVS14 + 1 G > A, DPYD*2A, is the most common mutation associated with DPD deficiency. A G > A base change at the splice recognition sequence of intron 14, leads to exon skipping and results in a 165-bp deletion in the DPD mRNA. We have previously demonstrated that a homozygote DPYD*2A genotype results in complete deficiency while the heterozygous DPYD*2A genotype results in partial deficiency of DPD. This work was supported in part by NIH grant CA 62164.     

Increased risk of grade IV neutropenia after administration of 5-fluorouracil due to a dihydropyrimidine dehydrogenase deficiency: high prevalence of the IVS14+1g>a mutation

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk of developing severe 5-FU-associated toxicity. We evaluated the importance of DPD deficiency, gender and the presence of the IVS14+1G>A mutation in the etiology of 5-FU toxicity. In 61% of cases, decreased DPD activity could be detected in peripheral blood mononuclear cells. Furthermore, the number of females (65%) in the total group of patients appeared to be higher than the number of males (35%) (p = 0.03). Patients with partial DPD deficiency appeared to have a 3.4-fold higher risk of developing grade IV neutropenia than patients with normal DPD activity. Analysis of the DPYD gene of patients suffering from grade IV neutropenia for the presence of the IVS14+1G>A mutation showed that 50% of the patients investigated were heterozygous or homozygous for the IVS14+1G>A mutation. Adopting a threshold level for DPD activity of 70% of that observed in the normal population, 14% of the population is prone to the development of severe 5-FU-associated toxicity. Below this threshold level, 90% of individuals heterozygous for a mutation in the DPYD gene can be identified. Considering the common use of 5-FU in the treatment of cancer, the severe 5-FU-related toxicities in patients with low DPD activity and the apparently high prevalence of the IVS14+1G>A mutation, screening of patients at risk before administration of 5-FU is warranted.     

Analysis of the DPYD gene implicated in 5-fluorouracil catabolism in a cohort of Caucasian individuals.

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.     

Dihydropyrimidine dehydrogenase pharmacogenetics in the Taiwanese population

Background/purpose 5-Fluorouracil (5-FU) remains the most frequently used chemotherapy agent in various human cancers. Over 80% of the 5-FU administered is metabolized by dihydropyrimidine dehydrogenase (DPD) in the liver. However, mutations in the DPD gene have been found to be associated with low DPD activity causing severe complications. The aim of this study was to determine the frequency of 11 known mutations in Taiwanese subjects and the relationship between mutation and DPD level.Methods Samples from a total of 300 subjects were investigated in this study. The PCR-RFLP method was used to identify 11 mutations of the DPYD gene, including 62G>A, 74A>G, 85T>C (DPYD*9A), 812delT, 1003G>T, 1156G>T, 1627A>G (DPYD*5), 1714C>G, 1897delC (DPYD*3), 2194G>A (DPYD*6), and IVS14+1G>A (DPYD*2A). DPD protein levels were determined using a DPD ELISA kit.Results Four mutations, including 74A>G, 85T>C (DPYD*9A), 1627A>G (DPYD*5), and 2194G>A (DPYD*6), were found in our 300 samples. The following mutations were not detected: 62G>A, 812delT, 1003G>T, 1156G>T, 1714C>G, 1897delC (DPYD*3), and IVS14+1G>A (DPYD*2A). The phenotype analysis by DPD protein level indicated that the 1627A>G (DPYD*5) mutation was not associated with the DPD protein level and might be a polymorphism in the DPD gene. The DPD level was also not correlated with gender.Conclusion No significant correlations between these 11 mutations and DPD protein level were found indicating that examination of these mutations is insufficient to provide a high-value prediction of the 5-FU pharmacogenetic syndrome in Taiwanese. Genotype and phenotype analysis indicated the 1627A>G (DPYD*5) mutation to be a polymorphism.     

Dihydropyrimidine dehydrogenase and the efficacy and toxicity of 5-fluorouracil

The identification of genetic factors associated with either responsiveness or resistance to 5-fluorouracil (5-FU) chemotherapy, as well as genetic factors predisposing patients to the development of severe 5-FU-associated toxicity, is increasingly being recognised as an important field of study. Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). Although the role of tumoral levels as a prognostic factor for clinical responsiviness has not been firmly established, there is ample evidence that a deficiency of DPD is associated with severe toxicity after the administration of 5-FU. Patients with a partial DPD deficiency have an increased risk of developing grade IV neutropenia. In addition, the onset of toxicity occurred twice as fast compared with patients with a normal DPD activity. To date, 39 different mutations and polymorphisms have been identified in DPYD. The IVS14+1G>A mutation proved to be the most common one and was detected in 24-28% of all patients suffering from severe 5-FU toxicity. Thus, a deficiency of DPD appears to be an important pharmacogenetic syndrome.     

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.     

Hypermethylation of the DPYD promoter region is not a major predictor of severe toxicity in 5-fluorouracil based chemotherapy

Background

The activity of dihydropyrimidine dehydrogenase (DPD), the key enzyme of pyrimidine catabolism, is thought to be an important determinant for the occurrence of severe toxic reactions to 5-fluorouracil (5-FU), which is one of the most commonly prescribed chemotherapeutic agents for the treatment of solid cancers. Genetic variation in the DPD gene (DPYD) has been proposed as a main factor for variation in DPD activity in the population. However, only a small proportion of severe toxicities in 5-FU based chemotherapy can be explained with such rare deleterious DPYD mutations resulting in severe enzyme deficiencies. Recently, hypermethylation of the DPYD promoter region has been proposed as an alternative mechanism for DPD deficiency and thus as a major cause of severe 5-FU toxicity.

Methods

Here, the prognostic significance of this epigenetic marker with respect to severe 5-FU toxicity was assessed in 27 cancer patients receiving 5-FU based chemotherapy, including 17 patients experiencing severe toxic side effects following drug administration, none of which were carriers of a known deleterious DPYD mutation, and ten control patients. The methylation status of the DPYD promoter region in peripheral blood mononuclear cells was evaluated by analysing for each patient between 19 and 30 different clones of a PCR-amplified 209 base pair fragment of the bisulfite-modified DPYD promoter region. The fragments were sequenced to detect bisulfite-induced, methylation-dependent sequence differences.

Results

No evidence of DPYD promoter methylation was observed in any of the investigated patient samples, whereas in a control experiment, as little as 10% methylated genomic DNA could be detected.

Conclusion

Our results indicate that DYPD promoter hypermethylation is not of major importance as a prognostic factor for severe toxicity in 5-FU based chemotherapy.     

Dihydropyrimidine dehydrogenase activity and its genetic aberrations

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases, uracil and thymine, and is also known to be the key enzyme catalyzing the metabolic degradation of the anti-cancer drug 5-fluorouracil (5-FU). 5-FU has been commonly and widely used as a chemotherapeutic agent for the treatment of cancer of the gastrointestinal tract, breast, and head and neck. More than 85% of the administered 5-FU is catabolized by DPD.The clinical importance of DPD has been demonstrated with the identification of severe or lethal toxicity in patients administered 5-FU who are deficient in or have low levels of DPD activity in their peripheral blood mononuclear cells (PBMC). The importance of the role of DPD in 5-FU chemotherapy also has been shown by studies with competitive and irreversible DPD inhibitors. Population studies of DPD activity in PBMC were reported in healthy volunteers and cancer patients to evaluate the incidence of complete or partial DPD deficiency. In these studies, considerable variation was observed, and the frequency of low or deficient DPD activity (<30% and <10% of the mean activity of the normal population, respectively), was estimated to be 3-5% and 0.1%,respectively. We also found one healthy volunteer (0.7% of the population) with very low PBMC-DPD activity due to heterozygosity for a mutant allele of the DPYD gene in a population of 150 healthy Japanese volunteers. To date, at least 34 DPYD variants have been reported. However, genotyping of cancer patients with reduced or normal DPD activity showed that only 17% of those patients had a molecular basis for their deficient phenotype, which emphasized the complex nature of the molecular mechanisms controlling polymorphic DPD activity in vivo,suggesting that it is difficult to identify DPD deficiency by genotyping. Therefore, it is important to develop methods for identifying DPD deficiency in cancer patients by phenotyping before 5-FU treatment.     

Severe fluoropyrimidine-related toxicity: clinical implications of DPYD analysis and UH2/U ratio evaluation

The fluoropyrimidines are commonly used in chemotherapeutic cancer medicine, but many patients still experience severe adverse side effects from these drugs. We observed a severe toxicity in a 50-year-old woman treated with capecitabine and docetaxel for a metastatic breast cancer. Since dihydropyrimidine dehydrogenase (DPD) is the main candidate for pharmacogenetic studies on 5-FU toxicity, the entire coding sequence and exon-flanking intronic regions of the DPYD gene were sequenced in the patient. None of the previously described deleterious variants were detected. Also, the haplotype-based analysis failed to reveal DPYD variations associated with 5-FU toxicity. We also evaluated the UH2/U ratio in plasma as an index of 5-FU pharmacokinetics. The UH2/U value did not demonstrate low DPD activity in the patient. We discuss the advantages and limitations of this approach, particularly concerning the clinical applications of 5-FU pharmacogenetics in the family setting.     

Multiple organ failure due to 5-fluorouracil chemotherapy in a patient with a rare dihydropyrimidine dehydrogenase gene variant

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the metabolism of the chemotherapeutic drug 5-fluorouracil (5-FU). Application of 5-FU is restricted by a narrow therapeutic index because of severe toxicity of WHO grades III-IV. The exon 14-skipping mutation (c.1905+1G>A) accounts for approximately a quarter of all severely toxic cases. However, numerous other polymorphisms have been identified within the DPYD gene in affected patients, and the pathophysiological significance of most of them is unclear. PATIENT AND METHODS: We report a patient with advanced caecum cancer who twice received 950 mg 5-FU and 45 mg folinic acid as adjuvant by bolus injection. 2 days after onset of chemotherapy, the patient developed a multiple organ dysfunction exhibiting a cardiogenic shock with severe left ventricular insufficiency, marked reduction of renal function, and beginning hepatic encephalopathy with somnolence, myoclonus, and a seizure. In order to investigate a possible defect within the DPYD gene direct sequencing of all 23 exons was carried out. RESULTS: Genotyping revealed a rare c.1601G>A polymorphism which causes a change in the protein sequence (S534N). Data regarding the clinical relevance are ambiguous. The polymorphism has been detected together with an intronic mutation and both polymorphisms have consistently been reported with reduced enzyme activity. CONCLUSION: The present case provides further evidence of an etiologic role of the c.1601G>A mutation for DPD deficiency and the occurrence of severe 5-FU-related toxicity and underlines the value of comprehensive pharmakogenetic diagnostics with respect to the dihydropyrimidine dehydrogenase.     

Toxic death-case after capecitabine + oxaliplatin (XELOX) administration: probable implication of dihydropyrimidine deshydrogenase deficiency

This report here is the case of a 52-year-old male patient who suffered from extremely severe haematological toxicities (G4 neutropenia, G4 thrombocytopenia) while undergoing Xelox (Xeloda + Oxaliplatin) treatment for his multifocal hepatocarcinoma. Despite appropriate supportive treatment, his condition quickly deteriorated and led to death. It was hypothesized that dihydropyrimidine deshydrogenase (DPD) gene polymorphism could be, at least in part, responsible for this fatal outcome. To test this hypothesis, both phenotypic and genotypic studies were undertaken, and fully confirmed the DPD-deficient status of this patient. Uracil to dihydrouracil ratio in plasma was evaluated as a surrogate marker for DPD deficiency, and showed values out of the range previously recorded from a reference, non-toxic population. Interestingly, the canonical IVS14+1G>A single nucleotide polymorphism, usually associated with the most severe toxicities reported with 5-fluorouracil (5-FU), was not found in this patient, but further investigations showed instead a heterozygosity for the 1896C>T mutation located in the exon 14 of the DPYD gene. Taken together, the data strongly suggest for the first time that a toxic-death case after capecitabine-containing protocol could be, at least in part, linked with a DPD-deficiency syndrome. The case reported here warrants therefore systematic detection of patients at risk, including when oral capecitabine is scheduled.     

Low Incidence of the DPD IVS14+1G>A Polymorphism in Jordanian Breast and Colorectal Cancer patients

Background: Dihydropyrimidine dehydrogenase (DPD) is a crucial enzyme in the catabolism of 5-fluorouracil (5-FU), a drug that is frequently used in cancer therapy. Patients with deficient DPD activity are at risk of developing severe 5-FU–associated toxicity. One possible cause of deficiency is genetic polymorphisms in the DPD gene, such as IVS14+1G>A. Aim: The present study was conducted to screen for the IVS14+1G>A polymorphism in cancer patients receiving 5-FU and a control group. Methods: A total of 40 cancer patients (30 colorectal cancer (CRC) and 10 breast cancer patients) were enrolled in this study. One hundred healthy controls were also tested using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). DNA sequence analysis was carried out to confirm the presence of the IVSI14+1G>A polymorphism. Results: Only one CRC patient showed heterozygous IVS14+1G>A polymorphism in the DPD gene. Conclusion: The results of this study demonstrated a very low frequency of the IVS14+1G>A polymorphism among Jordanian patients with colorectal and breast cancer.     

DPYD*5 gene mutation contributes to the reduced DPYD enzyme activity and chemotherapeutic toxicity of 5-FU

Background Dihydropyrimidine dehydrogenase (DPYD) plays an important role in the metabolism of 5-FU, which can directly influence the pharmacokinetics and toxicity of 5-FU in patients undergoing chemotherapy. However, little is known of the relationship between DPYD gene polymorphism and metabolism and chemotherapeutic toxicity of 5-FU in gastric carcinoma and colon carcinoma. The present genotyping study demonstrated the relationship between DPYD gene polymorphism among 75 gastric carcinoma and colon carcinoma patients and its impact on 5-FU pharmacokinetic and side effect. Methods We used a chemotherapy scheme based on 5-FU for the treatment of 75 patients with gastrointestinal carcinoma and detected the serum drug concentration and DPYD gene polymorphism (DPYD*2, *3, *4 *5 *9 *12). Results We found that there were no DPYD*2, *3, *4, *12 type mutation, in all patients. Of DPYD*9 gene polymorphism loci in 75 patients, 7 were heterozygote and 68 wild type; of DPYD*5 gene polymorphism loci in 75 patients, 11 were mutation and 23 heterozygote and 41 wild type. The elimination rate constant (Ke) value of DPYD*5 mutation group was statistically lower than the wild type (p=0.022). The incidence of middle-severe nausea and vomiting and white blood cell decreases in DPYD*5 gene type ranging from the highest to lowest can be listed as: mutation, heterozygote, wild type (p<0.05). The incidence of middle-severe nausea and vomiting was significantly higher in DPYD*9 heterozygous genotype than in DPYD*9 wild genotype (p<0.05). Conclusions DPYD*5 gene mutation contribute to reduced DPYD enzyme activity and 5-FU dysmetabolism, which is associated with the accumulation of 5-FU and the chemotherapeutic toxicity in gastric carcinoma and colon carcinoma.     

Profound dihydropyrimidine dehydrogenase deficiency resulting from a novel compound heterozygote genotype.

A familial approach was used to elucidate the genetic determinants of profound and partial dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) deficiency in an Alabama family. In 1988, our laboratory diagnosed profound DPD deficiency in a breast cancer patient with grade IV toxicity after cyclophosphamide/methotrexate/5-fluorouracil chemotherapy (R. B. Diasio et al., J. Clin. Investig., 81: 47-51, 1988). We now report the genetic analysis of archived genomic DNA that reveals that the proband was a compound heterozygote for two different mutations, one in each allele: (a) a G to A mutation in the GT 5' splicing recognition sequence of intron 14, which results in a 165-bp deletion (corresponding to exon 14) in the DPD mRNA (DPYD*2A); and (b) a T1679G mutation (now designated DPYD*13), which results in a I560S substitution. Sequence analysis revealed segregation of both mutations with the son and the daughter each inheriting one mutation. Phenotype analysis (DPD enzyme activity) confirmed that both children were partially DPD deficient. Plasma uracil and DPD mRNA levels were found to be within normal limits in both children. We conclude that profound DPD deficiency in the proband resulted from a combination of two mutations (one mutation in each allele) and that heterozygosity for either mutation results in partial DPD deficiency. Lastly, we identified two variant alleles reported previously as being associated with DPD enzyme deficiency [T85C resulting in a C29R substitution (DPYD*9A) and A496G (M166V) in a family member with normal DPD enzyme activity]. These data suggest that both variant alleles are unrelated to DPD deficiency and emphasize the need to perform detailed familial genotypic and phenotypic analysis while characterizing this pharmacogenetic syndrome.     

Prospective DPYD genotyping to reduce the risk of fluoropyrimidine-induced severe toxicity: Ready for prime time

5-Fluorouracil (5-FU) and capecitabine (CAP) are among the most frequently prescribed anticancer drugs. They are inactivated in the liver by the enzyme dihydropyrimidine dehydrogenase (DPD). Up to 5% of the population is DPD deficient and these patients have a significantly increased risk of severe and potentially lethal toxicity when treated with regular doses of 5-FU or CAP. DPD is encoded by the gene DPYD and variants in DPYD can lead to a decreased DPD activity. Although prospective DPYD genotyping is a valuable tool to identify patients with DPD deficiency, and thus those at risk for severe and potential life-threatening toxicity, prospective genotyping has not yet been implemented in daily clinical care. Our goal was to present the available evidence in favour of prospective genotyping, including discussion of unjustified worries on cost-effectiveness, and potential underdosing. We conclude that there is convincing evidence to implement prospective DPYD genotyping with an upfront dose adjustment in DPD deficient patients. Immediate benefit in patient care can be expected through decreasing toxicity, while maintaining efficacy.     

Genetic polymorphisms related to fluoropyrimidine sensitivity and toxicity

Fluoropyrimidine such as 5-fluorouracil(5-FU)exerts its antitumor activities via anabolism by several enzymes. Genetic polymorphisms of these enzymes related to sensitivity and toxicity of fluoropyrimidines are reviewed. Expression of thymidylate synthase(TS), a target enzyme of 5-FU, is regulated by variable number of a 28 bp tandem repeat in the enhancer region. The double tandem repeat is associated with low TS expression, and consequently, patients with double tandem repeat demonstrated higher sensitivity to 5-FU than those with triple tandem repeat. Single nucleotide polymorphism within the second tandem repeat and loss of heterozygosity are also reported to be related to fluoropyrimidine sensitivity. In addition, patients having a 6 bp deletion in 3'-UTR region showed remarkably high antitumor activity by 5-FU based chemotherapy. Genetic variations in 5-FU catabolic enzymes can also have a profound effect on 5-FU toxicity. So far 39 mutations/ polymorphisms have been identified in dihydropyrimidine dehydrogenase(DPD)gene, a major catabolic enzyme of 5- FU. Among them, IVS14+1G>A is reported to be highly associated with severe toxicity caused by chemotherapy with fluoropyrimidine. A polymorphism that may influence the efficacy of 5-FU by influencing folate pools is that of the methylenetetrahydrofolate reductase(MTHFR)gene. C677T mutation was associated with a higher response rate on 5-FU/folinic acid chemotherapy. Prospective clinical trials to confirm the predictability of genetic polymorphism for sensitivity and toxicity of 5-FU should be performed.     

Lethal outcome of a patient with a complete dihydropyrimidine dehydrogenase (DPD) deficiency after administration of 5-fluorouracil: frequency of the common IVS14+1G>A mutation causing DPD deficiency.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk from developing a severe 5FU-associated toxicity. In this study, we demonstrated that a lethal toxicity after a treatment with 5FU was attributable to a complete deficiency of DPD. Analysis of the DPD gene for the presence of mutations showed that the patient was homozygous for a G-->A mutation in the invariant GT splice donor site flanking exon 14 (IVS14+1G>A). As a consequence, no significant residual activity of DPD was detected in peripheral blood mononuclear cells. To determine the frequency of the IVS14+1G>A mutation in the Dutch population, we developed a novel PCR-based method allowing the rapid analysis of the IVS14+1G>A mutation by RFLP. Screening for the presence of this mutation in 1357 Caucasians showed an allele frequency of 0.91%. In our view, the apparently high prevalence of the IVS14+1G>A mutation in the normal population, with 1.8% heterozygotes, warrants genetic screening for the presence of this mutation in cancer patients before the administration of 5FU.