Complete Dihydropyrimidine Dehydrogenase Deficiency Research Articles

Assessing the Hepatotoxic Effects of Fluoropyrimidine Chemotherapy in Male Iraqi Colorectal Cancer Patients.

Colorectal cancer (CRC) is one the most frequently occurring cancer types among various populations. Fluoropyrimidine is the backbone of first-line chemotherapy, the oral capecitabine, or intravenous 5-fluorouracil (5-FU) in various combinations and schedules the chemotherapy regime in the treatment of a wide variety of gastrointestinal cancers. The enzyme dihydropyrimidine dehydrogenase (DPD) functions as the rate-limiting step in the metabolism of fluoropyrimidine chemotherapies, and patients with complete or partial DPD deficiency are at increased risk of severe and fatal toxicity during treatment with fluorouracil. This study aimed to examine thechemotoxicity of the 5-FU drug on hepatocytes in male Iraqi CRC patients. This research is a cross-sectional study conducted between November 2022 and April 2023. The study included 80 male participants who had undergone surgical intervention for stage III CRC under the care of the Misan Health Directorate, Misan Center for Tumors Treatment, located in Misan, Iraq. Based on their subsequent surgical treatment, the participants were divided into two groups. The first group, comprising 45 males aged between 41 and 71 years, experienced a relapse despite receiving adjuvant therapy, which involved a singular cycle of fluoropyrimidine-based chemotherapy (5-FU). The second group consisted of 35 male patients with CRC, aged between 40 and 57 years, who did not experience a relapse post-adjuvant therapy. Their adjuvant therapy involved a single round of fluoropyrimidine-based chemotherapy with 5-FU. Relapse in patients was determined by assessing the white blood cell count(WBC). Liver enzymes were significantly increased after 5-FU treatment, while the concentration of albumin was significantly decreased. The findings of our study clearly indicate that 5-FU induced hepatic injury, lowering the hepatocytefunction with elevated levels of hepatic enzymes and low concentration of albumin in the blood, which is an important predictive marker of chemotherapy toxicity.

Complete DPYD genotyping combined with dihydropyrimidine dehydrogenase phenotyping to prevent fluoropyrimidine toxicity: A retrospective study.

In April 2019, French authorities mandated dihydropyrimidine dehydrogenase (DPD) screening, specifically testing uracilemia, to mitigate the risk of toxicity associated with fluoropyrimidine-based chemotherapy. However, this subject is still of debate as there is no consensus on a standardized DPD deficiency screening test. We conducted a real-life retrospective study with the aim of assessing the impact of DPD screening on the occurrence of severe toxicity and exploring the potential benefits of complete genotyping using next-generation sequencing. All adult patients consecutively treated with 5-fluorouracil (5-FU) or its oral prodrug at six cancer centers between March 2018 and February 2019 were considered for inclusion. Dihydropyrimidine dehydrogenase deficiency screening included gene encoding DPD (DPYD) genotyping using complete genome sequencing and DPD phenotyping (uracilemia or dihydrouracilemia/uracilemia ratio) or both tests. Associations between each DPD screening method and (i) severe (grade ≥3) early toxicity and (ii) fluoropyrimidine dose reduction in the second chemotherapy cycle were evaluated using multivariable logistic regression analysis. Furthermore, we assessed the concordance between DPD genotype and phenotype using Cohen's kappa. A total of 551 patients were included. Most patients were tested for DPD deficiency (86%) including DPYD genotyping only (6%), DPD phenotyping only (8%), or both (72%). Complete DPD deficiency was not detected in the study population. Severe early toxicity events were observed in 73 patients (13%), with two patients (0.30%) presenting grade 5 toxicity. Despite the numerically higher toxicity rate in untested patients, the occurrence of severe toxicity was not significantly associated with the DPD screening method (p = 0.69). Concordance between the DPD genotype and phenotype was weak (Cohen's kappa of 0.14). Due to insufficient numbers, our study was not able to demonstrate any added value of DPYD genotyping using complete genome sequencing to prevent 5-FU toxicity. The optimal strategy for DPD screening before fluoropyrimidine-based chemotherapy requires further clinical evaluation.

Raltitrexed in Patients With Dihydropyrimidine Dehydrogenase Deficiency


 This review identified limited evidence regarding the clinical effectiveness of raltitrexed for patients who had previously experienced adverse events following treatment with fluoropyrimidines. These studies had several limitations, most notably lacking a separate control group; therefore, the effectiveness of raltitrexed in this population is uncertain.
 Limited evidence was found about the safety of raltitrexed for patients who had previously experienced adverse events with fluoropyrimidine treatment. Reported adverse events included cardiac or vascular adverse events, anemia, and nausea and vomiting. No treatment-associated deaths were reported in the studies included in this review.
 No studies were identified that compared the clinical effectiveness of raltitrexed to other therapies, placebo, or no treatment comparator groups for treatment of patients who had experienced adverse events from fluoropyrimidine therapy.
 This review identified evidence for people who had previously experienced severe adverse events, primarily cardiotoxicity, following treatment with fluoropyrimidines. It is unclear if these findings are appliable to patients with a complete dihydropyrimidine dehydrogenase deficiency.

Personalized therapy: the crucial role of the DPYD c.2194G>A (V732I) allele in the treatment of colorectal cancer patients candidates for therapy with fluoropyrimidines

5-Fluorouracil (5FU) is a chemotherapeutic agent belonging to the class of antimetabolite drugs, which exert a toxic action causing death of neoplastic cells. 5FU is mostly used as a standard treatment for colorectal cancer; the development of toxicity phenomena is related to the partial or complete deficiency of the enzyme dihydropyrimidine dehydrogenase (DPD), limiting factor of the catabolism of fluoropyrimidines. Only 3-5% of 5-FU is converted to an active metabolite, while 85% of the drug is inactivated by DPD to 5-fluoro-dihydrouracil (5-FDHU). A reduced enzymatic activity of the DPD can be the cause for the presence of adverse drug reactions and toxicity in the patient, with multiorgan involvement, which can sometimes lead to death. The variants of the DPYD gene recommended by the AIOM (Associazione Italiana di Oncologia Medica)guidelines are: DPYD*2A (IVS14+1G>A, c.1905+1G>A); DPYD*13 (c.1679T>G); DPYD c.2846A>T, D949V; DPYD c.1236G>A (HapB3); DPYD c.2194G>A (V732I).Patients with complete DPD deficiency are at high risk of life-threatening or fatal toxicity and should not be treated with fluoropyrimidines, but this is a rare condition; while patients with partial deficiency should be treated with a reduced dose of the drug. Before starting treatment it’s crucial to determine the genetic profile of the patients candidates to therapy with fluoropyrimidines. In our cohort of the 370 samples analyzed by Real Time PCR, 294(~80%) are wild type for each variant screened. : DPYD c.2194G>A (V732I) alleleis significantly represented in the population examinated: considering the 15% reduction in drug administration imposed by this genotype, molecular profiling is essential before starting therapy with 5FU.In our study we also found a rare variant DPYD F632F rs17376848 c.1896 T> C in a patient, whose relevancefor therapeutic purposes is currently of uncertain significance.

SO-29 Dihydropyrimidine dehydrogenase (DPD) genotype and phenotype among Danish cancer patients: Prevalence and correlation between DPYD-genotype mutations and P-uracil concentrations

Fluoropyrimidines (FP) are the cornerstone of chemotherapy of several, mainly gastrointestinal, solid tumors. FP includes 5-fluorouracil, capecitabine, and tegafur, all metabolized by dihydropyrimidine dehydrogenase (DPD) enzyme. Patients with decreased DPD-activity have a higher risk of getting severe toxicity if treated with standard FP doses. The DPD-activity is currently a standard test in Denmark with a DPYD-genotype-test and/or a measurement of the endogenous compound uracil (DPD-phenotype test). In Denmark, the recommended FP starting dose for patients diagnosed with partial DPD-deficiency is 50%. FP treatment is contraindicated in patients with complete DPD-deficiency due to a high risk of life-threatening adverse effects. Patients with one variant SNP and/or uracil concentrations above 16 ng/ml are categorized as having partial DPD deficiency. However, patients are categorized as having complete DPD deficiency if they are homozygous for one variant SNP or compound heterozygous (patients with≥ 2 different variant SNPs) and/or have uracil concentrations above 150 ng/ml. In France and the Netherlands, the prevalence of clinically relevant DPYD variants was 4.7% and 7.7% respectively. No studies have reported data on the prevalence of DPYD variants or P-uracil in Danish cancer patients. We collected data from all Danish public hospital laboratories performing DPYD-genotype and DPD-phenotype analysis from the 1st of July 2020 to the 31st of December 2021. Patients were genotyped for the four clinically relevant DPYD-variants: rs3918290, rs67376798, rs55886062, and rs56038477/rs75018182. Most of the samples were genotyped using the LAMP (loop-mediated isothermal amplification) technology (Lc-Dpd4mut-Lp-24-Lacar Mdx Technologies). Plasma uracil concentrations were measured using liquid chromatography-mass spectrometry (LC-MS). In total 4228 Danish cancer patients were tested. 1985 of the patients were only tested using one method (P-uracil=17, DPYD-genotype=1968). 2243 patients were tested using P-uracil and DPYD-genotyping. Of the 4215 DPYD-genotyped patients, 3895 (92.4%) was wild type and 316 (7.5%) was heterozygous for one of the tested variants (SNP'S). rs3918290 (1%, n=43), rs67376798 (1.4%, n=57), rs55886062 (0.2%, n=8) and rs56038477/rs75018182 (4.9%, n=208). Four patients (0.1%) were compound heterozygous. Among 2260 patients tested, 2132 (94.3%) had an uracil concentration below 16 ng/ml. The remaining 128 (5.7%) patients had uracil concentrations ≥ 16 ng/mL and < 150 ng/ml. No values above 150 ng/ml was found. Median uracil concentration was 8 ng/ml (range 5-82 ng/ml). Both geno- and phenotype were available for 2243 of the patients. Uracil concentrations among wild type patients were lower (mean 8.6 ng/ml) compared to patients with DPYD-variants (mean 11.2 ng/ml), mean difference of 2.7 ng/ml. (95% CI; 1.9-3.4 ng/ml). In 4.8% (n=99) of wild type patients, the uracil concentration was ≥ 16 ng/ml compared to 16.8% (n=29) in the group with DPYD-mutations. The prevalence of clinically relevant DPYD-mutations in Danish cancer patients is 7.6% and is in line with results from similar studies of other European populations. 5.7% of phenotyped patients had uracil concentrations ≥ 16 ng/ml. Patients with DPYD-variants had higher mean uracil concentrations compared to wild type, but the uracil concentrations varied substantially in both groups. Data regarding adverse events are needed to conclude on the clinical implications.

A comprehensive population-based study comparing the phenotype and genotype in a pretherapeutic screen of dihydropyrimidine dehydrogenase deficiency

BackgroundPretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended or required prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD-deficient patients remains elusive.MethodsAmong a nationwide cohort of 5886 phenotyped patients with cancer who were screened for DPD deficiency over a 3 years period, we assessed the characteristics of both DPD phenotypes and DPYD genotypes in a subgroup of 3680 patients who had completed the two tests. The extent to which defective allelic variants of DPYD predict DPD activity as estimated by the plasma concentrations of uracil [U] and its product dihydrouracil [UH2] was evaluated.ResultsWhen [U] was used to monitor DPD activity, 6.8% of the patients were classified as having DPD deficiency ([U] > 16 ng/ml), while the [UH2]:[U] ratio identified 11.5% of the patients as having DPD deficiency (UH2]:[U] < 10). [U] classified two patients (0.05%) with complete DPD deficiency (> 150 ng/ml), and [UH2]:[U] < 1 identified three patients (0.08%) with a complete DPD deficiency. A defective DPYD variant was present in 4.5% of the patients, and two patients (0.05%) carrying 2 defective variants of DPYD were predicted to have low metabolism. The mutation status of DPYD displayed a very low positive predictive value in identifying individuals with DPD deficiency, although a higher predictive value was observed when [UH2]:[U] was used to measure DPD activity. Whole exon sequencing of the DPYD gene in 111 patients with DPD deficiency and a “wild-type” genotype (based on the four most common variants) identified seven heterozygous carriers of a defective allelic variant.ConclusionsFrequent genetic DPYD variants have low performances in predicting partial DPD deficiency when evaluated by [U] alone, and [UH2]:[U] might better reflect the impact of genetic variants on DPD activity. A clinical trial comparing toxicity rates after dose adjustment according to the results of genotyping or phenotyping testing to detect DPD deficiency will provide critical information on the best strategy to identify DPD deficiency.

Prevention of 5-fluorouracil-induced early severe toxicity by pre-therapeutic dihydropyrimidine dehydrogenase deficiency screening: Assessment of a multiparametric approach

5-Fluorouracil (5-FU)–based treatments can lead to early-onset severe (4%–5%) even fatal (0.3%) toxicities in patients with dihydropyrimidine dehydrogenase (DPD) deficiency. This multicenter prospective cohort study aimed to assess the clinical benefit of pretherapeutic screening for DPD deficiency using a multiparametric approach. Two parallel cohorts of patients treated with 5-FU–based chemotherapy for colorectal carcinoma were compared in a prospective nonrandomized study. In arm A, patients had DPD deficiency screening before treatment, whereas in arm B no pretherapy screening was performed. Dosing was based on 5-FU administration guidelines of each institution. DPD deficiency screening was performed using a combined multiparametric approach (5-FUODPM Tox). The frequency of early grade 4–5 toxic events potentially induced by 5-FU was compared in the two groups. At total of 1,142 patients (n = 1,116 evaluable) were enrolled. In arm A, out of 718 evaluable patients, nine grade 4 early toxicities potentially related to 5-FU were reported in nine patients (1.2%) with no toxic death despite one complete DPD deficiency and 24 partial deficiencies. The 24 patients with partial deficiency had safe pharmacokinetics (PK)-monitored 5-FU. In arm B, among 398 evaluable patients, 17 grade 4–5 toxic early events potentially related to 5-FU were reported in 12 patients (4.2%). The incidence of early severe toxicity was significantly higher in arm B (P = .0019), confirming the positive impact of pretherapeutic DPD assessment. The percent of patients with a toxicity grade 3 or higher observed in arm A was 10.8% (n = 78) compared to 17.55% (n = 69) in arm B (P = .0497). The percentage of death was reduced from 2.5/1,000 in arm B to 0 in arm A. The time to occurrence of all grade ≥3 toxicities was determined in both arms and the difference between the two arms was significant (P = .047). Overall, one patient with complete DPD deficiency confirmed retrospectively died within 13 days from grade 5 multivisceral toxicity. Enrollment was prematurely closed after external experts’ decision. In conclusion, multiparametric pretherapeutic DPD deficiency screening significantly lowered the risk of early severe toxicity and avoided an early toxic death. This approach should be used for safe administration of 5-FU–based treatments.

Detection of complete Dihydropyrimidine Dehydrogenase deficiency in a Tunisian family using a simple phenotypic test

Detection of complete Dihydropyrimidine Dehydrogenase deficiency in a Tunisian family using a simple phenotypic test

Dual Diagnosis of Dihydropyrimidine Dehydrogenase Deficiency and GM1 Gangliosidosis

An 8-month-old girl, born to consanguineous parents, presented with developmental delay, decreased muscle tone, disinterest in her surroundings, and sleepiness. Tests revealed a marked excretion of thymine with significantly increased uracil excretion in the urine, indicating a pyrimidine catabolic disorder, i.e., dihydropyrimidine dehydrogenase deficiency. Plasma endogenous purines confirmed elevated plasma thymine (21 μmol/L) and uracil (29 μmol/L), also consistent with dihydropyrimidine dehydrogenase deficiency. Purine mutation analysis confirmed complete dihydropyrimidine dehydrogenase deficiency with a 16 [ corrected] base pair homozygous deletion in exon 16, corresponding to DPYD c.2043-2058del. Cranial magnetic resonance imaging at 14 months indicated severe hypomyelination with gliosis. Her basal ganglia were also involved. At age 15 months, she was hospitalized for aspiration pneumonia and seizures, and also manifested hepatosplenomegaly. White cell enzymes revealed a marked deficiency of β-galactosidase activity (4 μmol/g/hour) in white cells and an elevated chitotriosidase activity (443 μmol/L/hour) in plasma indicating GM(1) gangliosidosis. Mutation analysis confirmed c.841C>T (p.His281Tyr) homozygosity for GM(1) gangliosidosis. She died at age 19 months.

Uridine triacetate for prevention of 5FU toxicity due to dihydropyrimidine dehydrogenase (DPD) deficiency.

e19560 Background: Dihydropyrimidine dehydrogenase (DPD) catalyzes degradation of 5-fluorouracil (5FU). Patients with complete or partial DPD deficiency (0.1 and 3% of the population, respectively) can experience severe or lethal toxicities to 5FU, and impaired clearance may underlie many of the 1300 US 5FU toxicity deaths each year. Uridine triacetate prevents or diminishes toxicities when orally administered after 5FU overexposure, and has been used successfully as an investigational antidote in >50 patients following accidental overdoses. Methods: A colorectal cancer patient experienced Grade 3 and 4 GI and hematologic toxicities after a 96 hour infusion of 1000 mg/m2 5FU and was found to be DPD deficient (double mutation). The patient subsequently tolerated 50 and 100 mg 5FU bolus treatments in a modified FOLFOX regimen. However, during the second cycle, this DPD-deficient patient inadvertently received 1000 mg 5FU in 1 minute. Life-threatening toxicity was expected. Treatment with uridine triacetate (10 g q6h for 20 total doses) began within 8 hours. Results: In marked contrast to his first exposure to 5FU, the patient experienced no mucositis or additional cytopenia. Preclinical and mechanistic data predicted and support this observation. In a mouse model of DPD deficiency using ethynyluracil (2 mg/kg i.p.), a standard therapeutic 5FU dose of 100 mg/kg i.p. was lethal. Treatment with oral uridine triacetate (2 g/kg t.i.d. x 5 d) starting 2, 24 or 72 hours after 5FU resulted in 100%, 80% or 70% survival, respectively. Conclusions: Timely treatment with uridine triacetate appeared to prevent severe 5FU toxicity in a DPD-deficient patient and reduced mortality in DPD-inhibited mice receiving 5FU. Uridine triacetate has been shown previously to protect patients (and mice) from 5FU toxicities following 5FU overdose. Therefore, DPD-deficient patients who have received 5FU should also benefit from uridine triacetate treatment if the overexposure is identified soon enough after 5FU dosing. Therapeutic monitoring of 5FU during or after infusions would permit rapid detection of 5FU overexposure due to DPD-deficiency or other clearance defects, enabling the use of uridine triacetate as a potential antidote.

Abstract 5451: High-throughput detection of dihydropyrimidine dehydrogenase gene (DPYD) variants using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)

Abstract Catabolism of fluoropyrimidine drugs such as 5-fluorouracil (5-FU) and its prodrug capecitabine depends on an exclusive enzymatic step, dihydropyrimidine dehydrogenase (DPD), which results in drug inactivation. DPD exhibits wide interindividual variation, with partial to complete DPD deficiency occurring in approximately 3 to 5% of the general population. Patients with less pronounced, but decreased DPD activity may also be unable to efficiently inactivate 5-FU, such that this may contribute to 5-FU toxicity. Though numerous tests have been developed to phenotypically assess the DPD enzyme, they remain labor intensive and time consuming with difficulty in defining threshold values for separating DPD deficient patients from those with normal DPD enzyme activity. To date, attempts to develop genotypic assays have been complicated by the size and complex structure of the gene encoding the DPD enzyme (DPYD). Numerous sequence variants have been reported with varying frequencies across different populations. Unfortunately many DPYD variants have not been examined to determine whether they are associated with loss of function in the DPD enzyme. Current genotypic methods are limited due to the inability to rapidly assess multiple variants in a large number of samples at one time. In the current study, we have developed a rapid, high-throughput genotypic method using 6 multiplexed PCR assays covering 115 DPYD variants analyzed with the Sequenom matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Selected variants included previously identified and functionally characterized SNPs as well as newly identified mutations within the coding region from a previous mutation discovery analysis in Coriell cell lines and patient samples. Due to the potential importance of variants in the untranslated regions (UTRs), we have included known variants from the 5′- and 3′- UTRs (9 and 16 respectively) within our plexes. A total of 95 DNA samples derived from 4 colorectal cancer cell lines, 76 Coriell cell lines (40 Caucasian and 36 African American), and 7 Caucasian patients with clinically observed 5-FU toxicity and/or decreased DPD activity were utilized for initial assay testing. To assess “calling” efficiency and presence of false positives, 8 Coriell DNA samples plated in duplicate and 1 negative control were included. Our results showed that identified variants were 100% in agreement across duplicated samples, and only four variants within the 6 different plexes were removed due to PCR/extension failure. In summary, this method provides a comprehensive and rapid analysis of 111 DPYD variants within 6 different plexes with both sensitive and accurate detection suitable for high-throughput patient screening. Functional studies in a large patient population are underway (CA116964). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2011-5451

Lethal outcome of 5‐fluorouracil infusion in a patient with a total DPD deficiency and a double DPYD and UTG1A1 gene mutation

5-Fluorouracil (5-FU)-based chemotherapy has been widely used for almost 50 years in the treatment of solid malignancies, especially in digestive cancers. Its main effect consists in the inhibition, via its active metabolite, of thymidylate synthase, a major enzyme involved in DNA synthesis. In normal conditions, less than 20% of the administered 5-FU is used in this anabolic pathway while more than 80% is catabolized via dihydropyrimidine dehydrogenase (DPD) [1]. This is the initial and rate-limiting enzyme in the catabolic pathway and therefore a partial or complete DPD deficiency is associated with different degrees of 5-FU toxicity whose most frequent manifestations include neutropenia, mucositis and diarrhoea [2].

Uracil pharmacokinetics in a DPD-deficient patient with a novel DPYD mutation compared to volunteers with normal DPD activity.

e13134 Background: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the metabolism of 5-fluorouracil (5-FU). Patients with a partial or complete DPD deficiency are at risk to develop severe toxicity after 5-FU administration. Uracil (U) is degraded in dihydrouracil (DHU) in a similar way as 5-FU. An oral uracil test dose might be useful to determine the systemic DPD activity by measuring uracil and its metabolite dihydrouracil in plasma. DPD deficiency is hypothesized to result in higher uracil levels and a reduced turnover of uracil into dihydrouracil. Methods: Uracil (500 mg/m2) was administered to 11 healthy volunteers with normal DPD activity (≥ 6 nmol/mg/hour) and 1 patient with colorectal carcinoma with a novel DPYD mutation and decreased DPD activity (4.6 nmol/mg/hour). DPD activity was measured in PBMC, as determined as described earlier. Blood samples were taken at t= 15, 30, 45, 60, 80, 100, 120, 150, 180, and 220 or 240 min after oral uracil intake. U and DHU pla...

Uridine triacetate for lethal 5-FU toxicity due to dihydropyrimidine dehydrogenase (DPD) deficiency.

e13505 Background: The enzyme dihydropyrimidine dehydrogenase (DPD) catalyzes the first step in degradation of 5-fluorouracil (5FU). Patients with complete or partial DPD deficiency (about 0.1% and 3% of the population, respectively) can experience severe or lethal toxicities after receiving standard doses of 5FU, and impaired clearance may underlie a large fraction of the ∼1,300 deaths per year due to 5FU toxicity in the US. Orally-administered uridine triacetate (vistonuridine) prevents or diminishes toxicities when administered after 5FU overexposure, and has been used successfully as an antidote in more than 28 patients to date who had received accidental overdoses of 5FU. Because DPD deficiency may alter 5FU clearance kinetics (versus 5FU overdoses in the setting of normal DPD activity), studies were conducted on reversal of 5FU toxicity in mice pretreated with the potent DPD inhibitor ethynyluracil (eniluracil; EU) to model DPD deficiency. Methods: Balb/C mice received 2 mg/kg EU i.p., followed by 1...

Dihydropyrimidine dehydrogenase deficiency presenting at birth

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is a clinically heterogeneous autosomal recessive disorder of pyrimidine metabolism. DPD is the enzyme that catalyses the first and the rate-limiting step in the catabolism of uracil, thymine and the analogue 5-fluorouracil. To date, more than 30 patients have been diagnosed with a complete enzyme deficiency. Here, we describe the fifth case with a complete DPD deficiency presenting at birth with severe neurological abnormalities. The patient was homozygous for the common splice-site mutation IVS14+1G > A.

Novel disease-causing mutations in the dihydropyrimidine dehydrogenase gene interpreted by analysis of the three-dimensional protein structure.

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria in homozygous deficient patients. Cancer patients with a partial deficiency of DPD are at risk of developing severe life-threatening toxicities after the administration of 5-fluorouracil. Thus, identification of novel disease-causing mutations is of the utmost importance to allow screening of patients at risk. In eight patients presenting with a complete DPD deficiency, a considerable variation in the clinical presentation was noted. Whereas motor retardation was observed in all patients, no patients presented with convulsive disorders. In this group of patients, nine novel mutations were identified including one deletion of two nucleotides [1039-1042delTG] and eight missense mutations. Analysis of the crystal structure of pig DPD suggested that five out of eight amino acid exchanges present in these patients with a complete DPD deficiency, Pro86Leu, Ser201Arg, Ser492Leu, Asp949Val and His978Arg, interfered directly or indirectly with cofactor binding or electron transport. Furthermore, the mutations Ile560Ser and Tyr211Cys most likely affected the structural integrity of the DPD protein. Only the effect of the Ile370Val and a previously identified Cys29Arg mutation could not be readily explained by analysis of the three-dimensional structure of the DPD enzyme, suggesting that at least the latter might be a common polymorphism. Our data demonstrate for the first time the possible consequences of missense mutations in the DPD gene on the function and stability of the DPD enzyme.

Pitfalls in the Diagnosis of Patients with a Partial Dihydropyrimidine Dehydrogenase Deficiency

Dihydropyrimidine dehydrogenase (DPD) catalyzes the degradation of thymine, uracil, and the chemotherapeutic drug 5-fluorouracil. To identify patients suffering from complete or partial DPD deficiency and to identify pitfalls that can preclude the proper diagnosis of patients with partial DPD deficiency, a sensitive and accurate assay is necessary. The activity of DPD was measured using [4-(14)C]thymine followed by separation of substrate and products with reversed-phase HPLC with on-line detection of the radioactivity. Complete baseline separation of radiolabeled thymine and all degradation products was achieved within 15 min. The detection limit for dihydrothymine was 0. 4 pmol. In lymphocytes, the DPD activity deviated from linearity at low protein concentrations (<0.2 g/L). Profoundly decreased activity of DPD was detected in the peripheral blood mononuclear cells (PBM cells) of two tumor patients when measured at low protein concentrations. Low DPD activity comparable to that observed in obligate heterozygotes was initially detected in PBM cells, containing substantial amounts of myeloid cells, from a patient suffering from 5-fluorouracil toxicity. However, after the patient experienced full clinical recovery, normal DPD activity was observed in the PBM cells. No significant differences in DPD activity were observed between exponentially growing fibroblasts and those at confluence. The range of DPD activities of obligate heterozygotes overlaps the range of DPD activities of controls. The low activity of DPD measured in PBM cells containing myeloid cells or that measured at a low protein concentration in the assay mixture is not indicative of heterozygosity for a mutant DPD allele. Although fibroblasts are suitable to establish a complete deficiency of DPD, unambiguous detection of heterozygotes is not possible.

Profound variation in dihydropyrimidine dehydrogenase activity in human blood cells: major implications for the detection of partly deficient patients.

Dihydropyrimidine dehydrogenase (DPD) is responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5FU), thereby limiting the efficacy of the therapy. To identify patients suffering from a complete or partial DPD deficiency, the activity of DPD is usually determined in peripheral blood mononuclear cells (PBM cells). In this study, we demonstrated that the highest activity of DPD was found in monocytes followed by that of lymphocytes, granulocytes and platelets, whereas no significant activity of DPD could be detected in erythrocytes. The activity of DPD in PBM cells proved to be intermediate compared with the DPD activity observed in monocytes and lymphocytes. The mean percentage of monocytes in the PBM cells obtained from cancer patients proved to be significantly higher than that observed in PBM cells obtained from healthy volunteers. Moreover, a profound positive correlation was observed between the DPD activity of PBM cells and the percentage of monocytes, thus introducing a large inter- and intrapatient variability in the activity of DPD and hindering the detection of patients with a partial DPD deficiency. © 1999 Cancer Research Campaign

Dihydropyrimidine dehydrogenase (DPD) deficiency: identification and expression of missense mutations C29R, R886H and R235W.

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is an autosomal recessive disease characterized by thymine-uraciluria in homozygous-deficient patients and associated with a variable clinical phenotype. Cancer patients with this defect should not be treated with the usual dose of 5-fluorouracil because of the expected lethal toxicity. In addition, heterozygosity for mutations in the DPD gene increases the risk of toxicity in cancer patients treated with this drug. Sequence analysis in a patient with complete DPD deficiency, previously shown to be heterozygous for the delta C1897 frame-shift mutation, revealed the presence of a novel missense mutation, R235W. Expression of this novel mutation and previously identified missense mutations C29R and R886H in Escherichia coli showed that both C29R and R235W lead to a mutant DPD protein without significant residual enzymatic activity. The R886H mutation, however, resulted in about 25% residual enzymatic activity and is unlikely to be responsible for the DPD-deficient phenotype. We show that the E. coli expression system is a valuable tool for examining DPD enzymatic variants. In addition, two new patients who were both heterozygous for the C29R mutation and the common splice donor site mutation were identified. Only one of these patients showed convulsive disorders during childhood, whereas the other showed no clinical phenotype, further illustrating the lack of correlation between genotype and phenotype in DPD deficiency.

Urinary screening for pyrimidine metabolism disorders: Reference ranges for dihydrouracil, uracil and dihydrouraci/uracil ratio

5-Fluorouracil (5-FU)–based treatments can lead to early-onset severe (4%–5%) even fatal (0.3%) toxicities in patients with dihydropyrimidine dehydrogenase (DPD) deficiency. This multicenter prospective cohort study aimed to assess the clinical benefit of pretherapeutic screening for DPD deficiency using a multiparametric approach. Two parallel cohorts of patients treated with 5-FU–based chemotherapy for colorectal carcinoma were compared in a prospective nonrandomized study. In arm A, patients had DPD deficiency screening before treatment, whereas in arm B no pretherapy screening was performed. Dosing was based on 5-FU administration guidelines of each institution. DPD deficiency screening was performed using a combined multiparametric approach (5-FUODPM Tox). The frequency of early grade 4–5 toxic events potentially induced by 5-FU was compared in the two groups. At total of 1,142 patients (n = 1,116 evaluable) were enrolled. In arm A, out of 718 evaluable patients, nine grade 4 early toxicities potentially related to 5-FU were reported in nine patients (1.2%) with no toxic death despite one complete DPD deficiency and 24 partial deficiencies. The 24 patients with partial deficiency had safe pharmacokinetics (PK)-monitored 5-FU. In arm B, among 398 evaluable patients, 17 grade 4–5 toxic early events potentially related to 5-FU were reported in 12 patients (4.2%). The incidence of early severe toxicity was significantly higher in arm B (P = .0019), confirming the positive impact of pretherapeutic DPD assessment. The percent of patients with a toxicity grade 3 or higher observed in arm A was 10.8% (n = 78) compared to 17.55% (n = 69) in arm B (P = .0497). The percentage of death was reduced from 2.5/1,000 in arm B to 0 in arm A. The time to occurrence of all grade ≥3 toxicities was determined in both arms and the difference between the two arms was significant (P = .047). Overall, one patient with complete DPD deficiency confirmed retrospectively died within 13 days from grade 5 multivisceral toxicity. Enrollment was prematurely closed after external experts’ decision. In conclusion, multiparametric pretherapeutic DPD deficiency screening significantly lowered the risk of early severe toxicity and avoided an early toxic death. This approach should be used for safe administration of 5-FU–based treatments.