Dihydropyrimidinase Research Articles

Dihydropyrimidinase enzyme deficiency and congenital isolated adrenocorticotrophin deficiency: dual genetic diagnosis in a Sri Lankan boy.

We report on a male patient who was investigated for frequent apneic episodes, feeding problems, hypotonia, and left-sided middle cerebral artery infarction in the magnetic resonance imaging at 2 weeks of age. Primary diagnosis of dihydropyrimidinase (DPYS) deficiency was suspected following the analysis of urine for organic acid; DPYS deficiency was strongly suggested by the presence of dihydrouracil, thymine, and uracil. Subsequent genetic evaluation by whole exome sequencing revealed 2 separate mutations, homozygous pathogenic variant c.1010T>C p.Leu337Pro of the DPYS gene, resulting in DPYS deficiency, and homozygous pathogenic variant c.535C>T p.Arg179* of TBX19 gene, which is associated with autosomal recessive congenital isolated adrenocorticotrophic hormone deficiency. Currently, the patient is 2 years old, and he has gross motor retardation and seizure disorder. We suggest that the clinical phenotype of the proband can be a result of mixed expression of both mutations.

Common dihydropyrimidinase ( DPYS ) genetic variations do not predict fluoropyrimidine-related chemotherapy toxicity in a Canadian cohort.

Known genetic variations in dihydropyrimidine dehydrogenase (gene name DPYD ) do not fully predict patients at risk for severe fluoropyrimidine-associated chemotherapy toxicity. Dihydropyrimidinase (gene name DPYS ), the second catabolic enzyme in fluoropyrimidine metabolism, has been noted as a potential determinant of variation in fluoropyrimidine metabolism and response. In this study, we genotyped for DPYS c.-1T>C (rs2959023), c.265-58T>C (rs2669429) and c.541C>T (rs36027551) in a Canadian cohort of 248 patients who were wild type for Clinical Pharmacogenetics Implementation Consortium recommended DPYD variants and had received a standard dose of fluoropyrimidine chemotherapy. None of our patients were found to carry the DPYS c.541C>T variant, while the minor allele frequencies were 63% and 54% for c.-1T>C and c.265-58T>C, respectively. There was no association between DPYS c.-1T>C wild type and heterozygote [odds ratio (OR) (95% confidence interval, CI) = 1.10 (0.51-2.40)] or homozygote variant carriers [OR (95% CI) = 1.22 (0.55-2.70)], or between DPYS c.265-58T>C wild-type patients and heterozygote [OR (95% CI) = 0.93 (0.48-1.80)] or homozygote variant carriers [OR (95% CI) = 0.76 (0.37-1.55)] in terms of fluoropyrimidine-associated toxicity. Therefore, in our cohort of mostly Caucasian Canadians, genetic variations in DPYS do not appear to be a significant contributor to severe fluoropyrimidine-associated toxicity.

New Insights into rice pyrimidine catabolic enzymes.

Rice is a primary global food source, and its production is affected by abiotic stress, caused by climate change and other factors. Recently, the pyrimidine reductive catabolic pathway, catalyzed by dihydropyrimidine dehydrogenase (DHPD), dihydropyrimidinase (DHP) and β-ureidopropionase (β-UP), has emerged as a potential participant in the abiotic stress response of rice. The rice enzymes were produced as recombinant proteins, and two were kinetically characterized. Rice dihydroorotate dehydrogenase (DHODH), an enzyme of pyrimidine biosynthesis often confused with DHPD, was also characterized. Salt-sensitive and salt-resistant rice seedlings were subjected to salt stress (24 h) and metabolites in leaves were determined by mass spectrometry. The OsDHPD sequence was homologous to the C-terminal half of mammalian DHPD, conserving FMN and uracil binding sites, but lacked sites for Fe/S clusters, FAD, and NADPH. OsDHPD, truncated to eliminate the chloroplast targeting peptide, was soluble, but inactive. Database searches for polypeptides homologous to the N-terminal half of mammalian DHPD, that could act as co-reductants, were unsuccessful. OsDHODH exhibited kinetic parameters similar to those of other plant DHODHs. OsDHP, truncated to remove a signal sequence, exhibited a kcat/Km = 3.6 x 103 s-1M-1. Osb-UP exhibited a kcat/Km = 1.8 x 104 s-1M-1. Short-term salt exposure caused insignificant differences in the levels of the ureide intermediates dihydrouracil and ureidopropionate in leaves of salt-sensitive and salt-resistant plants. Allantoin, a ureide metabolite of purine catabolism, was found to be significantly higher in the resistant cultivar compared to one of the sensitive cultivars. OsDHP, the first plant enzyme to be characterized, showed low kinetic efficiency, but its activity may have been affected by truncation. Osb-UP exhibited kinetic parameters in the range of enzymes of secondary metabolism. Levels of two pathway metabolites were similar in sensitive and resistant cultivars and appeared to be unaffected by short-term salt exposure."

Structural investigation of pathogenic variants in dihydropyrimidinase using molecular dynamics simulations

Dihydropyrimidinase (DHP) is an enzyme that catabolizes the degradation of pyrimidine and fluoropyrimidine drugs such as 5-fluorouracil. DHP deficiency triggers various clinical symptoms and increases the risk of fluoropyrimidine drug toxicity. Various pathogenic variants of DHP cause DHP deficiency, and their catalytic activities have been well studied. However, the three-dimensional structures of DHP variants have not been clarified. In this study, we investigated the effects of mutations on DHP structures using the molecular dynamics simulations. Simulations of the wild type and 10 variants were performed and compared. In the T68R, D81G, G278D, and L337P variants, the flexibilities of structures related to the interaction for oligomer formation increased in comparison with those of the wild type. W117R, T343A, and R412 M mutations affected the structures of stereochemistry gate loops or the substrate-binding pocket. The three-dimensional structures of W360R and G435R variants were suggested to collapse. On the other hand, only slight structural changes were observed in the R181W variant, whose experimentally observed activity was similar to that of the wild type. The computational results are expected to clarify the relationship between clinical mutations and structural effects of drug-metabolizing enzymes.

In Vitro Assessment of Fluoropyrimidine-Metabolizing Enzymes: Dihydropyrimidine Dehydrogenase, Dihydropyrimidinase, and β-Ureidopropionase.

Fluoropyrimidine drugs (FPs), including 5-fluorouracil, tegafur, capecitabine, and doxifluridine, are among the most widely used anticancer agents in the treatment of solid tumors. However, severe toxicity occurs in approximately 30% of patients following FP administration, emphasizing the importance of predicting the risk of acute toxicity before treatment. Three metabolic enzymes, dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP), and β-ureidopropionase (β-UP), degrade FPs; hence, deficiencies in these enzymes, arising from genetic polymorphisms, are involved in severe FP-related toxicity, although the effect of these polymorphisms on in vivo enzymatic activity has not been clarified. Furthermore, the clinical usefulness of current methods for predicting in vivo activity, such as pyrimidine concentrations in blood or urine, is unknown. In vitro tests have been established as advantageous for predicting the in vivo activity of enzyme variants. This is due to several studies that evaluated FP activities after enzyme metabolism using transient expression systems in Escherichia coli or mammalian cells; however, there are no comparative reports of these results. Thus, in this review, we summarized the results of in vitro analyses involving DPD, DHP, and β-UP in an attempt to encourage further comparative studies using these drug types and to aid in the elucidation of their underlying mechanisms.

Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites.

Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA–protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.

Characterization of the Pyrimidine Catabolic Pathway of Oryza sativa

ObjectiveTo characterize plant pyrimidine catabolic enzymes and to identify potential candidate for additional enzymes participating in the pathwayRice is a primary global food source and is an important crop in Colombia. Its production is affected by abiotic stress, caused by climate change and other factors. Recently, genes of the pyrimidine catabolic pathway have emerged as potential participants in the abiotic stress response of plants.In mammals, three enzymes are involved in the degradation of uracil or thymine. The first is dihydropyrimidine dehydrogenase (DHPD), the second is dihydropyrimidinase (DHP) and the last is β‐ureidopropionase (β‐UP). Liu and coworkers (2009) found that overexpression of DHPD in rice plants (OsDHPD) results in tolerance to salt and drought stress, however, in that study OsDHPD was misidentified as a type I dihydroorotate dehydrogenase. DHP appears to be involved in cold stress‐response in grass (Xuan et al., 2013). In addition, the overexpression of β‐UP in transgenic tomatoes plants made them resistant to heat during anthesis (Shen et al., 2014). The role of pyrimidine catabolism in stress tolerance could be related to this pathway's production of precursors for proline biosynthesis. In plants, a complete characterization of the pathway is lacking. Comparison of plant and mammalian DHPD sequences showed that the plant protein lacks a domain needed for binding NADH(P), FAD, and Fe/S clusters, suggesting that another, unidentified, partner protein should be interacting with OsDHPD.ResultsWe cloned and expressed three enzymes of the pathway, OsDHPD, OsDHP and Osβ‐UP. The third enzyme, Osβ‐UP, was soluble, and preliminary kinetics revealed a Km 0.85 ± 0.11 mM for 3‐ureidopropionic acid, and a specific activity apparently 10‐fold faster (Vmax 384.3 ± 12.0 nmol/min/mg) than the Arabidopsis thaliana recombinant enzyme (30 nmol/min/mg) (Walsh et al., 2001). Truncations lacking chloroplast localization signals were required to produce soluble recombinant proteins of the first two enzymes. Truncated OsDHPD, was soluble, but inactive, presumably because it requires a partner protein for catalysis. By screening the rice database for proteins with the appropriate combination of cofactors we identified two potential candidates that may act as partners of OsDHPD; the first was a salt‐induced chloroplast ferredoxin, and the second was a rice chloroplast “fruit protein”. Both were cloned and expressed, and testing of interactions with OsDHPD on native gels are in progress. In addition, we produced polyclonal antibodies to OsDHPD to permit pull‐down assays of chloroplast extracts.ConclusionsThree rice pyrimidine catabolic enzymes were cloned, expressed and purified. Potential candidates for the missing protein in the pathway were identified using bioinformatics tools, and antibodies to OsDHPD were generated. Together these approaches may allow identification of the missing participant in pyrimidine catabolism in plants.Support or Funding InformationThe research was supported by funding from Colciencias project 120471250531, and from the Facultad de Ciencias and the Vicerrectoria de Investigaciones (Universidad de los Andes, Colombia).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dihydropyrimidinase deficiency in four East Asian patients due to novel and rare DPYS mutations affecting protein structural integrity and catalytic activity.

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyzes the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 31 genetically confirmed patients with a DHP deficiency have been reported and the clinical, biochemical and genetic spectrum of DHP deficient patients is, therefore, still largely unknown. Here, we show that 4 newly identified DHP deficient patients presented with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in urine and a highly variable clinical presentation, ranging from asymptomatic to infantile spasm and reduced white matter and brain atrophy. Analysis of the DHP gene (DPYS) showed the presence of 8 variants including 4 novel/rare missense variants and one novel deletion. Functional analysis of recombinantly expressed DHP mutants carrying the p.M250I, p.H295R, p.Q334R, p.T418I and the p.R490H variant showed residual DHP activities of 2.0%, 9.8%, 9.7%, 64% and 0.3%, respectively. The crystal structure of human DHP indicated that all point mutations were likely to cause rearrangements of loops shaping the active site, primarily affecting substrate binding and stability of the enzyme. The observation that the identified mutations were more prevalent in East Asians and the Japanese population indicates that DHP deficiency may be more common than anticipated in these ethnic groups.

From sequence and structure of sulfotransferases and dihydropyrimidinases to an understanding of their mechanisms of action and function

Importance of the field: Most enzymes that catalyze physiologically important reactions are known to be highly efficient and specific to their particular substrates. On the contrary, the enzymes of detoxification are known to catalyze unlimited number of substrates containing similar function groups that may have significant structural variation. This review explores nature's strategy to design enzymes with special properties.Areas covered in this review: We review articles from 1981 to 2009, with special focus on the relationships of sequence, structure and function of cytosolic sulfotransferases and dihydropyrimidinase (DHP).What the reader will gain: Specific amino acids responsible for substrate inhibition, substrate binding orientations, substrate specificity, quaternary structures and inactivation of sulfotransferases and DHP related enzymes are elucidated. Susceptibility to some diseases possibly resulted from the mutation of a single amino acid that causes dysfunction of these enzymes. Terminal deletion of amino acid that may affect surface interaction, subunit dissociation, stability alteration and then cause the syndrome of DHP deficiency is discussed.Take home message: Based on the multiple sequence/structure analysis and with sufficient information from other members of the same enzyme families, the origin and mechanism of specific enzyme actions and proteins assembly can be clarified and predicted.

Dihydropyrimidinase deficiency: Phenotype, genotype and structural consequences in 17 patients

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 11 individuals have been reported suffering from a complete DHP deficiency. Here, we report on the clinical, biochemical and molecular findings of 17 newly identified DHP deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological and gastrointestinal abnormalities and markedly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. Analysis of DPYS, encoding DHP, showed nine missense mutations, two nonsense mutations, two deletions and one splice-site mutation. Seventy-one percent of the mutations were located at exons 5–8, representing 41% of the coding sequence. Heterologous expression of 11 mutant enzymes in Escherichia coli showed that all but two missense mutations yielded mutant DHP proteins without significant activity. Only DHP enzymes containing the mutations p.R302Q and p.T343A possessed a residual activity of 3.9% and 49%, respectively. The crystal structure of human DHP indicated that the point mutations p.R490C, p.R302Q and p.V364M affect the oligomerization of the enzyme. In contrast, p.M70T, p.D81G, p.L337P and p.T343A affect regions near the di-zinc centre and the substrate binding site. The p.S379R and p.L7V mutations were likely to cause structural destabilization and protein misfolding. Four mutations were identified in multiple unrelated DHP patients, indicating that DHP deficiency may be more common than anticipated.

Contribution of dihydropyrimidinase gene alterations to the development of serious toxicity in fluoropyrimidine-treated cancer patients

Decreased 5-fluorouracil catabolism has been considered a major factor contributing to fluoropyrimidine (FP)-related toxicity. Alterations in the dihydropyrimidine dehydrogenase gene coding for the first and rate-limiting enzyme of FP catabolic pathway could explain toxicity in only a limited proportion of FP-treated patients. The importance of gene variants in dihydropyrimidinase (DPYS) coding for subsequent catabolic enzyme of FP degradation is not fully understood. We performed genotyping of DPYS based on denaturing high-performance liquid chromatography in 113 cancer patients including 67 with severe FP-related toxicity and 46 without toxicity excellently tolerating FPs treatment. We detected nine DPYS variants including four located in non-coding sequence (c.-1T>C, IVS1+34C>G, IVS1-58T>C, and novel IVS4+11G>T), four silent (c.15G>A, c.216C>T, and novel c.105C>T and c.324C>A), and one novel missense variant c.1441C>T (p.R481W). All novel alterations were detected once only in patients without toxicity. The c.-1T>C and IVS1-58T>C variants were found to modify the risk of toxicity. The CC carriers of the c.-1C alleles were at higher risk of mucositis (OR = 4.13; 95% CI = 1.51-11.31; P = 0.006) and gastrointestinal toxicity (OR = 3.54; 95% CI = 1.59-7.88; P = 0.002), whereas the presence of the IVS1-58C allele decreased the risk of gastrointestinal toxicity (OR = 0.4; 95% CI = 0.17-0.93; P = 0.03) and leucopenia (OR = 0.29; 95% CI = 0.08-1.01; P = 0.05). Our results indicate that missense and nonsense variants in DPYS are infrequent, however, the development of serious primarily gastrointestinal toxicity could be influenced by non-coding DPYS sequence variants c.-1T>C and IVS1-58T>C.

Children's toxicology from bench to bed - Liver Injury (1): Drug-induced metabolic disturbance - Toxicity of 5-FU for pyrimidine metabolic disorders and pivalic acid for carnitine metabolism -

Congenital disorders of metabolism show a wide spectrum of symptoms as a consequence of impairment of a certain metabolic pathway by mutated enzymes resulting in abnormal accumulation of enzyme substrates, deficiency of expected products, and abnormal burden to collateral metabolic pathways, etc. However, in some occasions, depending on which pathway up to what degree of disturbance, it can be asymptomatic until a certain kind of burden is placed on to the patient. Enzyme deficiency involved in pyrimidine degradation, such as Dihydropyrimidine dehydrogenase (DPD) and Dihydropyrimidinase (DHP), has been reported with convulsion or autism as symptoms, but many asymptomatic cases are also reported. However, when the patients are treated with 5-fluorouracil, a pyrimidine analogue anticancer drug, lethal side-effects can be seen even in asymptomatic patients. Some oral cephem antibiotics have pivalic acid side chain to increase absorption rate at intestine. These antibiotics degrade into active antibiotics and pivalic acid at the intestinal wall. This pivalic acid is carnitine-conjugated and excreted into urine. Carnitine acts as a carrier of long chain fatty acid to mitochondria and to beta-oxidation, thus an important molecule for energy production by beta-oxidation and maintenance of mitochondrial function. Because of this, long term administration of such antibiotics could induce depletion of carnitine from the body and lead to low ketotic hypoglycemia, convulsion and consciousness disturbance. This paper reports some possible serious side effects closely linked to drug metabolism.

ß-Alanine and ß-Aminoisobutyric Acid Levels in Two Siblings With Dihydropyrimidinase Deficiency

Dihydropyrimidinase (DHP) deficiency is an inborn error of the pyrimidine degradation pathway, affecting the hydrolytic ring opening of the dihydropyrimidines. In two siblings with a complete DHP deficiency and a variable clinical presentation, a normal concentration of ß-alanine and strongly decreased levels of ß-aminoisobutyric acid were observed in plasma, urine and CSF. No major differences were observed for the concentrations of the ß-amino acids in plasma and urine between the symptomatic and asymptomatic sibling. Thus, the relevance of the shortage of ß-aminoisobutyric acid for the onset of a clinical phenotype in patients with DHP deficiency remains to be established.

Genetic regulation of β-ureidopropionase and its possible implication in altered uracil catabolism

Approximately 30-40% of grade III-IV toxicity to 5-FU has been associated with partial or profound deficiency in dihydropyrimidine dehydrogenase (DPD), the first of three enzymes in the catabolic pathway of fluoropyrimidines. There remains, however, a subset of patients presenting with 5-FU-associated toxicity despite normal DPD activity, suggesting possible deficiencies in enzymes downstream of DPD: dihydropyrimidinase (DHP), encoded by the DPYS gene, and/or beta-ureidopropionase (BUP-1), encoded by the UPB1 gene. Previously, we reported the identification of inactivating mutations in the DPYS gene that could potentially alter the uracil catabolic pathway in healthy individuals with normal DPD enzyme activity. This study investigates the possible role of UPB1 genetic variations in the regulation of the uracil catabolic pathway in individuals presenting with a deficient uracil breath test (13C-UraBT) despite normal DPD enzyme activity. This study included 219 healthy asymptomatic volunteers with known DPD enzyme activity and [2-(13)C]-uracil breath test (UraBT). All samples were genotyped for sequence variations in the UPB1 gene using denaturing high performance liquid chromatography (DHPLC) and Surveyor enzyme digestion with confirmation of detected sequence variants by direct sequencing. Seven novel and six previously reported sequence variations were identified, including one nonconservative mutation, which demonstrated 97.3% reduction in BUP-1 activity when expressed in the RKO cell line. Data presented in this study demonstrate that alterations of uracil catabolism are not limited to DPD and/or DHP deficiency and that inactivating mutations in the UPB1 gene might impair uracil catabolism.

Genetic regulation of dihydropyrimidinase and its possible implication in altered uracil catabolism

Dihydropyrimidine dehydrogenase (DPD) deficiency accounts for approximately 43% of grade 3-4 toxicity to 5-fluorouracil. There, however, remain a number of patients presenting with 5-fluorouracil-associated toxicity despite normal DPD enzyme activity, suggesting possible deficiencies in dihydropyrimidinase (DHP), encoded by the DPYS gene, and/or beta-ureidopropionase (BUP-1), encoded by the UPB1 gene. This study investigates the role of DPYS sequence variations in individuals with unexplained molecular basis of altered uracil catabolism. This study included 219 asymptomatic healthy volunteers with known DPD enzyme activity and [2-13C]-uracil breath test (UraBT) profiles. All samples were genotyped for sequence variations in the DPYS gene using denaturing high-performance liquid chromatography (DHPLC) and Surveyor enzyme digestion with confirmation by direct sequencing. Site-directed mutagenesis and expression analysis were performed to determine the effect of the identified nonconservative mutations on DHP enzyme activity. Seven previously reported and 11 novel sequence variations were identified, including three nonconservative mutations; two of which (L7V and 1635delC) demonstrated decreased DHP activity when expressed in the RKO cell line (P=0.25). The P values were not significant due to the small sample size (n=3); however, a modified [2-13C]-uracil breath test, the 13C-dihydrouracil breath test, was administered to four volunteers to confirm that the 1635delC mutation does in fact reduce in-vivo DHP activity. Data presented in this study demonstrate that alterations of uracil catabolism are not limited to DPD deficiency, and that inactivating mutations in DHP might impair uracil catabolism in cases of normal DPD activity.

Clinical, biochemical and genetic findings in two siblings with a dihydropyrimidinase deficiency

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and it catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine to N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, respectively. To date, only nine individuals have been reported suffering from a complete DHP deficiency. We report two siblings presenting with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. One of the siblings had a severe delay in speech development and white matter abnormalities, whereas the other one was free of symptoms. Analysis of the DHP gene ( DPYS) showed that both patients were compound heterozygous for the missense mutation 1078T > C (W360R) in exon 6 and a novel missense mutation 1235G > T (R412M) in exon 7. Heterologous expression of the mutant enzymes in Escherichia coli showed that both missense mutations resulted in a mutant DHP enzyme without residual activity. Analysis of the crystal structure of eukaryotic DHP from the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum suggests that the W360R and R412M mutations lead to structural instability of the enzyme which could potentially impair the assembly of the tetramer.

Molecular basis of altered uracil catabolism in individuals with 5-FU toxicity and normal DPD enzyme activity

3070 Background: Dihydropyrimidine dehydrogenase (DPD) deficiency accounts for approximately 43% of grade 3–4 toxicity to 5-Fluorouracil (5-FU). However, a significant number of patients with normal DPD enzyme activity remain with unexplained molecular basis of 5-FU toxicity. It has been suggested by the few cases previously reported that deficiency of dihydropyrimidinase (DHP)enzyme encoded by the DPYS gene and/or beta-ureidopropionase enzyme, encoded by the BUP-1 gene, could also be implicated in 5-FU toxicity. Methods: This study included 40 volunteers with known 13C-UraBT and DPD enzyme activity, 25 cancer patients with 5-FU toxicity despite normal DPD enzyme activity, and 25 liver biopsies from cancer patients with different grades of toxicity. All samples were analyzed for molecular defects in the DPYS and BUP-1 genes, using DHPLC and RT-PCR techniques. Results: Molecular analysis of the DPYS gene revealed the presence of two non-conservative amino acid changes, one frame-shift mutation that leads to a stop codon and premature termination of the DHP protein, five silent mutations, nine intronic sequence variations, two sequence variations in the 5’UTR and one in the non-coding region of exon 10. Molecular analysis of the BUP-1 gene revealed the presence of two non-conservative amino acid changes, one of which (314C>A: A85E) has already been reported to abolish enzyme activity; six silent mutations, four intronic sequence variations, one polymorphism in the 5’upstream sequence, and one sequence variation in each of the 5’UTR and the 3’UTR. Conclusions: The molecular basis of 5-FU toxicity is not limited to DPD deficiency; since molecular defects in genes downstream of DPD can potentially also impair 5-FU catabolism. Genetic testing for molecular defects in DPYS and BUP-1 may predict patients at risk of developing 5-FU toxicity despite having normal DPD enzyme activity. Assessing the integrity of the entire uracil catabolic pathway might be crucial to avoid toxicity in a significant group of patients receiving 5-FU or a related drug (CA62164). No significant financial relationships to disclose.

Activity of Pyrimidine Degradation Enzymes in Normal Tissues

In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and ß-ureidopropionase (ß-UP), using radiolabeled substrates, in 16 different tissues obtained at autopsy from a single patient. The activity of DPD could be detected in all tissues examined, with the highest activity being present in spleen and liver. Surprisingly, the highest activity of DHP was present in kidney followed by that of liver. Furthermore, a low DHP activity could also be detected in 8 other tissues. The highest activity of ß-UP was detected in liver and kidney. However, low UP activities were also present in 8 other tissues. Our results demonstrated that the entire pyrimidine catabolic pathway was predominantly confined to the liver and kidney. However, significant residual activities of DPD, DHP and ß-UP were also present in a variety of other tissues, especially in bronchus.

Analysis of Pyrimidine Catabolism inDrosophila melanogasterUsing Epistatic Interactions With Mutations of Pyrimidine Biosynthesis and β-Alanine Metabolism

The biochemical pathway for pyrimidine catabolism links the pathways for pyrimidine biosynthesis and salvage with beta-alanine metabolism, providing an array of epistatic interactions with which to analyze mutations of these pathways. Loss-of-function mutations have been identified and characterized for each of the enzymes for pyrimidine catabolism: dihydropyrimidine dehydrogenase (DPD), su(r) mutants; dihydropyrimidinase (DHP), CRMP mutants; beta-alanine synthase (betaAS), pyd3 mutants. For all three genes, mutants are viable and fertile and manifest no obvious phenotypes, aside from a variety of epistatic interactions. Mutations of all three genes disrupt suppression by the rudimentary gain-of-function mutation (r(Su(b))) of the dark cuticle phenotype of black mutants in which beta-alanine pools are diminished; these results confirm that pyrimidines are the major source of beta-alanine in cuticle pigmentation. The truncated wing phenotype of rudimentary mutants is suppressed completely by su(r) mutations and partially by CRMP mutations; however, no suppression is exhibited by pyd3 mutations. Similarly, su(r) mutants are hypersensitive to dietary 5-fluorouracil, CRMP mutants are less sensitive, and pyd3 mutants exhibit wild-type sensitivity. These results are discussed in the context of similar consequences of 5-fluoropyrimidine toxicity and pyrimidine catabolism mutations in humans.

Rapid gas chromatographic–mass spectrometric diagnosis of dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency

A rapid yet reliable chemical diagnosis for dihydropyrimidine dehydrogenase (DHPD) deficiency, and possibly dihydropyrimidinase (DHP) deficiency in cancer patients, prior to therapy with pyrimidine analogues such as 5-fluorouracil, is desired for prevention of severe side-effects by these drugs. We have reported the basic separation and quantitation technology for pyrimidine metabolites using gas chromatography–mass spectrometry. A proposal to use the number ( n) of standard deviations (SD) above the normal mean, as the index of the excessive urinary excretion of the metabolites appears not to be commonly used. When used, the values were too small, such as two or three, even in genetic disorders. Here, we applied the method to 11 urine specimens from proven cases including two DHP carriers and proved how specific the method is, because “ n”-values were markedly large for thymine (T), uracil (U) and/or dihydrothymine (DHT) and dihydrouracil (DHU). In three cases with DHPD deficiency, two were siblings, one with symptoms and the other without, n was 12 for T and 5.9 for U, and 5-hydroxymethyluracil was distinctly detected. These values indicate that the nature of genetic mutation relates closely to the degree of metabolite accumulation in pyrimidine disorders. In six patients with DHP deficiency, n was 8.4–12 for DHT and 7.2–11 for DHU. Many mutations are known for both genes and the assay of residual enzyme activity may be time-consuming or invasive especially for those with DHP deficiency. Thus, this noninvasive yet comprehensive urinalysis has great value for those without a family history, as the first trial, before DNA or the enzyme assay. Our findings again raise the question whether the metabolic block really causes the symptoms found in pyrimidine disorders.