This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Kuilenburg, A. B. P. Articles by van Gennip, A. H. Search for Related Content PubMed PubMed Citation Articles by van Kuilenburg, A. B. P. Articles by van Gennip, A. H.

Dihydropyrimidinase Deficiency and Severe 5-Fluorouracil Toxicity

Academic Medical Center, University of Amsterdam, Emma Children’s Hospital, and Departments of Clinical Chemistry and Neurogenetics, 1100 DE Amsterdam, the Netherlands [A. B. P. v. K., R. M., L. Z., F. B., A. H. v. G.]; University Medical Center Utrecht, Department of Medical Oncology, 3508 GA Utrecht, the Netherlands [B. A. Z.]; and Kobe Gakuin University Igawadani-cho, Kobe, Japan [K. M., N. T.]

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Dihydropyrimidinase (DHP) is the second enzyme in the catabolism of 5-fluorouracil (5FU), and it has been suggested that patients with a deficiency of this enzyme are at risk from developing severe 5FU-associated toxicity. In this study, we demonstrated for the first time that in one patient the severe toxicity, after a treatment with 5FU, was attributable to a partial deficiency of DHP. Analysis of the DHP gene showed that the patient was heterozygous for the missense mutation 833G>A (G278D) in exon 5. Heterologous expression of the mutant enzyme in Escherichia coli showed that the G278D mutation leads to a mutant DHP enzyme without residual activity. An analysis for the presence of this mutation in 96 unrelated Dutch Caucasians indicates that the allele frequency in the normal population is <0.5%. Our results show that a partial DHP deficiency is a novel pharmacogenetic disorder associated with severe 5FU toxicity.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
5FU² is one of the most commonly used chemotherapeutic agents for the systemic treatment of cancers of the gastrointestinal tract, breast, head, and neck. Recent advances in our understanding of the metabolism of 5FU, and the key enzymes involved in the activation and degradation of 5FU have led to an increased awareness that the catabolic route of 5FU plays an important role in the determination of toxicity as well as the efficacy toward 5FU (1, 2, 3) . It has been reported that >80% of the administered 5FU is catabolized by three consecutive enzymes of the pyrimidine degradation pathway (4) .

DPD catalyzes the conversion of 5FU to FUH₂, which is the initial and rate-limiting step in this catabolism (Fig. 1) . FUH₂ can be additionally degraded to fluoro-ß-ureidopropionate and subsequently to fluoro-ß-alanine by DHP and ß-ureidopropionase, respectively. The pivotal role of DPD in chemotherapy using 5FU has been shown in cancer patients with a complete or partial deficiency of this enzyme. These patients suffered from severe toxicity, including death, after the administration of 5FU (5, 6, 7, 8) . A number of these patients proved to be heterozygous or homozygous for a mutant DPYD allele (5, 6, 7, 8, 9) .

View larger version (11K): [in this window] [in a new window]   Fig. 1. The catabolic route of 5FU. DPD catalyses the conversion of 5FU to FUH2. FUH2 can be additionally degraded to fluoro-ß-ureidopropionate (FUPA) and subsequently to fluoro-ß-alanine (FBAL) by DHP and ß-ureidopropionase, respectively.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Analysis of Dihydropyrimidines.
The concentrations of dihydrouracil and dihydrothymine in plasma were determined using reversed-phase HPLC combined with electrospray tandem mass spectrometry (13) .

Determination of the DPD and DHP Activity.
The activity of DPD in peripheral blood mononuclear cells was determined using a radiochemical assay with subsequent separation of radiolabelled thymine from radiolabelled dihydrothymine with reversed-phase HPLC (14) . The activity of DHP was determined in an assay mixture containing 100 mM Tris-HCl (pH 8.0), 1 mM DTT, and 500 µM [2-¹⁴C]-dihydrouracil (1.85–2.22 GBq/mmol; Moravek Biochemicals, Brea, CA). Separation of radiolabelled dihydrouracil from N-carbamyl-ß-alanine was performed isocratically [50 mM NaH₂PO₄ (pH 4.5) at a flow rate of 1 ml/min] by reversed-phase HPLC on a Supelcosil LC-18-S column (250 x 4.6 mm; 5 µm particle size) with on-line detection of radioactivity (15) . Protein concentrations were determined with a copper-reduction method using bicinchoninic acid, essentially as described by Smith et al. (16) .

PCR Amplification of Coding Exons.
DNA was isolated from granulocytes by standard procedures. The PCR amplification of all nine of the coding exons and flanking intronic regions was carried out using the primer sets as described previously (17) . However, for exon 5, the sequence of the reverse primer was 5'-GGATCCAGATGGGAGGAC-3'. Forward primers had a 5'-TGTAAAACGACGGCCAGT-3' extension, whereas reverse primers had an 5'-CAGGAAACAGCTATGACC-3' extension at their 5'-ends. These sequences were complementary to the labeled -21M13 and M13 reversed primers used in the dye-primer sequence reaction. Amplification of all of the exons was carried out in 50-µl reaction mixtures containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5–2.5 mM MgCl₂, 10 pmol of each primer, 200 µM of each dNTP, and 2 units of Taq polymerase (Promega Benelux B.V., Leiden, the Netherlands). After initial denaturation for 5 min at 96°C, amplification was carried out for 35 cycles (0.25 min 96°C, 0.5 min 60°C, and 0.5 min 72°C). PCR products were separated on 1% agarose gels, visualized with ethidium bromide, and purified using a Qiaquick Gel Extraction kit (Qiagen, Hilden, Germany) or used for direct sequencing.

Expression of DHP in E. coli.
An expression plasmid containing the wild-type human DHP cDNA (pSE420-DHP) was constructed by subcloning the EcoRI-BamHI insert of a plasmid, containing the complete coding region of the cDNA encoding human DHP, into the pSE420 vector (18) . The 833G>A mutation (G278D) was introduced into the human DHP sequence using the megaprimer technique and subsequently subcloned in a pSE420 vector (pSE420-DHP-G278D). The resulting clones were sequenced completely to verify the presence of the mutation and to exclude the presence of random mutations introduced by PCR artifacts. Expression plasmids were introduced into the E. coli strain BL21. A 5-ml Luria-Bertani broth culture, supplemented with 50 µg/ml ampicillin, was inoculated with 100 µl of an overnight preculture grown in Luria-Bertani broth. Cells were grown for 3 h at 37°C, and induction was performed by the addition of isopropyl-ß-D-thiogalactoside to a final concentration of 1 mM. Cells were sedimented after 3 h, washed with an isolation buffer [35 mM potassium phosphate (pH 7.4) and complete; Roche Diagnostics, Almere, the Netherlands] and resuspended in 500 µl of isolation buffer. The cell suspension was frozen for 16 h at -20°C, thawed on ice, and lysed by sonication. The crude lysate was centrifuged at 18,000 x g for 15 min, and the resulting supernatant was stored at -80°C.

Western Blot Analysis.
Cell extracts (2.5 µg) were fractionated on a 7.5% (w/v) SDS-polyacrylamide gel and transferred to a nitrocellulose filter. Blocking of the membrane was performed for 16 h with TBS [25 mM Tris, 137 mM NaCl, and 2.7 mM KCl (pH 7.4)] containing 5% (w/v) nonfat dry milk. Subsequently, the membrane was incubated for 1 h with a 1:1000 dilution of rabbit antirat DHP polyclonal antibody in TBS, supplemented with 0.05% (v/v) Tween 20. The membranes were washed three times (5 min each) with TBS containing 0.05% (v/v) Tween 20 and incubated for 45 min with TBS containing 0.05% (v/v) Tween 20, 5% (w/v) nonfat dry milk, and a 1:5000 dilution of a pig antirabbit secondary antibody conjugated to horseradish peroxidase (Dako, Copenhagen, Denmark). After rinsing the membrane three times (5 min each) with TBS containing 0.05% Tween 20, detection of DHP was performed with enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom).

DHPLC Analysis of the 833G>A Mutation.
PCR fragments containing exon 5 were analyzed on a Agilent 1100-DHPLC system (Agilent Technologies Netherlands B.V., Amstelveen, the Netherlands). The buffer system for the DHPLC consisted of buffer A [0.1 M triethylammonium acetate and 1 mM EDTA (pH 7.0)] and buffer B [buffer A supplemented with 25% (v/v) acetonitrile]. Before injection, PCR fragments were denatured at 95°C followed by slow renaturation. PCR fragments (5 µl) were loaded in 40% buffer B on a Zorbax ds DNA column (Agilent Technologies Netherlands B.V.) and subsequently eluted, within 6 min at 65°C, with a gradient from 55–65% buffer B.

Sequence Analysis.
Sequence analysis of genomic fragments amplified by PCR and expression plasmids was carried out on an Applied Biosystems model 3100 automated DNA sequencer using the dye-terminator and the dye-primer method (Perkin-Elmer Corp., Foster City, CA), respectively.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Clinical Evaluation.
The patient was female, born in 1943, and had a history of an appendectomy, middle ear surgery, bilateral removal of cervical ribs, multiple cysts, and abscesses in the vagina and a hysterectomy. In addition, she suffered from obesity and diabetes mellitus. In 1991, the patient underwent a mastectomy for a ductal carcinoma (pT₂N₁M₀) combined with five courses of CMF chemotherapy (60 mg methotrexate i.v., 900 mg 5FU i.v. on days 1 and 8, and p.o. 150 mg cyclophosphamide from days 1 to 14). The chemotherapy with CMF caused considerable mucosal toxicity and, therefore, the planned sixth course with CMF was cancelled. In 1994, the patient developed skeletal metastasis for which she was treated successively with tamoxifen, aminogluthetimide, and Re-186 etidronate. In 1997, lung metastasis was observed, and therapy was changed to chemotherapy with doxorubicine and cyclophosphamide. In April 1998, because of progression, she received radiotherapy and docetaxel. Because of disease progression, palliative treatment commenced with continuous infusion of 5FU (300 mg/m²/day). During the first week of treatment she noticed increased pain while swallowing, and after 14 days, a skin rash was observed that developed to skin ulcers on the thorax, palms of the hands, and soles of both feet. Her condition worsened, and she became confused and was hospitalized with leucopenic fever and sepsis. At this stage, treatment with 5FU was aborted. Antibiotic treatment with floxapen and gentamicin was administered and she recovered. However, after 2 weeks, her mental state worsened again and brain metastases were suspected. The patient declined additional diagnostic procedures and returned home for terminal care. She died 5 months later.

DPD Activity and Dihydropyrimidines in Plasma.
A normal activity of DPD was detected in peripheral blood mononuclear cells of the patient (13.1 nmol/mg/h; controls 9.9 ± 2.8; n = 54), thus excluding a (partial) deficiency of DPD as the underlying cause of the severe toxicity, after the administration of 5FU. To investigate whether the severe toxicity might have been caused by a complete deficiency of DHP, plasma was collected to determine the levels of 5,6-dihydrouracil and 5,6-dihydrothymine. A normal concentration of 5,6-dihydrouracil (3.9 µM; controls 2.2 ± 3.1 µM; n = 97) and a high-normal concentration of 5,6-dihydrothymine (1.44 µM; controls 0.7 ± 0.4 µM; n = 97) were detected in plasma. The concentration of 5,6-dihydrothymine was at the upper limit of the 95% distribution range of the control population.

Sequence Analysis of the DHP Gene.
Analysis of the genomic sequences of exons 1–9 of the DHP gene showed that the tumor patient was heterozygous for a missense mutation 833G>A in exon 5, leading to the amino acid substitution G278D (Fig. 2) . In addition, the patient proved to be homozygous for a -1T>C mutation and heterozygous for a silent mutation 216C>T (F72F) in exon 1. No missense mutations could be detected in the DHP gene of 22 other patients, with normal DPD activity, but who suffered nevertheless from severe 5FU associated-toxicity.

View larger version (79K): [in this window] [in a new window]   Fig. 2. Genomic organization and conservation of amino acid sequences of the DHP gene. The DHP gene consists of 10 exons encoding an open reading frame of 1560 bp (top panel). The different mutations identified in patients with a deficiency of DHP are indicated, numbers correspond to the cDNA position. The bottom panel shows the alignment of the amino-acid sequences of DHP, DRP, and Hydantoinase (Hy) from Agrobacterium radiobacter (Ar), Arabidopsis thaliana (A), Bacillus stearothermophilus (Bs), bovine (b), Burkholderia pickettii (bp), Caenorhabditis elegans (Ce), Chicken (c), Dictyostelium discoideum (Dd), Drosophila melanogaster (Dm), Human (h), Mouse (m), Pseudomonas aeruginosa (Pa), Pseudomonas putida (Pp), Rat (r), Saccharomyces kluyveri (Sk), Streptomyces coelicolor (Sc), and Xenopus laevis (x). The GenBank accession numbers are depicted in parentheses. In Saccharomyces kluyveri, the inserted sequences were removed before alignment (24) . Conserved amino-acid sequences are boxed, and the arrow indicates the position of the mutation G278D in human DHP.

Expression Analysis of the G278D Mutation.
To investigate the effect of the mutation G278D on the activity of DHP, the mutation was introduced into the pSE420-DHP vector by site-directed mutagenisis and expressed in E. coli. No endogenous DHP activity (< 0.01 nmol/mg/h) could be detected in the E. coli strain used for the expression of the constructs. Introduction of the wild-type DHP construct increased the DHP activity >61,000-fold above the background. Expression of the DHP construct containing the G287D mutation yielded no detectable activity of DHP (Fig. 3) .

View larger version (14K): [in this window] [in a new window]   Fig. 3. DHP activity and immunoblot analysis of wild-type and mutant DHP expressed in E. coli. The activity of DHP was assayed in E. coli lysates. The results represent the mean of four independent measurements; bar, ± 1 SD (left panel). For immunoblot analysis, equal amounts of protein (2.5 µg) were separated by 7.5% SDS-PAGE and analyzed on immunoblot with DHP-specific antibodies (right panel).

Population Screening for the 833G>A Mutation.
Screening of individuals for the presence of the 833G>A mutation was performed with DHPLC. Fig. 4 shows that there is a clear separation between heteroduplex DNA from homoduplex DNA. Therefore, distinctive chromatographic patterns were obtained for PCR fragments containing the 833G>A mutation or the wild-type sequence. An analysis for the presence of the 833G>A mutation in 96 Dutch Caucasians did not identify individuals either heterozygous or homozygous for this mutation. Thus, the allele frequency of the 833G>A mutation in the normal population is <0.5%.

View larger version (13K): [in this window] [in a new window]   Fig. 4. DHPLC screening for the 833G>A mutation. PCR fragments of exon 5 of the DHP gene containing the wild-type sequence (----) and from the patient heterozygous for the 833G>A mutation (———) were separated at 65°C with DHPLC.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

To investigate the role of DHP, the second enzyme of the 5FU catabolic pathway, in the etiology of 5FU toxicity, we have analyzed the DHP gene of 23 tumor patients with normal DPD activity but, nevertheless, suffering from severe toxicity for the presence of mutations. One of these patients proved to be heterozygous for a 833G>A mutation in exon 5, and heterologous expression of this mutation showed that the mutant DHP protein bore no residual activity.

DHP deficiency is an autosomal recessive disease characterized by dihydropyrimidinuria and has been associated with a variable clinical phenotype (10, 11, 12 , 17) . Loading studies with uracil, in patients suffering from a complete DHP deficiency, showed strongly elevated levels and prolonged retention of uracil and dihydrouracil in serum, with >80% of the administered dose being excreted either unchanged or in the form of dihydrouracil during the first 24 h after the load (10 , 11) . In individuals heterozygous for a mutant DHP allele, the urinary concentration of dihydrouracil was several fold higher compared with that observed in controls, after loading with uracil (12) . Under normal conditions, a low DHP activity is probably sufficient to maintain dihydrouracil and dihydrothymine homeostasis as heterozygotes do not excrete elevated levels of dihydropyrimidines. After the loading of such patients with uracil, the accumulation of dihydrouracil in urine increased several fold compared with normal individuals, indicating a decreased capacity of heterozygotes to degrade dihydropyrimidines (12) . In this respect, it is worthwhile to note that the coadministration of FUH₂ with 5FU attenuated the antitumor activity and increased the toxicity of 5FU (20) . Furthermore, in the presence of elevated concentrations of dihydropyrimidines, the reverse reaction, catalyzed by DPD, toward the pyrimidine bases is stimulated (10 , 21) . Thus, the decreased capacity to degrade FUH₂ and 5FU, because of a decreased DHP activity, might be directly responsible for the observed toxicity in the study patient. In this respect, it is worthwhile to note that neutropenia has also been associated with a partial deficiency of DPD (5 , 7 , 8) .

The identification of genetic factors predisposing patients for development of severe 5FU-associated toxicity is increasingly being recognized as an important field of study. It has been shown recently that the C677T polymorphism in the methylenetetrahydrofolate reductase gene might be responsible for the severe toxicity observed in some breast cancer patients receiving adjuvant treatment with cyclophosphamide, methotrexate, and 5FU (22) . In addition, it has been suggested that a polymorphism in the enhancer region of the thymidylate synthase gene promoter is associated with toxicity toward 5FU (23) .

Our results indicate that a partial DHP deficiency is not a major determinant in the etiology of 5FU toxicity. To date, only 9 individuals suffering from a complete DHP deficiency have been reported, which, to some extent, may be because of the lack of specific and efficient methods in most laboratories to detect the dihydropyrimidines. In fact, the prevalence of a DHP deficiency in Japan has been estimated to be 1 in 10,000, which is comparable with the estimated frequency of patients with a DPD deficiency in the Netherlands (6) . Therefore, a DHP deficiency might be less rare than generally assumed.

	ACKNOWLEDGMENTS

 
We thank Eva Beke and Dr. Doreen Dobritzsch for assistance with the sequence analysis, and Dr. Hans R. Waterham and Fiona Ward for critical reading of the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Academic Medical Center, Laboratory Genetic Metabolic Diseases, F0–224, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. Phone: 31-205665958; Fax: 31-206962596; E-mail: a.b.vankuilenburg{at}amc.uva.nl

² The abbreviations used are: 5FU, 5-fluorouracil; DHP, dihydropyrimidinase; DHPLC, denaturing high performance liquid chromatography; DPD, dihydropyrimidine dehydrogenase; DRP, dihydropyrimidinase-related protein; FUH₂, fluoro-5,6-dihydrouracil; HPLC, high-performance liquid chromatography; TBS, Tris-buffered saline; CMF, cyclophosphamide, methotrexate, and fluorouracil.

Received 3/21/03; revised 5/21/03; accepted 5/27/03.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Salonga D., Danenberg K. D., Johnson M., Metzger R., Groshen S., Tsao-Wei D. D., Lenz H-J., Leichman C. G., Leichman L., Diasio R. B., Danenberg P. Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase. Clin. Cancer Res., 6: 1322-1327, 2000.[Abstract/Free Full Text]
2. Ichikawa W., Uetaka H., Shirota Y., Yamada H., Nishi N., Nihei Z., Sugihara K., Hirayama R. Combination of dihydropyrimidine dehydrogenase and thymidylate synthase gene expressions in primary tumors as predictive parameters for the efficacy of fluoropyrimidine-based chemotherapy for metastatic colorectal cancer. Clin. Cancer Res., 9: 786-791, 2003.[Abstract/Free Full Text]
3. Van Kuilenburg A. B. P., De Abreu R. A., Van Gennip A. H. Pharmacogenetic and clinical aspects of dihydropyrimidine dehydrogenase deficiency. Ann. Clin. Biochem., 40: 41-45, 2003.[CrossRef][Medline]
4. Heggie G. D., Sommadossi J-P., Cross D. S., Huster W. J., Diasio R. B. Clinical pharmacokinetics of 5-fluorouracil and its metabolism in plasma, urine, and bile. Cancer Res., 47: 2203-2206, 1987.[Abstract/Free Full Text]
5. Van Kuilenburg A. B. P., Haasjes J., Richel D. J., Zoetekouw L., Van Lenthe H., De Abreu R. A., Maring J. G., Vreken P., van Gennip A. H. Clinical implications of dihydropyrimidine dehydrogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPD gene. Clin. Cancer Res., 6: 4705-4712, 2000.[Abstract/Free Full Text]
6. Van Kuilenburg A. B. P., Muller E. W., Haasjes J., Meinsma R., Zoetekouw L., Waterham H. R., Baas F., Richel D. J., van Gennip A. H. 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. Clin. Cancer Res., 7: 1149-1153, 2001.[Abstract/Free Full Text]
7. Raida M., Schwabe W., Häusler P., Van Kuilenburg A. B. P., Van Gennip A. H., Behnke D., Höffken K. Prevalence of a common point mutation in the dihydropyrimidine dehydrogenase (DPD) gene within the 5'-splice donor site of intron 14 in patients with severe 5-fluorouracil (5-FU)-related toxicity compared to controls. Clin. Cancer Res., 7: 2832-2839, 2001.[Abstract/Free Full Text]
8. Van Kuilenburg A. B. P., Meinsma R., Zoetekouw L., van Gennip A. H. Increased risk of grade IV neutropenia after the administration of 5-fluorouracil due to a dihydropyrimidine dehydrogenase deficiency: high prevalence of the IVS14+1G>A mutation. Int. J. Cancer, 101: 253-258, 2002.[CrossRef][Medline]
9. Van Kuilenburg A. B. P., Meinsma R., Zoetekouw L., van Gennip A. H. High prevalence of the IVS14+1G>A mutation in the dihydropyrimidine dehydrogenase gene of patients with severe 5-fluorouracil-associated toxicity. Pharmacogenetics, 12: 555-558, 2002.[CrossRef][Medline]
10. Duran M., Rovers P., de Bree P. K., Schreuder C. H., Beukenhorst H., Dorland L., Berger R. Dihydropyrimidiuria: a new inborn error of pyrimidine metabolism. J. Inher. Metab. Dis., 14: 367-370, 1991.[CrossRef][Medline]
11. Hayashi K., Kidouchi K., Sumi S., Mizokami M., Orito E., Kumada K., Ueda R., Wada Y. Possible predicition of adverse reactions to pyrimidine chemotherapy from urinary pyrimidine levels and a case of asymptomatic adult dihydropyrimidinuria. Clin. Cancer Res., 2: 1937-1941, 1996.[Abstract]
12. Sumi S., Imaeda M., Kidouchi K., Ohba S., Hamajima N., Kodama K., Togari H., Wada Y. Population and family studies of dihydropyrimidinuria: prevalence, inheritance moe and risk of fluorouracil toxicity. Am. J. Med. Genet., 78: 336-340, 1998.[CrossRef][Medline]
13. Van Lenthe H., van Kuilenburg A. B. P., Ito T., Bootsma A. H., van Cruchten A., Wada Y., van Gennip A. H. Defects in Pyrimidine degradation identified by HPLC-electrospray tandem mass spectrometry of urine specimens or urine-soaked filter paper strips. Clin. Chem., 46: 1916-1922, 2000.[Abstract/Free Full Text]
14. Van Kuilenburg A. B. P., Van Lenthe H., Tromp A., Veltman P. C. J., Van Gennip A. H. Pitfalls in the diagnosis of patients with a partial dihydropyrimidine dehydrogenase deficiency. Clin. Chem., 46: 9-17, 2000.[Abstract/Free Full Text]
15. Van Kuilenburg A. B. P., van Lenthe H., van Gennip A. H. Radiochemical assay for determination of dihydropyrimidinase activity using reversed-phase high-performance liquid chromatography. J. Chrom. B, 729: 307-314, 1999.
16. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem., 150: 76-85, 1985.[CrossRef][Medline]
17. Hamajima N., Kouwaki M., Vreken P., Matsuda K., Sumi S., Imaeda M., Ohba S., Kidouchi K., Nonaka M., Sasaki M., Tamaki N., Endo Y., De Abreu R., Rotteveel J., van Kuilenburg A., van Gennip A. H., Togari H., Wada Y. Dihydropyrimidinase deficiency: structural organisation, chromosomal localisation, and mutation analysis of the human dihydropyrimidinase gene. Am. J. Hum. Genet., 63: 717-726, 1998.[CrossRef][Medline]
18. Hamajima N., Matsuda K., Sakata S., Tamaki N., Sasaki M., Nonaka M. A Novel gene family defined by human dihydropyrimidinase and three related proteins with differential tissue distribution. Gene, 188: 157-163, 1996.
19. Meta-Analysis Group in Cancer. Toxicity of fluorouracil in patients with advanced colorectal cancer: effect of administration schedule and prognostic factors. J. Clin. Oncol., 16: 3537-3541, 1998.[Abstract]
20. Spector T., Cao S., Rustum Y. M., Harrington J. A., Porter D. J. T. Attenuation of the antitumor activity of 5-fluorouracil by (R)-5-fluoro-5, 6-dihydrouracil. Cancer Res., 55: 1239-1241, 1995.[Abstract/Free Full Text]
21. Porter D. J. T., Harrington J. A., Almond M. R., Lowen G. T., Spector T. (R)-5-Fluorouracil-5, 6-dihydrouracil: kinetics of oxidation by dihydropyrimidine dehydrogenase and hydrolysis by dihydropyrimidine aminohydrolase. Biochem. Pharmacol., 48: 775-779, 1994.[CrossRef][Medline]
22. Toffoli G., Veronesi A., Boiocchi M., Crivellari D. MTHFR gene polymorphism and severe toxicity during adjvant treatment of early breast cancer with cyclophosphamide, methotrexate, and fluorouracil (CMF). Ann Oncol., 11: 373-375, 2000.[Free Full Text]
23. Pullarkat S. T., Stoehlmacher J., Ghaderi V., Xiong Y-P., Ingles S. A., Sherrod A., Warren R., Tsao-Wei D., Groshen S., Lenz H-J. Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J., 1: 65-70, 2001.[Medline]
24. Gojkovic Z., Rislund L., Andersen B., Sandrini M. P. B., Cook P. F., Schnackerz K. D., Pikur J. Dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties. Nucl. Acids Res., 31: 1-9, 2003.[Abstract/Free Full Text]

This article has been cited by other articles:

				H. H. Ezzeldin and R. B. Diasio Predicting Fluorouracil Toxicity: Can We Finally Do It? J. Clin. Oncol., May 1, 2008; 26(13): 2080 - 2082. [Full Text] [PDF]

				L. K. Mattison, J. Fourie, R. A. Desmond, A. Modak, M. W. Saif, and R. B. Diasio Increased prevalence of dihydropyrimidine dehydrogenase deficiency in african-americans compared with Caucasians. Clin. Cancer Res., September 15, 2006; 12(18): 5491 - 5495. [Abstract] [Full Text] [PDF]

				H. H. Ezzeldin and R. B. Diasio Genetic testing in cancer therapeutics. Clin. Cancer Res., July 15, 2006; 12(14): 4137 - 4141. [Full Text] [PDF]

				B. Lohkamp, B. Andersen, J. Piskur, and D. Dobritzsch The Crystal Structures of Dihydropyrimidinases Reaffirm the Close Relationship between Cyclic Amidohydrolases and Explain Their Substrate Specificity J. Biol. Chem., May 12, 2006; 281(19): 13762 - 13776. [Abstract] [Full Text] [PDF]

				J. M. Rawls Jr. Analysis of Pyrimidine Catabolism in Drosophila melanogaster Using Epistatic Interactions With Mutations of Pyrimidine Biosynthesis and {beta}-Alanine Metabolism Genetics, March 1, 2006; 172(3): 1665 - 1674. [Abstract] [Full Text] [PDF]

				A. B.P. van Kuilenburg, R. Meinsma, E. Beke, B. Assmann, A. Ribes, I. Lorente, R. Busch, E. Mayatepek, N. G.G.M. Abeling, A. van Cruchten, et al. {beta}-Ureidopropionase deficiency: an inborn error of pyrimidine degradation associated with neurological abnormalities Hum. Mol. Genet., November 15, 2004; 13(22): 2793 - 2801. [Abstract] [Full Text] [PDF]

				L. K. Mattison, H. Ezzeldin, M. Carpenter, A. Modak, M. R. Johnson, and R. B. Diasio Rapid Identification of Dihydropyrimidine Dehydrogenase Deficiency by Using a Novel 2-13C-Uracil Breath Test Clin. Cancer Res., April 15, 2004; 10(8): 2652 - 2658. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Kuilenburg, A. B. P. Articles by van Gennip, A. H. Search for Related Content PubMed PubMed Citation Articles by van Kuilenburg, A. B. P. Articles by van Gennip, A. H.