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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Thomas
Holly R
HR
Division of Clinical Pharmacology and Toxicology, University of Alabama at Birmingham, Alabama, USA.
Ezzeldin
Hany H
HH
Guarcello
Vincenzo
V
Mattison
Lori K
LK
Fridley
Brooke L
BL
Diasio
Robert B
RB
D000328:Adult; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000378:metabolism*; D001944:Breath Tests; D002851:Chromatography, High Pressure Liquid; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000473:pathology; D003955:Diagnostic Tests, Routine; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D005838:Genotype; D006239:Haplotypes; D006801:Humans; D015810:Linkage Disequilibrium; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D017386:Sequence Homology, Amino Acid; D014498:Uracil / Q000378:metabolism*
D000970:Antineoplastic Agents; D014498:Uracil; D042943:Dihydrouracil Dehydrogenase (NADP)