Journal of Inherited Metabolic Disease

, Volume 19, Issue 5, pp 645–654 | Cite as

A point mutation in an invariant splice donor site leads to exon skipping in two unrelated Dutch patients with dihydropyrimidine dehydrogenase deficiency

  • P. Vreken
  • A. B. P. Van Kuilenburg
  • R. Meinsma
  • G. P. A. Smit
  • H. D. Bakker
  • R. A. De Abreu
  • A. H. van Gennip


Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria and associated with a variable clinical phenotype. In order to identify the molecular defect underlying complete DPD deficiency in a Dutch patient previously shown to have a 165 base pair deletion in the mature DPD mRNA, we cloned the genomic region encompassing the skipped exon and its flanking intron sequences. Sequence analysis revealed that the patient was homozygous for a single G→A point mutation in the invariant GT dinucleotide splice donor site downstream of the skipped exon. The same mutation was identified in another, unrelated, Dutch patient. Because this mutation destroys a uniqueMaeII restriction site, rapid screening using restriction enzyme cleavage of the amplified genomic region encompassing this mutation is possible. Analysis of 50 controls revealed no individuals heterozygous for this mutation


Dinucleotide Enzyme Cleavage Dehydrogenase Deficiency Splice Donor Site Molecular Defect 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bakker HD, Rubio Gozalbo ME, van Gennip AH (1994) Dihydropyrimidine dehydrogenase deficiency presenting with psychomotor retardation and ocular abnormalities.J Inher Metab Dis 17: 640–641.Google Scholar
  2. Bakkeren JAJM, De Abreu RA, Sengers RCA, Gabreels FJM, Maas JM, Renier WO (1984) Elevated urine, blood and cerebrospinal fluid levels of uracil and thymine in a child with dihydropyrimidine dehydrogenase deficiency.Clin Chim Acta 140: 247–257.Google Scholar
  3. Berger R, Stoker-De Vries SA, Wadman SK, et al (1984) Dihydropyrimidine dehydrogenase deficiency leading to thymine-uraciluria. An inborn error of pyrimidine metabolism.Clin Chim Acta 141: 227–234.Google Scholar
  4. Braakhekke JP, Renier WO, Gabreëls FJM, De Abreu RA, Bakkeren JAJM, Sengers RCA (1987) Dihydropyrimidine dehydrogenase deficiency; neurological aspects.J Neurol Sci 78: 71–77.Google Scholar
  5. Carstens RP, Fenton WA, Rosenberg LR (1991) Identification of RNA splicing errors resulting in human ornithine transcarbamylase deficiency.Am J Hum Genet 48: 1105–1114.Google Scholar
  6. Chomczynski P, Sacchi N (1987) Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.Anal Biochem 162: 156–159.Google Scholar
  7. Diasio RB, Beavers TL, Carpenter JT (1988) Familial deficiency of dihydropyrimidine dehydrogenase. Biochemical basis for familial pyrimidinemia and severe 5-fluorouracil induced toxicity.J Clin Invest 81: 47–51.Google Scholar
  8. Fleming RA, Milano G, Thyss A, et al (1992) Correlation between dihydropyrimidine dehydrogenase activity in peripheral mononuclear cells and systemic clearance of fluorouracil in cancer patients.Cancer Res 52: 2899–2902.Google Scholar
  9. Harris BE, Carpenter JT, Diasio RB (1991) Severe fluorouracil toxicity secondary to dihydropyrimidine dehydrogenase deficiency. A potentially more common pharmacogenetic syndrome.Cancer Res 68: 499–501.Google Scholar
  10. Hoar DI (1987) Molecular diagnosis: new horizons in medicine.Can Fam Physician 33: 401–404.Google Scholar
  11. Kikugawa M, Kaneko M, Fujimoto-Sakata S, et al (1994) Purification, characterization and inhibition of dihydropyrimidinase from rat liver.Eur J Biochem 219: 393–399.Google Scholar
  12. Krawczak M, Reiss J, Cooper DN (1992) The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences.Hum Genet 90: 41–54.Google Scholar
  13. Kuivaniemie H, Kontusaari S, Tromp G, Zhao M, Sabol C, Prockop DJ (1990) Identical G+1 to A mutations in three different introns of the type III procollagen gene (COL3A1) produce different patterns of RNA splicing in three variants of Ehlers-Danlos syndrome IV.J Biol Chem 265: 12067–12074.Google Scholar
  14. Lu Z-H, Zhang R, Diasio RB (1993) Comparison of dihydropyrimidine dehydrogenase from human, rat, pig and cow liver.Biochem Pharmacol 46: 945–952.Google Scholar
  15. Marvit J, Dilella AG, Brayton K, Ledley FD, Robson KJH, Woo SLC (1987) GT to AT transition at a splice donor site causes skipping of the preceding exon in phenylketonuria.Nucleic Acids Res 15: 5613–5628.Google Scholar
  16. Matsuura T, Hoshide R, Komaki S, et al (1995) Identification of two new aberrant splicings in the omithine carbamoyltransferase (OCT) gene in two patients with early and late onset OCT deficiency.J Inher Metab Dis 18: 273–282.Google Scholar
  17. Meinsma R, Fernandez-Salguero P, van Kuilenburg ABP, van Gennip AH, Gonzalez FJ (1995) Human polymorphism in drug metabolism: Mutation in the dihydropyrimidine dehydrogenase gene results in exon skipping and thymine uraciluria.DNA Cell Biol 14: 1–6.Google Scholar
  18. Robberson BL, Cote GJ, Berget SM (1990) Exon definition may facilitate splice site selection in RNAs with multiple exons.Mol Cell Biol 10: 84–94.Google Scholar
  19. Senepathy P, Shapiro MB, Harris NL (1990) Splice junctions, branch point sites and exons: sequence statistics, identification and applications to genome project.Methods Enzymol 183: 252–278.Google Scholar
  20. Shapiro MB, Senepathy P (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression.Nucleic Acids Res 15: 7155–7175.Google Scholar
  21. Siebert PD, Chenchik A, Kellog DE, Lukyanov KA, Lukyanov SA (1995a) An improved method for walking in uncloned genomic DNA.Nucleic Acids Res 23: 1087–1088.Google Scholar
  22. Siebert PD, Chen S, Kellog DE (1995b) The human PromoterFinder DNA walking kit: a new PCR method for walking in uncloned genomic DNA.CLONTECHniques X: 1–3.Google Scholar
  23. Tuchman M, Roemeling RV, Hrushesky WAM, Sothern RB, O'Dea RF (1989) Dihydropyrimidine dehydrogenase activity in human blood mononuclear cells.Enzyme 42: 15–24.Google Scholar
  24. Van Gennip AH, Bakker HD, Zoetekouw A, Abeling NGGM (1987) A new case of thymineuraciluria.Klin Wochenschr 65 (suppl X): 14.Google Scholar
  25. Van Gennip AH, Abeling NGGM, Elzinga-Zoetekouw L, Scholten LG, van Cruchten A, Bakker HD (1989) Comparative study of thymine and uracil metabolism in healthy persons and in a patient with dihydropyrimidine dehydrogenase deficiency.Adv Exp Med Biol 253A: 111–118.Google Scholar
  26. Van Gennip AH, Abeling NGGM, Stroomer AEM, van Lenthe H, Bakker HD (1994) Clinical and biochemical findings in six patients with pyrimidine degradation defects.J Inher Metab Dis 17: 130–132.Google Scholar
  27. Van Kuilenburg ABP, van Lenthe H, van Gennip AH (1996) Identification and tissue-specific expression of a NADH-dependent activity of dihydropyrimidine dehydrogenase in man.Anticancer Res 16: 389–394.Google Scholar
  28. Vreken P, Niessen RWLM, Peters M, Schaap MCL, Zuithoff-Rijntjes JGM, Sturk A (1995) A point mutation in an invariant splice acceptor site results in a decreased mRNA level in a patient with severe coagulation factor XIII subunit A deficiency.Thrombosis & Hemostasis 74: 584–589.Google Scholar
  29. Wasternack C (1980) Degradation of pyrimidine analogs — pathway and mutual influences.Pharm Ther 8: 629–651.Google Scholar
  30. Yokota H, Fernandez-Salguero P, Furuya H, et al (1994) cDNA cloning and chromosome mapping of human dihydropyrimidine dehydrogenase, an enzyme associated with 5-fluorouracil toxicity and congenital thymine uraciluria.J Biol Chem 269: 23192–23196.Google Scholar
  31. Zhuang Y, Weiner AM (1986) A compensatory base change in U1 snRNA suppresses a 5′ splice site mutation.Cell 46: 827–835.Google Scholar

Copyright information

© SSIEM and Kluwer Academic Publishers 1996

Authors and Affiliations

  • P. Vreken
    • 1
  • A. B. P. Van Kuilenburg
    • 1
  • R. Meinsma
    • 1
  • G. P. A. Smit
    • 2
  • H. D. Bakker
    • 1
  • R. A. De Abreu
    • 3
  • A. H. van Gennip
    • 1
  1. 1.Academic Medical CenterUniversity of Amsterdam, Department of Clinical Chemistry F0-224AmsterdamThe Netherlands
  2. 2.Department of PediatricsAcademic Hospital GroningenGroningen
  3. 3.Department of PediatricsAcademic Hospital NijmegenNjmegenThe Netherlands

Personalised recommendations