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Letters |
a Author for correspondence. Fax 33-2-40-679731; e-mail p-jezequel{at}gauducheau-nantes.fnclcc.fr
1
Département de Biologie Oncologique, Centre Régional, de Lutte Contre le Cancer, René Gauducheau, Boulevard Jacques Monod, 44805 Nantes-St. Herblain Cedex, France,
2
Laboratoire d’Oncopharmacologie, Centre Régional, de Lutte Contre le Cancer, Antoine Lacassagne, 33 Avenue de Valombrose, 06189 Nice Cedex, France
To the Editor:
The human dihydropyrimidine dehydrogenase gene (DPYD)
encodes dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2), the first
and rate-limiting enzyme in the three-step pathway of uracil and
thymine catabolism. DPD is also the principal enzyme involved in
detoxification of pyrimidine-based antimetabolic analogs, such as
5-fluorouracil (5-FU), a drug that is commonly used in the treatment of
solid tumors (colon, breast, head, neck, ovary, and skin). Because
>80% of the administered 5-FU is degraded by DPD (1), the
DPD catalytic activity in cancer patients could affect the efficacy of
5-FU treatment. In cancer patients with very low DPD activity, toxic
reactions (e.g., diarrhea, stomatitis, mucositis, myelosuppression, and
neurotoxicity) were reported that in some cases were life-threatening
and sometimes fatal (2). A frequency as high as 3% of
putative heterozygotes for DPD deficiency was also estimated based on
catalytic activities in population studies (3)(4). The identification and characterization
of the human DPD cDNA (5) made possible the identification
and molecular analysis of mutations that affect DPD expression and
catalytic activity. The most common mutation (6) associated
with severe toxicity is a G
A transition at the 5'-splicing donor
consensus sequence in intron 14 that leads to exon 14 skipping (7)(8)(9)(10):
c.1905+1GA [according to mutation nomenclature (11)]. By itself, the G-to-A nucleotide change destroys a
unique restriction site only for the expensive MaeII
endonuclease (isoschizomers, TaiI and TscI). We
used PCR-mediated site-directed mutagenesis (PSM) utilizing a PCR
primer with a single-base mismatch near the mutation site to introduce
into the amplified wild-type product an allele-specific
SnaBI restriction site. In this way, the amplification
product encompassing this polymorphic site can be restriction-digested
and electrophoresed to resolve alleles easily.
Our strategy was such that the enzyme we used cut the amplified wild
type once, but not the amplified homozygous mutant type. This
choice provides in most cases a positive control for the
SnaBI digestion. Using the Hardy-Weinberg equilibrium, the
frequency of heterozygotes allows the estimation of up to 1 in 1000
homozygotes for DPYD mutations. We designed in the 3' end of
exon 14 of the DPYD gene the forward primer
DPD-PSM1
(5'-CTAAAGGCTGACTTTCCAGACTAC-3')
to contain a single-base mismatch (AT), creating a novel
SnaBI restriction site (TAC
GTA) in the amplified
wild-type allele. DPD-PSM1 was designed based on the sequence from
GenBank accession no. U20938. The reverse primer DPD-del-R
(5'-CAGCAAAGCAACTGGCAGATTC-3') was located in intron 14 (10).
The amplification product length before digestion was 155 bp.
Digestion by SnaBI restriction endonuclease generated
two fragments (131 and 24 bp; the 24-bp fragment migrates quickly and
is not seen on the gel) in the wild-type allele (Fig. 1
) and does not cut the product from the homozygous mutant c.1905+1GA
allele (not shown). After electrophoresis in an agarose gel, a
heterozygote for the mutation theoretically shows three bands of 155,
131, and 24 bp, which correspond to the two alleles (Fig. 1
).
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Because 5-FU is one of the most commonly prescribed chemotherapeutic
drugs in cancer treatment (in monotherapy or polytherapy) and the
c.1905+1GA mutation is frequently linked to severe toxicity,
molecular screening of cancer patients could be done routinely, coupled
with analysis of DPD activity in peripheral blood mononuclear cells,
before the start of treatment to avoid the toxic effects of 5-FU.
Because economic problems are very important in health-screening
strategies, screening tests must be the least expensive. The use of
SnaBI in a PSM method produces an 18-fold decrease in the
enzyme cost ($0.70 vs $12.87 US per reaction) compared with the
previous PCR-restriction method using MaeII (7)(8).
Acknowledgments
This work was supported in part by a generous gift from Caisse des Professions Libérales, Province: CAMPLP. We thank Dr. Roch for his support. We are indebted to Paul Bennett for revision of our manuscript.
References
The following articles in journals at HighWire Press have cited this article:
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  M Steiner, M Seule, B Steiner, I Bauer, M Freund, C H Kohne, and P Schuff-Werner 5-Fluorouracil/irinotecan induced lethal toxicity as a result of a combined pharmacogenetic syndrome: report of a case J. Clin. Pathol., May 1, 2005; 58(5): 553 - 555. [Abstract] [Full Text] [PDF]
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 A. B. P. van Kuilenburg, E. W. Muller, J. Haasjes, R. Meinsma, L. Zoetekouw, H. R. Waterham, F. Baas, D. J. Richel, and A. H. van Gennip 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., May 1, 2001; 7(5): 1149 - 1153. [Abstract] [Full Text]
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