| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Letters to the Editor |
T Mutation within the Dihydropyrimidine Dehydrogenase Gene and Potential for Misclassification with the Exon 14-skipping Mutation
1 Department of Pharmacology and
2 Institute of Virology, University of Cologne, Gleueler Strasse 24, 50931 Cologne, Germany
aAuthor for correspondence. Fax 49-221-478-5022; e-mail andreas.lazar{at}medizin.uni-koeln.de.
To the Editor:
Dihydropyrimidine dehydrogenase (DPYD) is the initial and rate-limiting enzyme in the metabolism of the chemotherapeutic drug 5-fluorouracil (5-FU), thus affecting its pharmacokinetics, efficacy, and toxicity
(1). Application of 5-FU is restricted by a narrow therapeutic index because of severe toxicity of WHO grades III–IV
(2). Polymorphisms within the DPYD gene have been reported, with deficiency in enzyme activity leading to severe 5-FU-related toxicity in cancer patients
(3). The so-called exon 14-skipping mutation at the 5'-splice donor site of exon 14 (1905 + 1G
A) has been detected in 
25% of affected patients
(4). To identify patients at increased risk for severe 5-FU-induced toxicity, many medical centers routinely screen for the exon 14-skipping mutation before starting chemotherapy.
We directly compared DNA sequencing with a fluorescence-based technology, namely, the LightCycler. For this purpose, we used genomic DNA from a homozygous wild-type individual (sample A) and two DNA samples from persons believed to be heterozygous for the exon 14-skipping mutation from earlier LightCycler analyses (samples B and C). The LightCycler method was used as described previously (5) except that the transition from the annealing phase to the elongation phase was 5 °C/s (amplification was not always successful at a temperature transition rate of 20 °C/s). PCR conditions of the direct sequencing method are available from the authors on request.
Melting curve analysis of the homozygous sample A revealed a single peak at 60.5 °C in accordance with published data
(5) (Fig. 1
). The result has been confirmed by direct sequencing.
|
The melting curve profiles of samples B and C both exhibited a melting point at 60.5 °C for the wild-type allele. However, the lower melting temperature peaks were different (55.1 and 51.7 °C), indicating two different sequence alterations
(6). Direct sequencing confirmed that sample C was heterozygous for the exon 14-skipping mutation. However, direct sequencing of sample B did not reveal the expected 1905 + 1GA polymorphism but a novel 1905CT mutation 1 bp away from the guanidine of the 5'-splice donor site. According to our knowledge of splicing mechanisms to date, the 1905CT nucleotide substitution presumably does not affect RNA processing and is supposed to be silent
(7).
Melting curve profiles may be influenced by several factors, such as the concentrations of fluorophores, the rate of temperature transition during final denaturation, initial copy number, and product yield (8). The mentioned conditions were equal for all samples. Nonetheless, unequal amplification efficiency for both strands cannot always be ruled out.
The data demonstrate shortcomings in the unambiguous identification of mutations with hybridization probe methods on the basis of published melting curve profiles (5) and calculations based on the nearest-neighbor method (9). Rather, our data emphasize the use of external standards verified by direct sequencing during the validation process. Increased attention is required for detection of the exon 14-skipping mutation of the DPYD gene in routine diagnostics because of at least one existing nearby polymorphism.
Acknowledgments
We thank C. Marx, M. Schwab (both of Stuttgart, Germany), and M. Nauck (Freiburg, Germany) for kindly providing genomic DNA samples.
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
