Rapid and reliable method for cytochrome P450 2D6 genotyping.
BACKGROUND
Single-nucleotide polymorphisms and single-base deletions within the cytochrome P450 2D6 (CYP2D6) gene have been associated with a poor metabolizer (PM) phenotype and display a frequency of 7-10% in the Caucasian population.


METHODS
We developed a reliable and rapid procedure to identify five major PM-associated mutations (CYP2D6*4, *7, and *8) and deletions (CYP2D6*3 and *6) by real-time PCR with subsequent fluorometric melting point analysis of the PCR product. These polymorphisms within the CYP2D6 gene were detected by use of two primer pairs and five different pairs of hybridization probes. DNA extracted from whole blood of 323 individuals was analyzed, and results were compared with genotypes obtained by allele-specific multiplex PCR. In case of uncertain results, additional sequence analysis was performed.


RESULTS
Genotyping results by real-time PCR were 100% reliable, whereas conventional allele-specific multiplex PCR produced uncertain results for 12.1% of samples, as confirmed by sequence analysis. Costs for reagents and consumables were considerably higher for the real-time PCR technology, but labor time was reduced by 2 h compared with allele-specific PCR. The allele frequencies in the population investigated were 0.186 for allele *4, 0.026 for allele *5, 0.009 for allele *3, 0.031 for allele *6, and 0.002 for allele *8. The defective CYP2D6*7 allele was not found. In addition, three additional mutations were detected, one of them displaying a PM genotype.


CONCLUSIONS
Genotyping of CYP2D6 by real-time PCR with fluorometric melting point analysis is a rapid and reliable method.
Stamer
Ulrike M
UM
Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. ulrike.stamer@ukb.uni-bonn.de
Bayerer
Bettina
B
Wolf
Stephanie
S
Hoeft
Andreas
A
Stüber
Frank
F
D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D005456:Fluorescent Dyes; D005838:Genotype; D006801:Humans; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results
D005456:Fluorescent Dyes; D019389:Cytochrome P-450 CYP2D6