Determination of CYP2D6 gene copy number by pyrosequencing.
BACKGROUND
Identification of CYP2D6 alleles *5 (deletion of the whole CYP2D6 gene) and *2xN (gene duplication) is very important because they are associated with decreased or increased metabolism of many drugs. The most commonly used method for analysis of these alleles is, however, considered to be laborious and unreliable.


METHODS
We developed a method to determine the copy number of the CYP2D6*5 and CYP2D6*2xN alleles by use of Pyrosequencing technology. A single set of PCR and sequencing primers was used to coamplify and sequence a region in the CYP2D6 gene and the equivalent region in the CYP2D8P pseudogene, and relative quantification between these fragments was performed. The CYP2D8P-specific Pyrosequencing peak heights were used as references for the CYP2D6-specific peak heights.


RESULTS
Analysis of 200 pregenotyped samples showed that this approach reliably resolved 0-4 genome copies of the CYP2D6 gene. In 15 of these samples, the peak pattern from one analyzed position was unexpected but could be solved by conclusive results from a second position. The method was verified on 270 other samples, of which 267 gave results that corresponded to the expected genotype. One of the samples could not be interpreted. The reproducibility of the method was high.


CONCLUSIONS
CYP2D6 gene copy determination by Pyrosequencing is a reliable and rapid alternative to other methods. The use of an internal CYP2D8P control as well as generation of a sequence context ensures a robust method and hence facilitates method validation.
Söderbäck
Erik
E
Biotage AB, Uppsala, Sweden.
Zackrisson
Anna-Lena
AL
Lindblom
Bertil
B
Alderborn
Anders
A
D001483:Base Sequence; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D018628:Gene Dosage; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D016415:Sequence Alignment
D019389:Cytochrome P-450 CYP2D6