Received on September 20, 2004
Accepted on December 27, 2004

Molecular Diagnostics and Genetics

Determination of CYP2D6 Gene Copy Number by Pyrosequencing

¹ Biotage AB, Uppsala, Sweden
² National Board of Forensic Medicine, Department of Forensic Genetics, Linköping, Sweden

^* To whom correspondence should be addressed. E-mail: anders.alderborn{at}telia.com.

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^TM technology. A single set of PCR and sequencing primers was used to coamplify and sequences of a region in the CYP2D6 gene and the equivalent region in the CYP2D8P pseudogene, and relative quantification between these fragments was performed. The heights of specific Pyrosequencing peaks from the CYP2D8P fragment were used as references for the CYP2D6-specific peak heights.

Results: The 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 can be expected to ensure a robust method and hence facilitate method validation.