Pharmacogenetic Screening of the Gene Deletion and Duplications of CYP2D6

Abstract

Cytochrome P450 (CYP) 2D6 is one of the most important enzymes involved in the metabolism of drugs. Multiple, clinically relevant, genetic variants of this gene have been identified and, among them, a gene deletion as well as multiplications of the gene. These large structural mutations in CYP2D6 occur at a relatively high frequency in several populations. Genotyping of CYP2D6 could therefore be applied to individualize drug therapy to improve therapeutic efficacy and decrease adverse effects in patients. However, a prerequisite for the pharmacogenetic screening of CYP2D6 in a clinical setting is the development of fast, reliable and cost-effective techniques for the routine genotyping of patients. In the case of CYP2D6, besides the general problems that arise in the detection of large gene deletions and multiplications, the presence of two highly homologous pseudogenes, CYP2D7 and CYP2D8, forms an extra challenge.

This review provides an overview of the techniques that have been described to detect the CYP2D6 gene deletion and multiplication: Southern-blotting RFLP, long-template PCR, and real-time PCR. Of these techniques, real-time PCR is the only technique giving quantitative information about the exact copy number of the gene. Considering all of the other advantages of this method over other methods, such as cost-effectiveness and suitability for high throughput screening, real-time PCR is the most promising method for the genotyping of large structural alterations in the CYP2D6 gene in a routine clinical setting.

Key Words :

• Cytochrome 2D6
• Pharmacogenetics
• Gene deletion
• Gene duplication

Notes

Two pseudogenes, CYP2D7 and CYP2D8, are present upstream of the CYP2D6 gene. The sequences show a high homology, making the design of primers for a PCR difficult. The grey and dark areas are almost identical stretches of nucleotides located in the intergenic regions. The broken line shows the 3′ deletion breakpoint. Figure A represents the wild-type gene, Figure B the gene deletion. The dark arrows indicated with S are the primers designed by Steen et al. (1995): forward 5′-ACCGGGCACCTGTACTCCTCA-3′, reverse 5′-GCATGAGCTAAGGCACCCAGAC-3′; the arrows indicated with J, the primers designed by Johansson et al. (1996): forward 5′- GCCACTCTCGTGTCGTCAGCTTT-3′, reverse 5′-GGCATGAGCTAAGGCACC-3′; and H, are those described by Hersberger et al. (2000): for the wild-type (A) forward 5′-GTTATCCCAGAAGGCTTTGCAGGCTTCA-3′, reverse 5′-GCCGACTGAGCCTGGGAGGTAGGTA-3 and for the identification of the CYP2D6 gene deletion (B): forward 5′-CAGGCATGAGCTAAGGCACCCAGAC-3′.

Long-template PCR used to detect the duplication or multiplication of the CYP2D6 gene. Primers designed by Johansson et al. (1996) are indicated by J: forward 5′-GCCACCATGGTGTCTTTGCTTTC-3′, reverse 5′-ACCGGATTCCAGCTGGGAAATG-3′; those of Lundqvist et al. (1999) by L: forward 5′-CCTGGGAAGGCCCCATGGAAG-3′, reverse 5′-CAGTTACGGCAGTGGTCAGCT-3′.

Long-template PCR used to detect the duplication of the CYP2D6 gene according to the method of Lovlie et al. (1996). The grey and dark areas are almost identical stretches of nucleotides located in the intergenic regions. The grey arrows indicate the position of the CYP2D6 duplication specific primers: forward 5′-CCCTCAGCCTCGTCACCTCAC-3′, reverse 5′-CACGTGCAGGGCACCTAGAT-3′, the dark arrows represent the primers developed to amplify a internal control fragment of the PCR reaction: forward 5′-TCCCCCACTGACCCAACTCT-3′, reverse 5′-CACGTGCAGGGCACCTAGAT-3′.

The target-specific primers and probe bind to the target DNA during the annealing phase of the PCR reaction. During the extension phase, the 5′→ 3′ endonuclease activity of the Taq polymerase cleaves the probe, leading to a release of the fluorescence probe and emission of a fluorescent signal.

Bold underlined sequences represent forward and reverse primers, sequences in italic the TaqMan™ probe. Asterisks show nucleotides unique in the CYP2D6 sequence compared to CYP2D7 and CYP2D8. Dark grey areas represent identical sequences between the genes and the two pseudogenes, light grey where CYP2D6 is identical to either CYP2D7 or CYP2D8. Arrows point to positions of known polymorphisms in the CYP2D6 gene. The alignment was performed using T-coffee (http://igs-server.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi).

Bold underlined sequences represent forward and reverse primers, sequences in italic the TaqMan™ probe. Asterisks show nucleotides unique in the CYP2D6 sequence compared to CYP2D7 and CYP2D8. Dark grey areas represent identical sequences between the genes and the two pseudogenes, light grey where CYP2D6 is identical to either CYP2D7 or CYP2D8. The alignment was performed using T-coffee. (http://igs-server.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi).

*This manuscript was referred by Dr. H. Kroemer, Institute of Pharmacology, University of Greifswald, Germany.