TaqMan real-time PCR quantification strategy of CYP2D6 gene copy number for the LightCycler 2.0
Introduction
The cytochrome P450 2D6 (CYP2D6 GenBank M33388) is responsible for the metabolism of many endogenous as well as exogenous compounds [1]. Up to 25% of all therapeutic drugs, particularly antidepressants and antipsychotics are catalyzed and degraded by this enzyme [2], [3], [4]. The metabolism for these substrates is highly dependent on the amount of active CYP2D6 enzymes expressed in the liver. At present, more than 80 relevant alleles of the CYP2D6 gene have been identified on the chromosome 22 [5], with varying frequencies between ethnic groups [6]. They produce null, decreased, normal or increased functional CYP2D6 enzymes. The metabolic capacity of these enzymes produces four distinct phenotypes which can be distinguished as: poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (EM) and ultrarapid metabolizers (UM). Unless pro-drugs are applied PM and IM have the highest risk of adverse drug reactions due to elevated drug concentration in the system. In contrast, UMs have elevated level of drug metabolites and decreased response to drug therapy. Adverse drug reactions and low therapeutic efficacy can be avoided by adjusting drug selection and dosage to each patient's individual need [7], [8]. Given the fact that fatal drug reactions are still one of the most common causes of death [9], predicting those phenotypes prior to therapy can result in a significant reduction of avoidable complications in the clinic [10].
Due to the close relationship between CYP2D6 genotype and phenotype [11], [12], [13], genotyping CYP2D6 variants is a reliable method to predict individual phenotypes. In Caucasians, CYP2D6⁎5 (gene deletion) is the second most frequent cause of poor metabolism just after CYP2D6⁎4. In Asians and Africans it is the most common nonfunctional genotype. For ultrarapid metabolism, gene amplifications (CYP2D6⁎1 × N and CYP2D6⁎2 × N) are responsible for approximately 10–30% of the cases [11], [13], [14], [15]. Thus, together with SNP genotyping, detecting CYP2D6 GCN is crucial in predicting extreme phenotypes.
Conventional methods to detect CYP2D6 GCN, like Southern blot RFLP and long-range PCR, are well-established but very laborious and inefficient for routine use. Published TaqMan real-time PCR quantification strategies, on the other hand, are efficient and rapid but have little sample sizes, i.e. 64 and 44 samples respectively [16], [17], to demonstrate their adequacy for routine application. Both methods were also realized solely for the Abi Prism PCR system and have not yet been adapted on other platforms.
Therefore, we established two TaqMan PCR methods to quantify CYP2D6 gene copy number on the LightCycler 2.0 platform, and by analyzing a large number of samples, we tried to assess the reliability of these methods for routine application.
Section snippets
DNA samples
A total of 617 collected DNA samples were used to test these assays. These samples have been collected from patients with psychiatric disorders since 2003 for previous population studies and extracted by different standard methods from leukocytes (Promega and Qiagen). The study was approved by the Institutional Review Boards (Technische Universät München) and informed consents were given by all donors.
All samples were previously genotyped for CYP2D6⁎1,⁎2, ⁎3, ⁎4, ⁎6, ⁎7, ⁎8, ⁎9, ⁎10, ⁎35, ⁎41
Assay design and validation
These assays were designed such that the CYP2D6 gene copy number was examined with the reference gene albumin (ALB GenBank: M12523). One primer pair and probe target the exon 9 region of the CYP2D6, the other the intron 6 region. By redesigning the probes, it was possible to use the previously described primers for the LightCycler 2.0 platform. The characteristic lengths of the PCR products for CYP2D6 exon 9 at 89 bp, CYP2D6 intron 6 at 78 bp and albumin at 72 bp were identified with gel
Discussion
Functional CYP2D6 gene copies are an important indicator of drug clearance for many important drugs [2], [3], [4]. Poor metabolism as well as ultrarapid metabolism could lead to serious adverse drug reactions and lack of therapeutic efficacy [24], [25], [26], [27]. Determination of the exact CYP2D6 gene copy number in combination with genotyping is inevitable in predicting the correct phenotype. Many methods are available to do so, but up till now the TaqMan real-time PCR quantification
Conclusion
All in all, this work established two TaqMan real-time PCR assays to quantify CYP2D6 gene copy numbers on the LightCycler 2.0 platform. By testing 617 samples, these methods are established to be efficient, reliable and accurate for a broad spectrum of samples with different conditions and concentrations. Even though further verifications are required, contrasting exon 9 and intron 6 gene ratio results may even allow screening for CYP2D6⁎16 and exon 9 gene conversions. Many laboratories with
Acknowledgements
We thank Dr. Julia Bachofer, Tobias Müller and Christian Schuol for collecting DNA samples and Christine Grubmüller for her technical assistance.
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