Robust CYP2D6 genotype assay including copy number variation using multiplex single-base extension for Asian populations
Introduction
Inter-individual genetic variation in drug responses may cause therapeutic failure or toxicity [1], [2]. Cytochrome P450 (CYP) super family consists of > 50 enzymes such as CYP2C9, CYP2C19, CYP2D6, and CYP3A4 [3]. They metabolize many endogenous compounds, drugs, and xenobiotics. CYP2D6 (GenBank accession number M33388) is involved in metabolizing > 100 drugs, including those that have relatively narrow therapeutic ranges, such as antipsychotic, antidepressant, and antiarrhythmic agents [4], [5]. It is also one of the most genetically variable of the CYPs, with 78 currently identified allelic variants [6]. In addition, there are unique ethnic differences in CYP2D6 genetic variance, which lead to differences in patterns of drug metabolism according to ethnicity [7]. Individuals are classified as ultra-rapid metabolizers (UMs), extensive metabolizers (EMs), intermediate metabolizers (IMs), and poor metabolizers (PMs) according to their enzymatic metabolic ability [8]. Therefore, the drug metabolizing ability determined by the genotype may affect the success or failure of therapy.
Although useful clinically, available methods used for CYP2D6 genotyping, such as PCR-RFLP, AS-PCR, sequencing, and microarray technology, such as AmpliChip® and DrugMEt™, are both costly and time-consuming to perform. Therefore, it is necessary to develop a new method that will allow the testing of all the important functional genetic polymorphisms of CYP2D6 simultaneously and rapidly in a cost-effective manner. A method involving multiplex primer extension reaction, proposed by Sistonen et al. [9], has been developed to overcome these problems. However, this method does not include the detection of copy number variation (CNV), such as duplication and deletion, and cannot be applied to Asian populations, including Koreans, as a result of ethnic differences. Therefore, we have developed additional techniques to perform CYP2D6 genotyping rapidly and simultaneously, to detect significant polymorphisms including CNV in Koreans and other Asians, using a multiplex single-base extension (SBE) assay. In this genotyping method, we also included recently identified functional alleles [10].
Section snippets
Materials and methods
Haplotype tagging SNP (htSNP) combinations were selected using SNPtagger [11], which covers most of the Korean haplotypes, based on our previous frequency results in 758 healthy volunteers [10]. CYP2D6 genotype to phenotype relationship was also evaluated in the previous study. All volunteers gave informed consent to participate in the study which was approved by the Institutional Review Board of Busan Paik Hospital (Busan, Korea). Molecular haplotyping was performed to determine the actual
Results
The electrograms of results with nine different genotypes are shown in Fig. 3. The duplication haplotype case (CYP2D6*2xN/*10) was confirmed by the published method [15]. The origin of the duplication could be recognized by peak height ratio of the polymorphism position compared with non-duplication cases (Fig. 3) in many cases. However there are no genotypes with two duplication alleles from the 758 samples. The combination of selected haplotype tagging SNP (htSNP) in this study was evaluated
Discussion
Individual CYP2D6 genotyping could be helpful in determining dosage at the start of treatment. Several previously developed methods did not cover many of the common or important Korean or Asian alleles, such as CYP2D6*18, *21, *49, *52, and *60 (Table 2), but did contain many alleles not observed among Koreans or other Asians, such as CYP2D6*3, *6, and *17. Therefore, we developed a cost-effective, rapid, and accurate CYP2D6 genotyping method using SBE reactions not only for Koreans, but also
Acknowledgement
This study was supported by a grant of the Korea health 21 R&D Project, Ministry of Health, Welfare, and Family Affairs (A030001) and by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Ministry of Education, Science, and Engineering (MOEST) (No. R13-2007-023-00000-0).
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2016, Drug Metabolism and PharmacokineticsCitation Excerpt :This study appears to confirm previous reports by Suwannarat et al., 2013 which detected CYP2D6 polymorphisms in Thai population [7]. Moreover, our study also compared the allele frequencies with other studies of Thai population and other Asian populations (Chinese, Japanese, and Korean) [7,30,31]. We found that the most common allele was CYP2D6*10 in Chinese (51.60%), Korean (45.60%) and Thai population in different studies (48.07%and 44.6%), which are concordance with the present study (51.79%).
Direct assessment of cytochrome P450 2D6 genotypes by high-resolution melting analysis and DNA sequencing
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2012, Analytica Chimica ActaCitation Excerpt :Developing methods for CYP2D6*10 genotyping is of considerable important due to the clinical significance of CYP2D6*10 in pharmacogenomics. Many strategies are known for the genotyping of CYP2D6, such as single-strand conformation polymorphism (SSCP) [11], allele-specific polymerase chain reaction (AS-PCR) [12], PCR with restriction fragment length polymorphism (PCR-RFLP) analysis [13], real-time PCR [14], pyrosequencing [15], oligonucleotide microarray technology [16], and multiplex single-base extension assay [17]. Though these techniques have been demonstrated to be useful tools for the assay of CYP2D6 typing, the shortages such as limited throughput, poor selectivity, low sensitivity, and the requirement of costly instruments and specific immobilizing reagents, may limit their use in genotyping of CYP2D6*10 in clinics.
Comments on "Analysis of 50 SNPs in CYP2D6, CYP2C19, CYP2C9, CYP3A4 and CYP1A2 by MALDI-TOF mass spectrometry in Chinese Han population"
2011, Forensic Science InternationalCombination of multiplex PCR and DHPLC-based strategy for CYP2D6 genotyping scheme in Thais
2011, Clinical BiochemistryCitation Excerpt :All discordant cases were resolved and revealed the presence of CNV, most of them are in tandem duplication (CYP2D6*36-*10B) and a few with four copies (CYP2D6*10Bx2/*36 × 2). We also compared the allele frequencies derived from our method with other Asian population (Korean, Chinese, and Japanese) genotyped by other methods in Table 5[27]. Allele coverage is over 97%, only a few SNPs has not been characterized here but found at very low frequency in other Asians.
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These authors contributed equally to this work.