Abstract
CYP2C19 is a polymorphic enzyme involved in the metabolism of clinically important drugs. Genotype-phenotype association studies of CYP2C19 have reported wide ranges in the metabolic ratios of its substrates. These discrepancies could be attributed to the variations in the promoter region and this aspect has been reported recently. The observations in the recent reports on the influence of promoter region variants on the metabolism of CYP2C19 substrates might also have been influenced by the copy number variations of CYP2C19. In this paper, we describe copy number variations of CYP2C19 using real-time polymerase chain reaction by comparative Ct method. No copy number variations were observed in the south Indian population indicating the observed discrepancies in genotype-phenotype association studies might be due to the regulatory region polymorphisms as reported earlier.
1. Introduction
The CYP2C19 is a clinically important drug metabolizing enzyme encoded by polymorphic CYP2C19 gene on chromosome 10, playing a major role in metabolizing about 5% of the clinically used drugs [1–5]. Owing to genetic polymorphisms, considerable interindividual variability exists in the metabolic activity of this enzyme [6]. About 43 variant alleles of CYP2C19 have been reported till date (http://www.imm.ki.se/CYPalleles, access date: 28th March 2012). Most of the genotype-phenotype association studies of CYP2C19 in different ethnic groups were focused on exonic region variants, but the promoter region variants and copy number variations were not explored to a greater extent [7–11]. Based on the findings of genotype-phenotype association studies, subjects were categorized as poor metabolizers (PMs), having either of the most commonly seen defective alleles of CYP2C19, namely, c.636G>A (*3 allele; rs4986893) or c.681G>A (*2 allele; rs4244285); extensive metabolizers (EMs) carrying no variant alleles; ultra-rapid metabolizers (UMs) carrying −806C>T and −3402C>T variations (*17 allele) in the promoter region of CYP2C19 [12]. Thus, the activity of CYP2C19 varies with the presence or absence of certain variations in its gene which also varies in its distribution among different ethnic groups [8, 13–18].
CYP2C19 activity differs among extensive metabolizers (EM), demonstrated by a wide range in the metabolic ratios (MR) of probe drugs, and some discrepancies were commonly noted in these reports [11–20]. These differences could be attributed to rare defective alleles or to polymorphisms in the regulatory region of CYP2C19 gene and copy number variants of the gene [20, 21]. Few studies have reported the promoter region variations of CYP2C19 in different populations [13, 14, 22–24]. Functional characterization of the promoter region polymorphisms has been performed in a recent study from our laboratory describing the influence of promoter region polymorphisms on proguanil oxidation to an active compound mainly by CYP2C19 [23, 24]. In this paper we concluded that the discrepancies observed in genotype-phenotype association studies of CYP2C19 were due to promoter region polymorphisms influencing the expression of CYP2C19. However, the existence of copy number variations and the variability in the basal levels of expression of CYP2C19 between individuals was not considered, which might have affected the observations. Thus an alternative explanation for these discrepancies could be the copy number variations of this gene. Therefore, here in this paper we describe the results of an explorative study analyzing copy number variations of CYP2C19 in the study subjects of the previous report.
2. Methods
The relative copy number was estimated using a real-time PCR (comparative ΔΔCt method of relative quantification) approach based on existing methodologies with gene-specific primers using IL-2 as reference gene using genomic DNA obtained from the peripheral white blood cells [25, 26]. Cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to cross the threshold, that is, exceeding the background level. The efficiency and specificity of primer pairs for CYP2C19 (F: 5′-GCC ATT TCC CAC TGG CTG AAA G-3′; R: 5′-ACG AAA CTA GGA GGG AG TCC-3′) and IL-2 (F: 5′-TGC ACA TAA TCT CAT CTT TCT AAC ACT CTT-3′; R: 5′-TTG AAA GCG CAA TAG ATG GAC AT-3′) from genomic sequence were evaluated using serial 10-fold dilutions of human genomic DNA (n = 50) ranging from 0.2 to 200 ng. The demographic details of the subjects were discussed elsewhere [23]. The primers used for CYP2C19 amplification are specific to prevent the amplification of other homologous genes such as CYP2C9 located on the same chromosome. Total reaction volume consists of 25 μL and contained the following: 12.5 μL of 2X SYBR Green Master Mix (Sigma-Aldrich, St. Louis, USA), human gDNA (0.2 to 200 ng with equal volume, that is, the total amount in 2 μL) and the appropriate primer pair (400 nM). The PCR thermoprofile consisted of initial activation of the polymerase at 95°C for 15 min followed by 40 PCR cycles of 95°C for 15 s, and 60°C for 1 min (40 cycles) followed by a melting curve to verify the presence of a single amplification product. Assays were performed in duplicate on an Applied Biosystems 7300 Fast Real-Time PCR system using genomic DNA of 2 μL (approximately 20 ng). The PCR was performed in a 96-well clear optical reaction plate MicroAmp (Applied Biosystems). IL-2 gene was coamplified with CYP2C19 gene and served as internal standard (reference gene). CYP2C19 copy number was calculated relative to the human gDNA sample with no genetic variations in exonic and promoter regions, showing a good genotype-phenotype correlation (standard). The following equation was used: relative copy number = 2∗2−ΔΔCt, where −ΔΔCt = (Ct of reference gene − Ct of target gene) of standard − (Ct of reference gene − Ct of target gene) of sample. Mean Ct values were taken for each sample derived from duplicate reactions. Efficiency of the assay was calculated using the formula E = 10(−1/slope) − 1 × 100. The calculated relative copy numbers were confirmed by recalculating the haploid copy number from the observed C t values, and then multiplied by 2 to give the diploid copy number. The alternate formula used for calculation of haploid copy number was 2−ΔΔCt = (1+E)−ΔCttest gene/(1=E)−ΔCt reference gene, where E = Efficiency of the PCR reaction; ΔCt test gene = Difference in the threshold cycle value between the test sample and calibrator sample for the gene (test gene) under investigation and ΔCt reference gene = Difference in the threshold cycle value between the test sample and calibrator sample for reference gene.
GraphPad Instat version 3.06 (San Diego, USA) was used for statistical analysis. Data were expressed as mean ± SD. The copy number range from 1.7–2.1 was considered as the normal diploid copy number.
3. Results and Discussion
The efficiency of the assay for IL-2 and CYP2C19 was between 87–100% and 94–100%, respectively. The efficiencies were similar and the assay stability was good. The slope of the standard curve for IL-2 was in the range of −2.7 to −4.3 with R 2 (mean) of 0.99. The slope of the standard curve for CYP2C19 was in the range of −3.2 to −4.5 with R 2 (mean) of 0.99. The product sizes for CYP2C19 and IL-2 were 193 bp and 93 bp, respectively. Dissociation curves have shown the specific melting temperatures for both IL-2 and CYP2C19 products and/or any primer dimer formed during amplification. The mean Ct values observed for the CYP2C19 and IL-2 standards are given in Table 1. The mean Ct values of CYP2C19 and IL-2 from healthy volunteers are given in Table 2. The amplification plots, dissociation curves, and standard curves of CYP2C19 and IL-2 are given in the Supplementary Material available online at doi: 10.1155/2012/643856. No copy number variations of CYP2C19 were detected in south Indian population. All the samples studied were found to have two copies of the gene. The CYP2C19 copy numbers were in the range of 1.7–2.17 with slight deviation from the set value 1.7–2.1 (Table 2). The values were obtained after taking mean values of ΔΔCt from 2 reactions of the same sample and calculating copy number of each sample using formula 2∗2−ΔΔCt. The results of this study are in accordance with the observations made by using comparative genomic hybridization technique with bacterial artificial chromosome of an individual (personal communication from Manipal Life Sciences Center, Manipal, India).
Table 1.
Mean Ct values for standard CYP2C19 and IL-2 gene amplification.
| Amount of genomic DNA in standards (ng) | CYP2C19 Ct value (mean ± SD; n = 2) |
IL-2 Ct value (mean ± SD; n = 2) |
|---|---|---|
| 200 | 20.77 ± 0.084 | 22.55 ± 0.19 |
| 20 | 23.59 ± 0.076 | NA |
| 2 | 26.43 ± 0.092 | 28.81 ± 0.092 |
| 0.2 | 28.90 ± 0.074 | 32.30 ± 0.096 |
NA: not included in the analysis. The standard curve was obtained using three points.
Table 2.
Mean Ct values of CYP2C19 and IL-2 genes amplified from genomic DNA of 50 healthy volunteers.
| Ct value for CYP2C19 (mean ± SD; n = 50) | Ct value for IL-2 (mean ± SD; n = 50) | Number of copies calculated from 2−ΔCt (range) |
|---|---|---|
| 22.23 ± 0.995 | 25.15 ± 0.965 | 1.7–2.171 |
Absence of the copy number variations of CYP2C19 in south Indian population indicates that the genetic variations such as single-nucleotide polymorphisms may predict the changes in the activity of the enzyme. This observation also indicates that the discrepancies in genotype-phenotype association studies and wide ranges in the metabolic ratios of CYP2C19 substrates in EMs are due to other variations in the regulatory region. The involvement of other CYP isoforms in the metabolism of CYP2C19 substrates may also contribute to the discrepancies observed in genotype-phenotype association studies. Further studies are needed on a larger scale to investigate the copy number variations of CYP2C19 in different disease states in comparison to the healthy volunteers.
4. Conclusion
We here by conclude that no copy number variations of CYP2C19 were found in south Indian population suggesting that the previously observed discrepancies of phenotype-genotype studies could be due to the variations in the regulatory region as reported earlier.
Supplementary Material
The amplification plots for CYP2C19, IL-2 gene amplification are depicted in the supplementary figures 1 and 2 (where x-axis represents the cycle number and y-axis represents ΔRn reflecting the magnitude of the signal generated by PCR conditions used for the experiment). The dissociation curves specific for CYP2C19 and IL-2 amplification are given in the supplementary figures 3 and 4 (where x-axis represents temperature in °C and y-axis represents the derivative of fluorescence with respect to temperature). The standard curves for CYP2C19 and IL-2 gene quantification are given in the figures 5 and 6 (where x-axis represents the log transformed concentrations of the standards used and y-axis represents the cycle threshold (Ct) reflecting the cycle number at which the fluorescence generated within a reaction crossing the threshold level.
Acknowledgments
The study was funded by Indian Council of Medical Research (ICMR) and Institut National de La Santea et de La Recherche Medicale (INSERM) collaborative research project (ICMR Sanction Order no. 50/6/2004 dated 05/10/06).
References
- 1.Goldstein JA. Clinical relevance of genetic polymorphisms in the human CYP2C subfamily. British Journal of Clinical Pharmacology. 2001;52(4):349–355. doi: 10.1046/j.0306-5251.2001.01499.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Ingelman-Sundberg M, Sim SC, Gomez A, Rodriguez-Antona C. Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects. Pharmacology and Therapeutics. 2007;116(3):496–526. doi: 10.1016/j.pharmthera.2007.09.004. [DOI] [PubMed] [Google Scholar]
- 3.Bertilsson L, Dahl ML, Ingelman-Sundberg M, Johansson I, Sjöqvist F. Interindividual and interethnic differences in polymorphic drug oxidation-implications for drug therapy with focus on psychoactive drugs. In: Pacific GM, Fracchia GN, editors. Advances in Drug Metabolism in Man. Luxembourg: Office for Official Publications of the European Communities; 1995. pp. 85–136. [Google Scholar]
- 4.Brøsen K, de Morais SMF, Meyer UA, Goldstein JA. A multifamily study on the relationship between CYP2C19 genotype and S-mephenytoin oxidation phenotype. Pharmacogenetics. 1995;5(5):312–317. doi: 10.1097/00008571-199510000-00007. [DOI] [PubMed] [Google Scholar]
- 5.Ellis KJ, Stouffer GA, McLeod HL, Lee CR. Clopidogrel pharmacogenomics and risk of inadequate platelet inhibition: US FDA recommendations. Pharmacogenomics. 2009;10(11):1799–1817. doi: 10.2217/pgs.09.143. [DOI] [PubMed] [Google Scholar]
- 6.Murayama N, Imai N, Nakane T, Shimizu M, Yamazaki H. Roles of CYP3A4 and CYP2C19 in methyl hydroxylated and N-oxidized metabolite formation from voriconazole, a new anti-fungal agent, in human liver microsomes. Biochemical Pharmacology. 2007;73(12):2020–2026. doi: 10.1016/j.bcp.2007.03.012. [DOI] [PubMed] [Google Scholar]
- 7.He N, Yan FX, Huang SL, et al. CYP2C19 genotype and S-mephenytoin 4′-hydroxylation phenotype in a Chinese Dai population. European Journal of Clinical Pharmacology. 2002;58(1):15–18. doi: 10.1007/s00228-002-0425-x. [DOI] [PubMed] [Google Scholar]
- 8.Desta Z, Zhao X, Shin JG, Flockhart DA. Clinical significance of the cytochrome P450 2C19 genetic polymorphism. Clinical Pharmacokinetics. 2002;41(12):913–958. doi: 10.2165/00003088-200241120-00002. [DOI] [PubMed] [Google Scholar]
- 9.Chang M, Dahl ML, Tybring G, Götharson E, Bertilsson L. Use of omeprazole as a probe drug for CYP2C19 phenotype in swedish caucasians: comparison with S-mephenytoin hydroxylation phenotype and CYP2C19 genotype. Pharmacogenetics. 1995;5(6):358–363. doi: 10.1097/00008571-199512000-00004. [DOI] [PubMed] [Google Scholar]
- 10.Hoskins JM, Shenfield GM, Gross AS. Concordance between proguanil phenotype and CYP2C19 genotype in Chinese. European Journal of Clinical Pharmacology. 2003;59(8-9):611–614. doi: 10.1007/s00228-003-0665-4. [DOI] [PubMed] [Google Scholar]
- 11.Chang M, Tybring G, Dahl ML, et al. Interphenotype differences in disposition and effect on gastrin levels of omeprazole suitability of omeprazole as a probe for CYP2C19 . British Journal of Clinical Pharmacology. 1995;39(5):511–518. doi: 10.1111/j.1365-2125.1995.tb04488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Sim SC, Risinger C, Dahl ML, et al. A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants. Clinical Pharmacology and Therapeutics. 2006;79(1):103–113. doi: 10.1016/j.clpt.2005.10.002. [DOI] [PubMed] [Google Scholar]
- 13.Blaisdell J, Mohrenweiser H, Jackson J, et al. Identification and functional characterization of new potentially defective alleles of human CYP2C19 . Pharmacogenetics. 2002;12(9):703–711. doi: 10.1097/00008571-200212000-00004. [DOI] [PubMed] [Google Scholar]
- 14.Arefayene M, Skaar TC, Zhao X, et al. Sequence diversity and functional characterization of the 5′-regulatory region of human CYP2C19 . Pharmacogenetics. 2003;13(4):199–206. doi: 10.1097/00008571-200304000-00004. [DOI] [PubMed] [Google Scholar]
- 15.Hanioka N, Tsuneto Y, Saito Y, et al. Functional characterization of two novel CYP2C19 variants (CYP2C19∗18 and CYP2C19∗19) found in a Japanese population. Xenobiotica. 2007;37(4):342–355. doi: 10.1080/00498250601127038. [DOI] [PubMed] [Google Scholar]
- 16.Furuta T, Shirai N, Sugimoto M, Nakamura A, Hishida A, Ishizaki T. Influence of CYP2C19 pharmacogenetic polymorphism on proton pump inhibitor-based therapies. Drug Metabolism and Pharmacokinetics. 2005;20(3):153–167. doi: 10.2133/dmpk.20.153. [DOI] [PubMed] [Google Scholar]
- 17.Taubert D, Bouman HJ, Van Werkum JW. Cytochrome P-450 polymorphisms and response to clopidogrel. The New England Journal of Medicine. 2009;360(21):2249–2250. doi: 10.1056/NEJMc090391. [DOI] [PubMed] [Google Scholar]
- 18.Norgard NB, Mathews KD, Wall GC. Drug-drug interaction between clopidogrel and the proton pump inhibitors. Annals of Pharmacotherapy. 2009;43(7-8):1266–1274. doi: 10.1345/aph.1M051. [DOI] [PubMed] [Google Scholar]
- 19.Kim MJ, Bertino JS, Jr., Gaedigk A, Zhang Y, Sellers EM, Nafziger AN. Effect of sex and menstrual cycle phase on cytochrome P450 2C19 activity with omeprazole used as a biomarker. Clinical Pharmacology and Therapeutics. 2002;72(2):192–199. doi: 10.1067/mcp.2002.126174. [DOI] [PubMed] [Google Scholar]
- 20.Rosemary J, Adithan C, Padmaja N, Shashindran CH, Gerard N, Krishnamoorthy R. The effect of the CYP2C19 genotype on the hydroxylation index of omeprazole in South Indians. European Journal of Clinical Pharmacology. 2005;61(1):19–23. doi: 10.1007/s00228-004-0833-1. [DOI] [PubMed] [Google Scholar]
- 21.Pastinen T, Hudson TJ. Cis-acting regulatory variation in the human genome. Science. 2004;306(5696):647–650. doi: 10.1126/science.1101659. [DOI] [PubMed] [Google Scholar]
- 22.Fukushima-Uesaka H, Saito Y, Maekawa K, et al. Genetic variations and haplotypes of CYP2C19 in a Japanese population. Drug Metabolism and Pharmacokinetics. 2005;20(4):300–307. doi: 10.2133/dmpk.20.300. [DOI] [PubMed] [Google Scholar]
- 23.Satyanarayana CR, Devendran A, Jayaraman M, et al. Influence of the genetic polymorphisms in the 5′ flanking and exonic regions of CYP2C19 on proguanil oxidation. Drug Metabolism and Pharmacokinetics. 2009;24(6):537–548. doi: 10.2133/dmpk.24.537. [DOI] [PubMed] [Google Scholar]
- 24.Satyanarayana CRU, Devendran A, Sundaram R, Gopal SD, Rajagopal K, Chandrasekaran A. Genetic variations and haplotypes of the 5′ regulatory region of CYP2C19 in South Indian population. Drug Metabolism and Pharmacokinetics. 2009;24(2):185–193. doi: 10.2133/dmpk.24.185. [DOI] [PubMed] [Google Scholar]
- 25.Hoebeeck J, Speleman F, Vandesompele J. Real-time quantitative PCR as an alternative to southern blot or fluorescence in situ hybridization for detection of gene copy number changes. Methods in Molecular Biology. 2007;353:205–226. doi: 10.1385/1-59745-229-7:205. [DOI] [PubMed] [Google Scholar]
- 26.Löfgren S, Baldwin RM, Hiratsuka M, et al. Generation of mice transgenic for human CYP2C18 and CYP2C19: characterization of the sexually dimorphic gene and enzyme expression. Drug Metabolism and Disposition. 2008;36(5):955–962. doi: 10.1124/dmd.107.019349. [DOI] [PubMed] [Google Scholar]
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Supplementary Materials
The amplification plots for CYP2C19, IL-2 gene amplification are depicted in the supplementary figures 1 and 2 (where x-axis represents the cycle number and y-axis represents ΔRn reflecting the magnitude of the signal generated by PCR conditions used for the experiment). The dissociation curves specific for CYP2C19 and IL-2 amplification are given in the supplementary figures 3 and 4 (where x-axis represents temperature in °C and y-axis represents the derivative of fluorescence with respect to temperature). The standard curves for CYP2C19 and IL-2 gene quantification are given in the figures 5 and 6 (where x-axis represents the log transformed concentrations of the standards used and y-axis represents the cycle threshold (Ct) reflecting the cycle number at which the fluorescence generated within a reaction crossing the threshold level.