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Malaria Journal
Published in: Malaria Journal 1/2018

Open Access 01-12-2018 | Methodology

Real time PCR detection of common CYP2D6 genetic variants and its application in a Karen population study

Authors: Kanokpich Puaprasert, Cindy Chu, Naowarat Saralamba, Nicholas P. J. Day, Francois Nosten, Nicholas J. White, Arjen M. Dondorp, Mallika Imwong

Published in: Malaria Journal | Issue 1/2018

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Abstract

Background

Plasmodium vivax malaria is characterized by relapses arising from the hypnozoite stages in the liver. The only currently registered drug for radical treatment to prevent relapse is primaquine. Primaquine, a prodrug, requires metabolism through the liver cytochrome CYP2D6 isoenzyme to its active metabolite. Mutations in the CYP2D6 gene may thus affect primaquine efficacy. A SNPs genotyping technique was developed to characterize the CYP2D6 genetic variants and tested this in the patients with Plasmodium vivax infection collected in a Karen population on the Thailand–Myanmar border, where P. vivax malaria is endemic.

Methods

Direct sequencing of PCR-reamplified products (DSP) was used to uncover exonic CYP2D6 sequence variations. Subsequently, an allele-specific oligonucleotide probe real-time SNPs genotyping (ASO) assay was developed for rapid detection of the four clinically relevant CYP2D6 variants occurring in this population. These two in-house developed assays were used to genotype CYP2D6 mutations in blood samples obtained from 70 Karen adults.

Results

Results showed a high degree of concordance between the DSP and ASO methods. Six CYP2D6 point mutations were identified within the Karen population: C100T, C1039T, G1661C, G1846A, C2850T and G4180C, at frequencies of 0.43, 0.43, 0.76, 0.02, 0.32 and 0.76, respectively. The CYP2D6*2, *4, *5, *10 and *36 allelic frequencies were 0.33, 0.02, 0.03, 0.40 and 0.01, respectively. Alleles conferring an intermediate CYP2D6 metabolizer phenotype comprised 46% of the total number of alleles.

Conclusion

The newly developed ASO assay is a reliable and rapid tool for large-scale CYP2D6 genotyping. The high frequency of the CYP2D6*10 allele in the Karen population warrants further assessment of its association with the radical curative efficacy of primaquine.
Appendix
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Literature
1.
Fernando D, Rodrigo C, Rajapakse S. Primaquine in vivax malaria: an update and review on management issues. Malar J. 2011;10:351.CrossRef
2.
Mihaly GW, Ward SA, Edwards G, Orme ML, Breckenridge AM. Pharmacokinetics of primaquine in man: identification of the carboxylic acid derivative as a major plasma metabolite. Br J Clin Pharmacol. 1984;17:441–6.CrossRef
3.
Constantino L, Paixao P, Moreira R, Portela M, Do Rosario V, Iley J. Metabolism of primaquine by liver homogenate fractions: evidence for monoamine oxidase and cytochrome P450 involvement in the oxidative deamination of primaquine to carboxyprimaquine. Exp Toxicol Pathol. 1999;51:299–303.CrossRef
4.
Pybus BS, Sousa JC, Jin X, Ferguson JA, Christian RE, Barnhart R, et al. CYP450 phenotyping and accurate mass identification of metabolites of the 8-aminoquinoline, anti-malarial drug primaquine. Malar J. 2012;11:259.CrossRef
5.
Vale N, Moreira R, Gomes P. Primaquine revisited six decades after its discovery. Eur J Med Chem. 2009;44:937–53.CrossRef
6.
Vasquez-Vivar J, Augusto O. Hydroxylated metabolites of the antimalarial drug primaquine. Oxidation and redox cycling. J Biol Chem. 1992;267:848–54.
7.
Idowu O, Peggins J, Brewer T, Kelley C. Metabolism of a candidate 8-aminoquinoline antimalarial agent, WR 238605, by rat liver microsomes. Drug Metab Dispos. 1995;23:1–17.PubMed
8.
Jin X, Pybus B, Marcsisin S, Logan T, Luong T, Sousa J, et al. An LC–MS based study of the metabolic profile of primaquine, an 8-aminoquinoline antiparasitic drug, with an in vitro primary human hepatocyte culture model. Eur J Drug Metab Pharmacokinet. 2014;39:139–46.CrossRef
9.
Pybus BS, Marcsisin SR, Jin X, Deye G, Sousa JC, Li Q, et al. The metabolism of primaquine to its active metabolite is dependent on CYP 2D6. Malar J. 2013;12:212.CrossRef
10.
Potter BM, Xie LH, Vuong C, Zhang J, Zhang P, Duan D, et al. Differential CYP 2D6 metabolism alters primaquine pharmacokinetics. Antimicrob Agents Chemother. 2015;59:2380–7.CrossRef
11.
Marcsisin SR, Reichard G, Pybus BS. Primaquine pharmacology in the context of CYP 2D6 pharmacogenomics: current state of the art. Pharmacol Ther. 2016;161:1–10.CrossRef
12.
Bennett JW, Pybus BS, Yadava A, Tosh D, Sousa JC, McCarthy WF, et al. Primaquine failure and cytochrome P-450 2D6 in Plasmodium vivax malaria. N Engl J Med. 2013;369:1381–2.CrossRef
13.
Silvino ACR, Costa GL, de Araujo FCF, Ascher DB, Pires DEV, Fontes CJF, et al. Variation in human cytochrome P-450 drug-metabolism genes: a gateway to the understanding of Plasmodium vivax relapses. PLoS ONE. 2016;11:e0160172.CrossRef
14.
Kimura S, Umeno M, Skoda R, Meyer U, Gonzalez F. The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene. Am J Hum Genet. 1989;45:889–904.PubMedPubMedCentral
15.
Meijerman I, Sanderson LM, Smits PH, Beijnen JH, Schellens JH. Pharmacogenetic screening of the gene deletion and duplications of CYP2D6. Drug Metab Rev. 2007;39:45–60.CrossRef
16.
Gaedigk A, Fuhr U, Johnson C, Bérard A, Bradford LD, Leeder JS. CYP2D7–2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction. Pharmacogenomics. 2010;11:43–53.CrossRef
17.
Kramer WE, Walker DL, O’Kane DJ, Mrazek DA, Fisher PK, Dukek BA, et al. CYP2D6: novel genomic structures and alleles. Pharmacogenet Genomics. 2009;19:813–22.CrossRef
18.
Gonzalez FJ, Skodat RC, Kimura S, Umeno M, Zanger UM, Nebert DW, et al. Characterization of the common genetic defect in humans deficient in debrisoquine metabolism. Nature. 1988;331:442–6.CrossRef
19.
Heim MH, Meyer UA. Evolution of a highly polymorphic human cytochrome P450 gene cluster: cYP2D6. Genomics. 1992;14:49–58.CrossRef
20.
Gaedigk A, Bradford LD, Alander SW, Leeder JS. CYP2D6* 36 gene arrangements within the cyp2d6 locus: association of CYP2D6* 36 with poor metabolizer status. Drug Metab Dispos. 2006;34:563–9.CrossRef
21.
Gaedigk A, Bertino JS Jr, Bérard A, Pratt V, Bradford L, Leeder JS. Identification of novel CYP2D7-2D6 hybrids: non-functional and functional variants. Front Pharmacol. 2010;1:121.CrossRef
22.
Hastings P, Lupski JR, Rosenberg SM, Ira G. Mechanisms of change in gene copy number. Nat Rev Genet. 2009;10:551–64.CrossRef
23.
Gaedigk A, Ndjountche L, Divakaran K, Dianne Bradford L, Zineh I, Oberlander T, et al. Cytochrome P4502D6 (CYP2D6) gene locus heterogeneity: characterization of gene duplication events. Clin Pharmacol Ther. 2007;81:242–51.CrossRef
24.
Steen VM, Molven A, Aarskog NK, Gulbrandsen A-K. Homologous unequal cross-over involving a 2.8 kb direct repeat as a mechanism for the generation of allelic variants of the human cytochrome P450 CYP2D6 gene. Hum Mol Genet. 1995;4:2251–7.CrossRef
25.
Gaedigk A. Complexities of CYP2D6 gene analysis and interpretation. Int Rev Psychiatry. 2013;25:534–53.CrossRef
26.
Evans WE, Relling MV. Moving towards individualized medicine with pharmacogenomics. Nature. 2004;429:464–8.CrossRef
27.
Ma MK, Woo MH, Mcleod HL. Genetic basis of drug metabolism. Am J Health Syst Pharm. 2002;59:2061–9.PubMed
28.
Bertilsson L. Geographical/interracial differences in polymorphic drug oxidation. Clin Pharmacokinet. 1995;29:192–209.CrossRef
29.
Dorado P, Caceres M, Pozo-Guisado E, Wong ML, Licinio J, Llerena A. Development of a PCR-based strategy for CYP2D6 genotyping including gene multiplication of worldwide potential use. Biotechniques. 2005;39:S571–4.CrossRef
30.
St Jean PL, Xue Z, Carter N, Koh GC, Duparc S, Taylor M, et al. Tafenoquine treatment of Plasmodium vivax malaria: suggestive evidence that CYP2D6 reduced metabolism is not associated with relapse in the Phase 2b DETECTIVE trial. Malar J. 2016;15:97.CrossRef
31.
Vuong C, Xie LH, Potter BM, Zhang J, Zhang P, Duan D, et al. Differential CYP 2D metabolism alters tafenoquine pharmacokinetics. Antimicrob Agents Chemother. 2015;59:3864–9.CrossRef
32.
De Gregori M, Allegri M, De Gregori S, Garbin G, Tinelli C, Regazzi M, et al. How and why to screen for CYP2D6 interindividual variability in patients under pharmacological treatments. Curr Drug Metab. 2010;11:276–82.CrossRef
33.
Heim M, Meyer U. Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification. Lancet. 1990;336:529–32.CrossRef
34.
Gaedigk A, Gotschall RR, Forbes NS, Simon SD, Kearns GL, Leeder JS. Optimization of cytochrome P4502D6 (CYP2D6) phenotype assignment using a genotyping algorithm based on allele frequency data. Pharmacogenet Genomics. 1999;9:669–82.CrossRef
35.
Broly F, Marez D, Sabbagh N, Legrand M, Millecamps S, Guidice JL, et al. An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism analysis. Pharmacogenet Genomics. 1995;5:373–84.CrossRef
36.
Marez D, Legrand M, Sabbagh N, Guidice JL, Spire C, Lafitte J, et al. Polymorphism of the cytochrome P450 CYP2D6 gene in a European population: characterization of 48 mutations and 53 alleles, their frequencies and evolution. Pharmacogenet Genomics. 1997;7:193–202.CrossRef
37.
Stüven T, Griese E-U, Kroemer HK, Eichelbaum M, Zanger UM. Rapid detection of CYP2D6 null alleles by long distance-and multiplex-polymerase chain reaction. Pharmacogenet Genomics. 1996;6:417–21.CrossRef
38.
Hersberger M, Marti-Jaun J, Rentsch K, Hänseler E. Rapid detection of the CYP2D6* 3, CYP2D6* 4, and CYP2D6* 6 alleles by tetra-primer PCR and of the CYP2D6* 5 allele by multiplex long PCR. Clin Chem. 2000;46:1072–7.PubMed
39.
Labuda D, Krajinovic M, Richer C, Skoll A, Sinnett H, Yotova V, et al. Rapid detection of CYP1A1, CYP2D6, and NAT variants by multiplex polymerase chain reaction and allele-specific oligonucleotide assay. Anal Biochem. 1999;275:84–92.CrossRef
40.
Stamer UM, Bayerer B, Wolf S, Hoeft A, Stüber F. Rapid and reliable method for cytochrome P450 2D6 genotyping. Clin Chem. 2002;48:1412–7.PubMed
41.
Müller B, Zöpf K, Bachofer J, Steimer W. Optimized strategy for rapid cytochrome P450 2D6 genotyping by real-time long PCR. Clin Chem. 2003;49:1624–31.CrossRef
42.
Arneth B, Shams M, Hiemke C, Härtter S. Rapid and reliable genotyping procedure for detection of alleles with mutations, deletion, or/and duplication of the CYP2D6 gene. Clin Biochem. 2009;42:1282–90.CrossRef
43.
Langaee T, Hamadeh I, Chapman AB, Gums JG, Johnson JA. A novel simple method for determining CYP2D6 gene copy number and identifying allele (s) with duplication/multiplication. PLoS ONE. 2015;10:e0113808.CrossRef
44.
Suwannasri P, Thongnoppakhun W, Pramyothin P, Assawamakin A, Limwongse C. Combination of multiplex PCR and DHPLC-based strategy for CYP2D6 genotyping scheme in Thais. Clin Biochem. 2011;44:1144–52.CrossRef
45.
Vanwong N, Ngamsamut N, Hongkaew Y, Nuntamool N, Puangpetch A, Chamnanphon M, et al. Detection of CYP2D6 polymorphism using Luminex xTAG technology in autism spectrum disorder: CYP2D6 activity score and its association with risperidone levels. Drug Metab Pharmacokinet. 2016;31:156–62.CrossRef
46.
Murphy GM, Pollock BG, Kirshner MA, Pascoe N, Cheuk W, Mulsant BH, et al. CYP2D6 genotyping with oligonucleotide microarrays and nortriptyline concentrations in geriatric depression. Neuropsychopharmacology. 2001;25:737–43.CrossRef
47.
Leon Jd, Susce MT, Johnson M, Hardin M, Maw L, Shao A, et al. DNA microarray technology in the clinical environment: the AmpliChip CYP450 test for CYP2D6 and CYP2C19 genotyping. CNS Spectr. 2009;14:19–34.CrossRef
48.
Burmester JK, Sedova M, Shapero MH, Mansfield E. DMET™ microarray technology for pharmacogenomics-based personalized medicine. Methods Mol Biol. 2010;632:99–124.CrossRef
49.
Sukasem C, Sirachainan E, Chamnanphon M, Pechatanan K, Sirisinha T, Ativitavas T, et al. Impact of CYP2D6 polymorphisms on tamoxifen responses of women with breast cancer: a microarray-based study in Thailand. Asian Pac J Cancer Prev. 2012;13:549–53.CrossRef
50.
Chamnanphon M, Pechatanan K, Sirachainan E, Trachu N, Chantratita W, Pasomsub E, et al. Association of CYP2D6 and CYP2C19 polymorphisms and disease-free survival of Thai post-menopausal breast cancer patients who received adjuvant tamoxifen. Pharmgenomics Pers Med. 2013;6:37–48.PubMedPubMedCentral
51.
Suwannarat P, Chamnanphon M, Ngamsamut AS, Chamkrachchangpada B, Tan-kam T, Puangpetch A, et al. Molecular genetic analysis of CYP2D6and HLA-B* 15: 02 in Thai autistic spectrum disorder children: implication for pharmacogenetics testing and optimization of drug treatments. Thai J Genet. 2013;6:82–7.
52.
Ji L, Pan S, Marti-Jaun J, Hänseler E, Rentsch K, Hersberger M. Single-step assays to analyze CYP2D6 gene polymorphisms in Asians: allele frequencies and a novel* 14B allele in mainland Chinese. Clin Chem. 2002;48:983–8.PubMed
53.
Qin S, Shen L, Zhang A, Xie J, Shen W, Chen L, et al. Systematic polymorphism analysis of the CYP2D6 gene in four different geographical Han populations in mainland China. Genomics. 2008;92:152–8.CrossRef
54.
Garcia-Barceló M, Chow LY, Chiu HFK, Wing YK, Lee DTS, Lam KL, et al. Genetic analysis of the CYP2D6 locus in a Hong Kong Chinese population. Clin Chem. 2000;46:18–23.PubMed
55.
Kim E-Y, Lee S-S, Jung H-J, Jung H-E, Yeo C-W, Shon J-H, et al. Robust CYP2D6 genotype assay including copy number variation using multiplex single-base extension for Asian populations. Clin Chim Acta. 2010;411:2043–8.CrossRef
56.
Tateishi T, Chida M, Ariyoshi N, Mizorogi Y, Kamataki T, Kobayashi S. Analysis of the CYP2D6 gene in relation to dextromethorphan O-demethylation capacity in a Japanese population. Clin Pharmacol Ther. 1999;65:570–5.CrossRef
57.
Nishida Y, Fukuda T, Yamamoto I, Azuma J. CYP2D6 genotypes in a Japanese population: low frequencies of CYP2D6 gene duplication but high frequency of CYP2D6* 10. Pharmacogenet Genomics. 2000;10:567–70.CrossRef
58.
Ishiguro A, Kubota T, Sasaki H, Yamada Y, Iga T. Common mutant alleles of CYP2D6 causing the defect of CYP2D6 enzyme activity in a Japanese population. Br J Clin Pharmacol. 2003;55:414–5.CrossRef
59.
Lee S-Y, Sohn KM, Ryu JY, Yoon YR, Shin JG, Kim J-W. Sequence-based CYP2D6 genotyping in the Korean population. Ther Drug Monit. 2006;28:382–7.CrossRef
60.
Lee S-J, Lee SS, Jung H-J, Kim H-S, Park S-J, Yeo C-W, et al. Discovery of novel functional variants and extensive evaluation of CYP2D6 genetic polymorphisms in Koreans. Drug Metab Dispos. 2009;37:1464–70.CrossRef
61.
Park HS, Choi J-Y, Lee M-J, Park S, Yeo C-W, Lee SS, et al. Association between genetic polymorphisms of CYP2D6 and outcomes in breast cancer patients with tamoxifen treatment. J Korean Med Sci. 2011;26:1007–13.CrossRef
62.
Naveen AT, Adithan C, Soya SS, Gerard N, Krishnamoorthy R. CYP2D6 genetic polymorphism in South Indian populations. Biol Pharm Bull. 2006;29:1655–8.CrossRef
63.
Sachse C, Brockmöller J, Bauer S, Roots I. Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences. Am J Hum Genet. 1997;60:284–95.PubMedPubMedCentral
64.
Mendoza R, Wan YJY, Poland RE, Smith M, Zheng Y, Berman N, et al. CYP2D6 polymorphism in a Mexican American population. Clin Pharmacol Ther. 2001;70:552–60.CrossRef
65.
Menoyo A, del Rio E, Baiget M. Characterization of variant alleles of cytochrome CYP2D6 in a Spanish population. Cell Biochem Funct. 2006;24:381–5.CrossRef
66.
Wan Y-JY, Poland RE, Han G, Konishi T, Zheng Y-P, Berman N, et al. Analysis of the CYP2D6 gene polymorphism and enzyme activity in African-Americans in southern California. Pharmacogenet Genomics. 2001;11:489–99.CrossRef
67.
Cai W, Nikoloff D, Pan R, De Leon J, Fanti P, Fairchild M, et al. CYP2D6 genetic variation in healthy adults and psychiatric African-American subjects: implications for clinical practice and genetic testing. Pharmacogenomics J. 2006;6:343–50.CrossRef
68.
Yee MM, Josephson C, Hill CE, Harrington R, Castillejo M-I, Ramjit R, et al. Cytochrome P450 2D6 polymorphisms and predicted opioid metabolism in African-American children with sickle cell disease. J Pediatr Hematol Oncol. 2013;35:e301–5.CrossRef
Metadata
Title
Real time PCR detection of common CYP2D6 genetic variants and its application in a Karen population study
Authors
Kanokpich Puaprasert
Cindy Chu
Naowarat Saralamba
Nicholas P. J. Day
Francois Nosten
Nicholas J. White
Arjen M. Dondorp
Mallika Imwong
Publication date
01-12-2018
Publisher
BioMed Central
Published in
Malaria Journal / Issue 1/2018
Electronic ISSN: 1475-2875
DOI
https://doi.org/10.1186/s12936-018-2579-8

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