Skip to main content

Advertisement

Springer Nature Link
Log in

Alternative Reliable Method for Cytochrome P450 2D6 Poor Metabolizers Genotyping

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

High-resolution melting curve analysis (HRM) of polymerase chain reaction (PCR) amplicons has been described as a fast, cheap, and reliable closed-tube method of genotyping with no need for labeled primers or labeled probes. We adapted this melting analysis assay for the detection of the most common nonfunctional alleles of cytochrome P-450 (CYP) 2D6 in the Caucasian population that affect the metabolism of many commonly used drugs. We used this method to genotype 91 patients under paroxetine therapy. The presence and the constitution of the most common single-nucleotide polymorphisms (1846G>A, 2988G>A, 100C>T, 2549delA, 2615_2617delAAG, and 1707delT) in poor and intermediate metabolizers from the Caucasian population were detected in short amplicons (≤148 bp). After fluorescence normalization, the wild-type, homozygous, and heterozygous samples were easily distinguishable from each other by their specific melting curve shape. A total of 92.6% of the 1846G>A heterozygotes, 96% of the 100C>T heterozygotes, and 100% of the 2988G>A, 2549delA, 2615_2617delAAG, and 1707delT heterozygotes have been correctly distinguished from the wild types. One hundred percent of all the homozygotes in this group of patients have been detected without any error. HRM of short amplicons is a simple tool for effective, rapid, and reliable CYP2D6 genotyping that does not require real-time PCR, labeled probes, processing or any separations after PCR. The reaction is performed in a closed-tube system and is highly specific and sensitive. We proved that this technique is highly reliable for use in routine diagnostics.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+
from $39.99 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Explore related subjects

Discover the latest articles, books and news in related subjects, suggested using machine learning.

References

  1. Sundberg, M. I., Sim, S. C., Gomez, A., & Antona, C. R. (2007). Influence of cytochrome P450 polymorphisms on drug therapies: Pharmacogenetic, pharmacoepigenetic and clinical aspects. Pharmacology and Therapeutics, 116(3), 496–526.

    Article  Google Scholar 

  2. Zourková, A., Hadasova, E., Pulkrabková, L., Ravcuková, B., & Kaspsrek, T. (2006). Clinical impact of CYP2D6 activity in long-term paroxetine treatment in new developments in anxiety disorder research (pp. 177–198). New York: Nova Science Publishers.

    Google Scholar 

  3. Anzenbacher, P., & Anzenbacherová, E. (2001). Cytochromes P450 and metabolism of xenobiotics. Cellular and Molecular Life Sciences, 58, 737–747.

    Article  CAS  Google Scholar 

  4. Lovlie, R., Daly, A. K., Molven, A., Idle, J. R., & Steen, V. M. (1996). Ultrarapid metabolizers of debrisoquine: Characterization and PCR-based detection of alleles with duplication of the CYP2D6 gene. FEBS Letters, 392, 30–34.

    Article  CAS  Google Scholar 

  5. Ingelman-Sundberg, M. (2005). Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): Clinical consequences, evolutionary aspects and functional diversity. The Pharmacogenomics Journal, 5, 5–16.

    Article  Google Scholar 

  6. Omari, A. A., & Murry, D. J. (2007). Pharmacogenetics of the cytochrome P450 enzyme system: Review of current knowledge and clinical significance. Journal of Pharmacy Practice, 20, 206–218.

    Article  Google Scholar 

  7. Lanfear, D., & McLeod, H. (2007). Using DNA to optimize drug therapy. Pharmacogenetics, 76, 8.

    Google Scholar 

  8. Fischer, S. G., & Lerman, L. S. (1980). Separation of random fragments of DNA according to properties of their sequences. Proceedings of the National Academy of Sciences USA, 1980(77), 4420–4424.

    Article  Google Scholar 

  9. Botstein, D., White, R. L., Skolnick, M., & Davis, R. W. (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics, 32(3), 314–331.

    CAS  Google Scholar 

  10. Hardyman, G. (2008). Pharmacogenomics in drug discovery and development. Methods in Molecular Biology, 448, 21–30.

    Article  Google Scholar 

  11. Baron, H., Fung, S., Aydin, A., et al. (1997). Oligonucleotide ligation assay for detection of apolipoprotein E polymorphisms. Clinical Chemistry, 43(10), 1984–1986.

    CAS  Google Scholar 

  12. Li, J., Butler, J. M., Tan, Y., et al. (1999). Single nucleotide polymorphism determination using primer extension and time-of-flight mass spectrometry. Electrophoresis, 20(6), 258–265.

    Article  CAS  Google Scholar 

  13. Schaeffeler, E., Schwab, M., Eichelbaum, M., & Zanger, U. M. (2003). CYP2D6 genotyping strategy based on gene copy number determination by TaqMan real-time PCR. Human Mutation, 22(6), 476–485.

    Article  CAS  Google Scholar 

  14. Murugesan, G., Aboudola, S., Szpurka, H., et al. (2006). Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis. American Journal of Clinical Pathology, 125(4), 625–633.

    CAS  Google Scholar 

  15. Ahmadian, A., Gharizadeh, B., Gustafsson, A. C., et al. (2000). Single-nucleotide polymorphism analysis by pyrosequencing. Analytical Biochemistry, 280(1), 103–110.

    Article  CAS  Google Scholar 

  16. Reed, G. H., & Wittwer, C. T. (2004). Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clinical Chemistry, 50, 1748–1754.

    Article  CAS  Google Scholar 

  17. Roche Diagnostics GmbH and Roche Applied Science. LightCycler® 480 Operator’s Manual. Gene Scanning Software. Germany, 2007.

  18. Wittwer, C. T., Reed, G. H., Gundry, C. N., Vandersteen, J. G., & Pryor, R. J. (2003). Highresolutiongenotyping by amplicon melting analysis using LCGreen. Clinical Chemistry, 49, 853–860.

    Article  CAS  Google Scholar 

  19. Reed, G. H., Kent, J. O., & Wittwer, C. T. (2007). High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics, 8(6), 597–608.

    Article  CAS  Google Scholar 

  20. Palais, R. A., Liew, M. A., & Wittwer, C. T. (2005). Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping. Analytical Biochemistry, 346(1), 167–175.

    Article  CAS  Google Scholar 

  21. Erali, M., Voelkerding, K. V., & Wittwer, C. T. (2008). High resolution melting applications for clinical laboratory medicine. Experimental and Molecular Pathology, 85(1), 50–58.

    Article  CAS  Google Scholar 

  22. Liew, M., Pryor, R., Palais, R., et al. (2004). Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clinical Chemistry, 50, 1156–1164.

    Article  CAS  Google Scholar 

  23. Reed, G. H., & Wittwer, C. T. (2004). Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clinical Chemistry, 50(10), 1748–1754.

    Article  CAS  Google Scholar 

  24. Zourkova, A., & Hadasová, E. (2003). Paroxetine-induced conversion of cytochrome P450 2D6 phenotype and occurence of adverse effects. General Physiology and Biophysics, 22, 103–113.

    CAS  Google Scholar 

  25. Zanger, U. M., Raimundo, S., & Eichelbaum, M. (2004). Cytochrome P450 2D6: Overview and update on pharmacology, genetics, biochemistry. Naunyn Schmiedebergs Archives of Pharmacology, 369, 23–37.

    Article  CAS  Google Scholar 

  26. Sistonen, J., Fuselli, S., Levo, A., & Sajantila, A. (2005). CYP2D6 genotyping by a multiplex primer extension reaction. Clinical Chemistry, 51(7), 1291–1294.

    Article  CAS  Google Scholar 

  27. Stamer, U. M., Bayerer, B., Wolf, S., Hoeft, A., & Stüber, F. (2002). Rapid and reliable method for cytochrome P450 2D6 genotyping. Clinical Chemistry, 48, 1412–1417.

    CAS  Google Scholar 

  28. Daly, A. K., & Steward, A. (1995). Use of the Expand™ Long Template PCR system in genotyping for polymorphisms in the human cytochrome P450 CYP2D6 gene. Biochemica, 4, 31–32.

    Google Scholar 

  29. American College of Medical Genetics. Standards and Guidelines for Clinical Genetics Laboratories. (2006). http://www.acmg.net/Pages/ACMG_Activities/stds-2002/c.htm.

  30. ICH Harmonized Tripartite Guideline. Validation of analytical procedures: Text and methodology. (1994). www.ich.org.

  31. Brdicka, R., Otahalova, E., Camajova, J., Stambergova, A., & Vrana, M. (2007). An overview of guidelines for diagnostic validation of genetic tests for the Czech and Slovak molecular genetic community. Clinical Biochemistry and Metabolism, 1, 63–64.

    Google Scholar 

  32. Prence, E. M. (1999). A practical guide for the validation of genetic tests. Genetic Testing, 3, 201–205.

    Article  CAS  Google Scholar 

  33. Norambuena, P. A., Copeland, J. A., Křenková, P., Štambergová, A., & Macek, M. (2009). Diagnostic method validation: High resolution melting (HRM) of small amplicons genotyping for the most common variants in the MTHFR gene. Clinical Biochemistry, 42(12), 1308–1316.

    Article  CAS  Google Scholar 

  34. Buzková, H., Pechandová, K., Slanař, O., & Perlík, F. (2006). Genetic polymorphism of cytochrome P450 and methods for its determination. Prague Medical Report, 107(4), 383–393.

    Google Scholar 

  35. Jin, Y., Desta, Z., Stearns, V., Ward, B., Ho, H., & Hoon, K. (2005). CYP2D6 genotype, antidepressant use, and tamoxifen metabolism during adjuvant. Journal of the National Cancer Institute, 97, 1.

    Article  Google Scholar 

  36. Vandel, P., Talon, J. M., Haffen, E., & Sechter, D. (2007). Pharmacogenetics and drug therapy in psychiatry—the role of the CYP2D6 polymorphism. Current Pharmaceutical Design, 13, 241–250.

    Article  CAS  Google Scholar 

  37. Grievink, H., & Stowell, K. M. (2008). Identification of ryanodine receptor 1 singlenucleotide polymorphisms by high-resolution melting using the light-cycler 480 system. Analytical Biochemistry, 374(2), 396–404.

    Article  CAS  Google Scholar 

  38. Gundry, C. N., Dobrowolski, S. F., Martin, Y. R., et al. (2008). Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons. Nucleic Acids Research, 36(10), 3401–3408.

    Article  CAS  Google Scholar 

  39. Krypuy, M., Newnham, G. N., Thomas, D. M., Conron, M., & Dobrovic, A. (2006). High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer, 6, 295.

    Article  Google Scholar 

  40. Zhou, L., Wang, L., Palais, R., Pryor, R., & Wittwer, C. T. (2005). High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clinical Chemistry, 51, 01770–01777.

    Article  CAS  Google Scholar 

  41. Fortini, D., Ciammaruconi, A., De Santis, R., et al. (2007). Optimization of high resolution melting analysis for low-cost and rapid screening of allelic variants of Bacillus anthracis by multiple-locus variable-number tandem repeat analysis. Clinical Chemistry, 53(7), 1377–1380.

    Article  CAS  Google Scholar 

  42. Chou, L. S., Lyon, E., & Wittwer, C. T. (2005). A comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning cystic fibrosis transmembrane conductance regulator gene as a model. American Journal of Clinical Pathology, 124, 330–338.

    Article  CAS  Google Scholar 

  43. Hill, C. E., Duncan, A., Wirth, D., & Nolte, F. S. (2006). Detection and identification of cytochrome P-450 2C9 alleles *1, *2, and *3 by high-resolution melting curve analysis of PCR amplicons. American Journal of Clinical Pathology, 125, 584–591.

    CAS  Google Scholar 

  44. Kalendar, R. (2007). FastPCR: A PCR primer and probe design and repeat sequence searching software with additional tools for the manipulation and analysis of DNA and protein. www.biocenter.helsinki.fi/bi/programs/fastpcr.htm.

  45. Bastien, R., Lewis, T. B., Hawkes, J. E., et al. (2008). High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation calling. Human Mutation, 29(5), 757–764.

    Article  CAS  Google Scholar 

  46. Kristensen, L. S., & Dobrovic, A. (2008). Direct genotyping of single nucleotide polymorphisms in methyl metabolism genes using probe-free high-resolution melting analysis. Cancer Epidemiology, Biomarkers and Prevention, 17(5), 1240–1247.

    Article  CAS  Google Scholar 

  47. Wittwer, C. T., Reed, G. H., Gundry, C. N., Vandersteen, J. G., & Pryor, R. J. (2003). High-resolution genotyping by amplicon melting analysis using LCGreen. Clinical Chemistry, 49(6), 853–860.

    Article  CAS  Google Scholar 

  48. Zhou, L., Wang, L., Palais, R., Pryor, R., & Wittwer, C. T. (2005). High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clinical Chemistry, 51(10), 1770–1777.

    Article  CAS  Google Scholar 

  49. Montgomery, J., Wittwer, C. T., Palais, R., & Zhou, L. (2007). Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis. Nature Protocols, 2(1), 59–66.

    Article  CAS  Google Scholar 

  50. Graham, R., Liew, M., Meadows, C., Lyon, E., Wittwer, C. T. (2005). Distinguishing different DNA heterozygotes by high-resolution melting. Clinical Chemistry 51(7), 1295–1298.

    Google Scholar 

  51. James, A. H. (2008). Thromboembolism in pregnancy: Recurrence risks, preventive and management. Current Opinion in Obstetrics and Gynecology, 20(6), 550–556.

    Article  Google Scholar 

  52. Nomoto, K., Tsuta, K., Takano, T., et al. (2006). Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high resolution melting analysis. American Journal of Clinical Pathology, 126(4), 608–615.

    Article  CAS  Google Scholar 

  53. Hung, C. C., Lee, C. N., Chang, C. H., et al. (2008). Genotyping of the G1138A station of the EGFR3 gene in patients with achondroplasia using high-resolution melting analysis. Clinical Biochemistry, 41(3), 162–166.

    Article  CAS  Google Scholar 

  54. Audrezet, M. P., Dabricot, A., Le Marechal, C., & Ferec, C. (2008). Validation of high resolution DNA melting analysis for mutation scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The Journal of Molecular Diagnostics, 10(5), 424–434.

    Article  CAS  Google Scholar 

  55. Sachse, Ch., Brockmoller, J., Bauer, S., & Roots, I. (1997). Cytochrome P450 2D6 variants in a Caucasian population: Allele frequencies and phenotypic consequences. American Journal of Human Genetics, 60, 284–295.

    CAS  Google Scholar 

  56. http://www.cigna.com/assets/docs/health-care-professionals/coverage_positions/mm_0381_coveragepositioncriteria_AmpliChip.pdf.

  57. http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=cap_today%2Ffeature_stories%2F0706pharma.html&_state=maximized&_pageLabel=cntvwr.

  58. Wu, L., Williams, P. M., & Koch, W. H. (2005). Clinical applications of microarray-based diagnostic tests. BioTechniques, 39, 577–582.

    Article  Google Scholar 

  59. Brosen, K., Nielsen, R. N., Brusgaard, K., & Gram, L. E. (1994). CYP2D6 genotype determination in the Danish population. European Journal of Clinical Pharmacology, 47, 221–225.

    Article  CAS  Google Scholar 

  60. http://www.autogenomics.com/pharma_2D6.php.

  61. http://www.luminexcorp.com/Products/Assays/ClinicalDiagnostics/xTAGCYP2D6/index.htm.

  62. http://www.healthanddna.com/healthcare-professional/p450-2d6-genotyping.html.

  63. http://www.starseq.com/?page=7&PHPSESSID=0913811ab4ded05fcff6b0cb52ca1843.

  64. Dahl, M. L., Johansson, I., Palmertz, M. P., Ingelman-Sundberg, M., & Sjöqvist, F. (1992). Analysis of the CYP2D6 gene in relation to debrisoquin and desipramine hydroxylation in a Swedish population. Clinical Pharmacology and Therapeutics, 51, 12–17.

    Article  CAS  Google Scholar 

  65. Eriksson, S., Berg, S. L., Wadelius, M., & Alderborn, A. (2002). Cytochrome P450 genotyping by multiplexed real-time DNA sequencing with Pyrosequencing™ technology. ASSAY and Drug Development Technologies, 1, 49–59.

    Article  CAS  Google Scholar 

  66. Zeppegno, P., Rolla, R., Dalo, V., Ressico, F., Parafioriti, A., Prosperini, P., et al. (2011). Venlafaxine and CYP2D6 in clinical practice: An observational study. European Psychiatry, 26, 1051.

    Article  Google Scholar 

  67. Vaart, S., Berger, H., Sistonen, J., Madadi, P., Matok, I., Gijsen, V. M. G. J., et al. (2011). CYP2D6 polymorphisms and codeine analgesia in postpartum pain management: A pilot study. Therapeutic Drug Monitoring, 33, 425–432.

    Article  Google Scholar 

  68. http://www.cyprotex.com/cloescreen/invitro-metabolism/cytochrome-p450inhibition/?gclid=CNmk48mgoK0CFUUPfAodYFhsmg.

Download references

Acknowledgments

The study was supported by the grant of the Czech Ministry of Health NS 9676-4/2008. This work was supported by the project “CEITEC—Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.

Author information

Authors and Affiliations

  1. Department of Applied Neuroscience, Faculty of Medicine, Central European Institute of Technology (CEITEC), Brno, Czech Republic

    E. Pindurová & A. Žourková

  2. Department of Psychiatry, Faculty of Medicine, Masaryk University, Brno, Czech Republic

    E. Pindurová & A. Žourková

  3. Faculty Hospital Brno-Bohunice, Brno, Czech Republic

    E. Pindurová & A. Žourková

  4. Kamenice 5, 625 00, Brno, Czech Republic

    E. Pindurová

  5. Department of Medical Genetics, Faculty Hospital Brno, Brno, Czech Republic

    J. Zrůstová

  6. Experimental and Applied Neuropsychopharmacology Group, Central European Institute of Technology (CEITEC), Brno, Czech Republic

    J. Juřica

  7. Department of Pharmacology, Faculty of Medicine, Masaryk University Brno, Brno, Czech Republic

    J. Juřica

  8. Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

    A. Pavelka

  9. Department of Computer Graphics and Design, Faculty of Informatics, Masaryk University, Brno, Czech Republic

    A. Pavelka

Authors
  1. E. Pindurová
    View author publications

    Search author on:PubMed Google Scholar

  2. A. Žourková
    View author publications

    Search author on:PubMed Google Scholar

  3. J. Zrůstová
    View author publications

    Search author on:PubMed Google Scholar

  4. J. Juřica
    View author publications

    Search author on:PubMed Google Scholar

  5. A. Pavelka
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to E. Pindurová.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pindurová, E., Žourková, A., Zrůstová, J. et al. Alternative Reliable Method for Cytochrome P450 2D6 Poor Metabolizers Genotyping. Mol Biotechnol 53, 29–40 (2013). https://doi.org/10.1007/s12033-012-9510-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-012-9510-2

Keywords