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Technical Briefs |
1 Fraunhofer Institute of Toxicology and Aerosol Research, Center of Drug Research and Medical Biotechnology, 30625 Hannover, Germany
aaddress correspondence to this author at: Fraunhofer Institute of Toxicology and Aerosol Research, Center of Drug Research and Medical Biotechnology, D-30659 Hannover, Germany; fax 49-511-5350-573, e-mail Borlak{at}ita.fhg.de
CYP2C9 and CYP2C19 monooxygenases (EC 1.14.14.1) are responsible for the metabolism of a variety of drugs and other xenobiotics, including proton pump inhibitors, certain tricyclic antidepressants, barbiturates, beta-blockers, nonsteroidal antiinflammatory drugs, warfarin, and others (1). More than 12 variants of CYP2C9 and CYP2C19 are known, some of which can be linked to altered drug metabolism and to potential severe side effects (2)(3). CYP2C9*2 (430C
T), CYP2C9*3 (1075AC), CYP2C19*2 (681GA), CYP2C19*3 (636GA), and CYP2C19*4 (1AG) account for >90% of Caucasian poor-metabolizer alleles (nucleotide changes in parentheses after the allele) (4)(5). The nucleotide changes in the CYP2C9*2 and CYP2C9*3 alleles lead to changes in the amino acid sequence (R144C for CYP2C9*2 and I359L for CYP2C9*3) and thus to decreased enzyme activity. In the case of CYP2C19*2, *3, and *4, the nucleotide changes lead to a splicing defect, stop codon, and GTG initiation codon, respectively, and therefore to a protein with no activity.
We developed a new assay based on fluorescence resonance energy transfer (FRET). We labeled oligonucleotides with donor and acceptor fluorophores for mutation detection and applied this assay to the LightCycler (Roche Diagnostics). Single base alterations can be identified on the basis of different melting temperatures (Tms), and we used this method to screen genotypes of healthy unrelated individuals from Southern Germany. We report a robust and swift genotyping assay to permit analysis of major CYP2C9 and CYP2C19 alleles within 60 min of blood collection for each allele. This assay can be used in routine clinical practice to provide guidance for dose adjustments of drugs metabolized by CYP2C9/19.
We examined 189 healthy males and females from a Human Pharmacology Unit for participation in various clinical research trials. After giving written informed consent, these individuals were genotyped for CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, and CYP2C19*4.
DNA from whole blood was isolated using the NucleoSpin Blood DNA Extraction Kit (Macherey-Nagel) according to the manufacturer’s instructions.
CYP2C9 alleles were genotyped as follows. The fluorogenic adjacent hybridization probes were obtained from TIB-MOLBIOL. The sequences for the various PCR oligonucleotides are shown in Table 1
. Hybridization probes were designed in such a way that their Tms were marginally higher than the Tms of the primers. The sensor probes of the CYP2C9*2 and CYP2C9*3 alleles were labeled with fluorescein at the 3' end, and the anchor probes were coupled with LightCycler Red 640 (CYP2C9*2) or LightCycler Red 705 (CYP2C9*3) at the 5' end (see Table 1
). Each of the corresponding probes recognized adjacent sequences, with the shorter probe lying over the mutation site, and probes were separated by one base. Fluorescein was used as the donor fluorophore and blocked extension from the probe during PCR. LightCycler Red 640 and LightCycler Red 705 were used as acceptors in the FRET process, with the 3' ends of the anchor probes phosphorylated to block extension. The greater stability of the longer anchor probe meant that loss of fluorescence occurred as the shorter probe (sensor) melted off the template. The probes were designed such that the two different mutation sites could be detected simultaneously (duplex PCR).
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PCR was performed with 100 nM CYP2C9 primers (see Table 1
) in a standard PCR reaction containing 400 nM each of the anchor and sensor hybridization probes, 100 ng of DNA, 4.0 mM MgCl2, and 2 µL of LightCycler DNA master hybridization mixture (LightCycler-DNA Master Hybridization Probes; Roche Diagnostics Inc.) in a total of 20 µL. The reaction was started with a denaturation step at 95 °C for 30 s, and amplification was performed for 50 cycles of denaturation (95 °C for 0 s; ramp rate, 20 °C/s), annealing (55 °C for 7 s; ramp rate, 20 °C/s), and extension (72 °C for 12 s; ramp rate, 20 °C/s).
PCR products were identified by monitoring DNA melting curves in the glass capillary. DNA was denatured at 95 °C for 30 s, and maximum fluorescence was acquired by holding the reaction at 52 °C for 30 s. Data for the melting curves were generated by heating slowly to 80 °C with a ramp rate of 0.1 °C/s, and were collected continuously during that time. When the shorter probe melted off the template, FRET no longer took place, and fluorescence was converted to melting peaks by software that plotted the negative derivative of fluorescence with respect to temperature (-dF/dT vs T). The sequence-specific hybridization probes melted off the target sequences at characteristic temperatures: 68 °C (variant allele) and 73 °C (wild-type allele) in the case of CYP2C9*2 (channel F2 in the LightCycler), and 60 °C (wild-type allele) and 66 °C (variant allele) for CYP2C9*3 (channel F3 in the LightCycler). The mutations produced a minimum Tm shift of 5 °C, allowing easy detection of a wild-type from a variant allele. A typical example is given in Fig. 1
.
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Reverse complementary oligonucleotides of the anchor and sensor probes were used as positive controls (see Table 1
). Certain amplification products were also sequenced (Genetic Analyzer 3100; ABI) to verify correct genotyping with this assay.
To detect the various CYP2C19 alleles, we used the same principle as described above with the following modifications: The sequences for PCR oligonucleotides and hybridization probes are shown in Table 1
. The sensor probe for CYP2C19*2 was labeled with LightCycler Red 640 at the 5' end, and the anchor probe was labeled with fluorescein at the 3' end. For the CYP2C19*3 and CYP2C19*4 alleles, the sensor probe was labeled with fluorescein at the 3' end, and the anchor probe was labeled with LightCycler Red 640 at the 5' end (see Table 1
). Hybridization probes were separated by one (CYP2C19*3), two (CYP2C19*2), or three (CYP2C19*4) bases.
PCR was performed with 400 nM CYP2C19 primers (see above) in a standard PCR reaction containing 100 nM each of the anchor and sensor hybridization probes, 100 ng of DNA, 4.0 mM MgCl2, and 2 µL of LightCycler DNA master hybridization mixture (LightCycler-DNA Master Hybridization Probes) in a total of 20 µL. To detect the CYP2C19*2 and CYP2C19*3 alleles, the reaction started with denaturation at 95 °C for 30 s, and amplification was performed for 50 cycles of denaturation (95 °C for 0 s; ramp rate, 20 °C/s), annealing (48 °C for 7 s; ramp rate, 20 °C/s), and extension (72 °C for 14 s; ramp rate, 20 °C/s). To detect the CYP2C19*4 allele, the reaction started with denaturation at 95 °C for 30 s, and amplification was performed for 50 cycles of denaturation (95 °C for 0 s; ramp rate, 20 °C/s), annealing (55 °C for 10 s; ramp rate, 20 °C/s), and extension (72 °C for 14 s; ramp rate, 20 °C/s). Melting point analysis for all CYP2C19 alleles was done by heating the amplification products slowly from 40 to 80 °C with a ramp rate of 0.1 °C/s.
Hybridization probes melted off the target sequences at characteristic temperatures that were detected by channel F2 in the LightCycler. For CYP2C19*2, the Tms were 56 °C for the wild-type and 62 °C for the variant allele; for CYP2C19*3, the Tms were 61 °C for the wild-type and 67 °C for the variant allele; and for CYP2C19*4, the Tms were 59 °C for the variant and 65 °C for the wild-type allele.
Reverse complementary oligonucleotides of the anchor and sensor probes were used as positive controls (see Table 1
).
Representative melting curves for the CYP2C9*2 and CYP2C19*2 alleles are depicted in Fig. 1
. The differences in the Tms among individual genotypes were sufficient to permit reliable discrimination of single alleles (dT, +5–7 °C). There was no difference between the theoretically predicted and sequenced PCR products (data not shown). We thus show corroborative and conclusive evidence for accurate DNA amplification of individual alleles.
In our cohort of unrelated individuals from Southern Germany, the allelic frequencies were 0.125 for the CYP2C9*2 and 0.083 for the CYP2C9*3 allele (n = 24), and 0.158, 0.003, and 0.000 for the CYP2C19*2, *3, and *4 alleles, respectively (n = 165).
We report a new genotyping assay for identification of the CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, and CYP2C19*4 alleles, which contribute substantially to the genetic variability in the pharmacokinetics of drugs and other xenobiotics that are metabolized by CYP2C9 and CYP2C19 in Caucasians (4)(5). Other molecular biology methods may be used to detect genetic polymorphisms, including direct sequencing (6), restriction fragment length polymorphism analysis (7), and single-strand conformation polymorphism analysis (8), but these methods are laborious and cumbersome, which is a major drawback for their routine use in clinical practice. Others have developed a fluorescence-based assay that can be performed with the TaqMan System, but not with the LightCycler technology (9)(10).
FRET provides a powerful tool for the rapid, inexpensive, and reliable determination of certain genetic polymorphisms, as recently shown by us for the molecular diagnosis of the Gilbert syndrome (11).
Recently, the clinical significance of cytochrome P450 genotyping before drug treatment has been shown for patients treated with the antiepileptic drug phenytoin: patients carrying at least one variant CYP2C9 allele required dose adjustment approximately two-thirds of standard doses to achieve a therapeutic serum concentrations (12).
In conclusion, our assay can be used in routine clinical practice to provide guidance on dose adjustments for drugs that are metabolized by CYP2C9 and CYP2C19.
References
The following articles in journals at HighWire Press have cited this article:
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  S. P. Yip, S. Y. Lee, S. S. T. To, and M. L. Wong Improved Real-Time PCR Assay for Homogeneous Multiplex Genotyping of Four CYP2C9 Alleles with Hybridization Probes Clin. Chem., December 1, 2003; 49(12): 2109 - 2111. [Full Text] [PDF]
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