( Surekha Karudapuram Ph...)Rapid SNP Genotyping of the Pharmacogenomically Important Allele CYP2D6*4 Using Real-Time qPCR on the DNA Engine Opticon 2 System

Surekha Karudapuram, Ph.D. and David Batey Ph.D.
MJ Research, Inc., South San Francisco, CA

Abstract
A two-color real-time quantitative PCR (qPCR) protocol was used on the MJ Research DNA Engine Opticon 2 system to genotype the CYP2D6*4 allele in 211 patient samples. Two hydrolysis probes, one labeled with VIC and the other labeled with FAM, were used to discriminate between the wild-type G nucleotide and the variant A nucleotide at position 1934 (*4 allele) of the CYP2D6 gene. An analysis feature within the Opticon Monitor software assigned one of the three possible genotypes to each sample. The results of this study highlight the ability of the Opticon 2 fluorescence detection system to accurately distinguish between the three SNP genotypes: GG homozygote, AA homozygote, and GA heterozygote.

Introduction
CYP2D6, or debrisoquine 4-hydroxylase, a member of the cytochrome P450 class of enzymes, is involved in the metabolism of clinically important drugs including cardiovascular agents and psychotropic drugs.¹ The human CYP2D6 gene contains numerous Single Nucleotide Polymorphisms (SNPs) that alter enzyme activity, with effects ranging from no activity to ultra rapid activity.^{2, 3} The different levels of enzyme activity result in individual differences in the ability to metabolize specific drugs. Based on these differences, individuals can be classified as poor metabolizers (PMs) who cannot metabolize CYP2D6-dependent drugs, extensive metabolizers (EMs) who metabolize CYP2D6 substrates normally, intermediate metabolizers (IMs) who have a metabolic rate between those of EMs and PMs, and ultrarapid metabolizers (UMs) who have a supernormal metabolic rate.^{1, 2}

In a clinical setting, CYP2D6 metabolic status can be determined from the patients phenotype or genotype. An appropriate drug regimen that takes into account CYP2D6 activity can then be prescribed.¹ For phenotyping, a CYP2D6-specific probe substrate is administered to a patient, and the ratio of unchanged drug to metabolite is measured by a method such as high-performance liquid chromatography or gas chromatography.⁴ Although this is the traditional method for determining metabolic status, it has several disadvantages, including lengthy assay times, intra-individual variability, and possible differences in the rate of metabolism of the probe substrate versus the actual drug.¹ For the genotyping method, the SNPs are characterized at one or more loci within the CYP2D6. The SNP genotype provides an accurate and reliable prediction of CYP2D6 activity.⁵ The genotyping method is preferred because it has short assay times, has lower intra-individual variability, and requires a very small amount of patient sample for DNA isolation.¹

In this Application Note, we use a two-color real-time qPCR protocol on the DNA Engine Opticon 2 fluorescence detection system to genotype one of the CYP2D6 SNPs that contains the *4 allele, in DNA from 211 patients. The *4 allele is a SNP at position 1934 (GenBank Accession #M33388), where a variant A nucleotide replaces the wild-type G nucleotide. Our assay was able to identify several individuals homozygous for the variant A allele who would be poor metabolizers of many clinical drugs.5

Materials and Methods
The Human CYP2D6*4 Allelic Discrimination Kit (4312555) and the TaqMan 2X Universal PCR Master Mix (4304437) were obtained from Applied Biosystems. The genotypes of the following DNA templates were analyzed: Control DNA for Allele 1 (G) and Allele 2 (A), a mixed control DNA that contained equal amounts of Allele 1 and 2, and 211 human DNA samples. DNA was isolated from patient blood samples by MaximBio (So. San Francisco, CA) using their genomic DNA isolation kit (EXT-0001). Reaction components were assembled in Hard-Shell microplates with white wells and white skirts (MJ Research, HSP-9655), and sealed with ultra-clear strip caps (MJ Research, TCS-0803). Volumes of individual components are shown in Table 1.

Following reaction assembly, the microplates were transferred to the DNA Engine Opticon 2 real-time system (MJ Research) and thermal cycling was performed in calculated mode according to the program listed in Table 2.

The fluorescence-threshold line was set as described in the DNA Engine Opticon 2 System Operation Manual. Briefly, the threshold line was set manually such that the line intersected the fluorescence traces at the point where signals surpassed background noise and began to increase. The threshold line was used to determine cycle threshold (C(t)) values for each well. The same threshold was applied to all wells, thereby ensuring accurate comparison of samples and controls.

To validate the results of the real-time qPCR protocol, a 613bp region spanning the SNP site was amplified from each patients sample DNA using primers with the sequence: forward 5-AGA-GAC-GAG-GTGCCC- C-3 and reverse 5-AAA-TCC-TGC-TCT-TCC-GAG-3. The presence of the wild-type (G) nucleotide was then determined by assaying for the absence of a Bst NI site in the PCR product following restriction digestion (data not shown). The recognition sequence of Bst NI [CC(A/T)GG] encompasses the site of the G to A allele at the third nucleotide.

Results
In this assay, two differentially labeled hydrolysis probes were used to discriminate between the wild-type G nucleotide and the variant A nucleotide at position 1934. The probe specific for the G nucleotide was labeled with VIC dye at the 5 end, while the probe specific for the A nucleotide contained a FAM label at the 5 end. DNA that is homozygous for the wild-type G nucleotide will be detected by the VIC-labeled probe and should generate only a VIC signal. DNA that is homozygous for the variant A nucleotide will be detected by the FAM probe and will only generate a FAM signal. DNA that is heterozygous will generate both a FAM and a VIC signal. The Opticon 2 system detects FAM fluorescence in channel 1 and VIC fluorescence in channel 2.

As a test of the assay, three control DNA templates were genotyped: a GG homozygote, an AA homozygote, and a GA heterozygote. As expected, reactions with the GG homozygote control DNA template showed only VIC fluorescence, the AA homozygote control DNA template showed only FAM fluorescence, and the GA heterozygote control DNA template showed both FAM and VIC fluorescence (Figure 1). The absence of carryover of FAM signal into channel 2 for the GG homozygote control template and VIC signal into channel 1 for the AA homozygote control template illustrates the excellent color-separation capability of the Opticon 2 system.

Having demonstrated the ability of the assay to distinguish the three genotypes, we next tested genomic DNA isolated from patient blood samples. A total of 211 patient DNA samples were genotyped in duplicate, along with the GG and AA homozygous control DNA, GA heterozygous control DNA, and a no-template control. The data were then analyzed by plotting the C(t) value in channel 1 (FAM) against the C(t) value in channel 2 (VIC) for each sample, using the Opticon Monitor software. The data for a 96-well plate are shown in Figure 2. Four tightly clustered groups were generated. Group 1 (blue) contained samples homozygous for the G allele, group 2 (red) contained samples homozygous for the A allele, group 3 (green) contained samples that were GA heterozygotes and group 4 (orange) were no-template control (NTC) reactions.

The results of genotyping the patient samples are summarized in Table 3. Of the 211 samples, 153 patients (72.5%) were homozygous for the wild-type allele G, while 9 (4.3%) were homozygous for the variant allele A. The remaining 49 samples (23.2%) were heterozygotes. The patients with a heterozygous genotype containing one copy of the wild-type allele would be extensive metabolizers, while those with the homozygous AA genotype would be poor metabolizers. The genotypes were present at frequencies consistent with previous studies.5

The results of the real-time qPCR assay were confirmed by restriction enzyme digestion of the PCR product at a BstN1 site that spans the SNP site. For each of the 211 patient samples, the CYP2D6*4 genotype determined by the real-time qPCR assay was identical to that determined by restriction digestion (data not shown).

Discussion
In this study, we accurately genotyped 211 patient samples for the CYP2D6*4 allele using a 2-color real-time protocol on the DNA Engine Opticon 2 system. The protocol uses two hydrolysis probes labeled with two different fluorophores, FAM and VIC, to distinguish between the wild-type G nucleotide and the variant A nucleotide at position 1934 of the CYP2D6 gene. After completion of the assay, each sample was automatically assigned to one of three groups, homozygous GG, homozygous AA, or heterozygous GA, by an analysis feature of the Opticon Monitor software.

This assay is a valuable tool for pharmacogenomics, in which genotype information can be used to design the most effective drug therapy for an individual patient. The short time (total of 3 hours) required for the qPCR run and data analysis, and the ability to perform reactions in a 96-well format should allow this assay to be used for routine genotyping in a clinical setting. In our patient population, the nine patients who carried two copies of the variant allele were classified as poor metabolizers. For these patients, a poorly metabolized drug could be avoided, or the dose and course of drug administration could be reduced, to avoid any adverse effects that might occur with a standard course of drug therapy. The ability of the Opticon 2 system to unequivocally distinguish between the three SNP genotypes in both the control and the patient DNA samples, and the availability of TaqMan assays for SNP analysis of other alleles in the CYP2D6 gene and other genes, demonstrate that the Opticon 2 system is an effective platform for a wide range of SNP genotype assays.⁶

References
1. McElroy, S., Sachse, C., Brockmoller, J., Richmond, J., Lira, M., Friedman, D., Roots, I., Silber, B. M. and Milos, P. M. CYP2D6 genotyping as an alternative to phenotyping for determination of metabolic status in a clinical trial setting. AAPS Pharmsci 2(4):1-11 (2000).

2. Marez, D., Legrand, M., Sabbagh, N., Lo Guidice, J.M., Spire, C., Lafitte, J.J., Meyer, U.A. and Broly, F. Polymorphism of the cytochrome P450 CYP2D6 gene in a European population: characterization of 48 mutations and 53 alleles, their frequencies and evolution. Pharmacogenomics 7:193-202 (1997).

3. Daly et al. Nomenclature for human CYP2D6 alleles. Pharmacogenetics 6:193-201 (1996)

4. Chen, Z.R., Somogyi, A.A. and Bochner, F. Simultaneous determination of dextromethorphan and three metabolites in plasma and urine using high-performance liquid chromatography with application to their disposition in man. Ther Drug Monit 12:97-104 (1990).

5. Hersberger, M, Marti-Jaun, J, Rentsch, K and Hanseler, E. Rapid detection of the CYP2D6*3, CYP2D6*4, CYP2D6*6 alleles by tetra-primer PCR and of the CYP2D6*5 allele by multiplex long PCR. Clinical Chemistry 46(8):1072-1077 (2000).

6. De La Vega, F.M., Dailey, D., Ziegle, J., Williams, J., Madden, D. and Gilbert, D.A. New generation pharmacogenomic tools: a SNP linkage disequilibrium Map, validated SNP assay resource, and high-throughput instrumentation system for large-scale genetic studies. BioTechniques 32:548-554 (2002).

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