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Technical Briefs |
1 College of Pharmacy, University of Florida, Gainesville, FL
aaddress correspondence to this author at: College of Pharmacy, University of Florida, PO Box 100486, Gainesville, FL 32610
The cytochrome P450 enzymes CYP2C8 and CYP2C9 play important roles in drug metabolism (1)(2)(3). Single-nucleotide polymorphisms that have been identified in the genes encoding for these enzymes may contribute to variability in drug response through alterations in metabolism. CYP2C8 expression includes five alleles: CYP2C8*1 (wild type), CYP2C8*2, CYP2C8*3 (1), CYP2C8*4(2), and CYP2C8*5 (4). The variant alleles CYP2C8*2 and *3 have been shown to encode for an enzyme with decreased activity in vitro (1), whereas CYP2C8*4 expression appears to cause decreased CYP2C8 activity (2). The allele frequency of CYP2C8*2 is 0.18 in the African-American population, whereas CYP2C8*3 and CYP2C8*4 are expressed primarily in the Caucasian population at frequencies of 0.13 and 0.075, respectively (1)(2).
Thirteen CYP2C9 alleles have been identified to date, and the majority of studies on these have focused on the importance of CYP2C9*2 (5) and CYP2C9*3 (6). CYP2C9*4 is expressed in the Asian population at an allele frequency of 0.02, but does not appear to be expressed in Caucasians or African Americans (7). CYP2C9*5 and *6 are found primarily in African Americans at frequencies of 0.017 (8) and 0.006 (9), respectively. Blaisdell et al.(10) recently identified variants designated as CYP2C9*7 through *12, which have allele frequencies ranging from 0.006 to 0.03 in African populations. The CYP2C9*8, *11, and *12 alleles have altered in vitro metabolic activities compared with the wild-type allele, CYP2C9*1 (10). Finally, CYP2C9*13 was identified in a Chinese population with an allele frequency of 0.01 and was associated with decreased CYP2C9 activity (11).
Given the importance of these enzymes in the metabolism of multiple drugs, a reliable genotyping method that is amenable to high-throughput applications would help facilitate research in this area. Several restriction fragment length polymorphism (RFLP) methods have been developed to identify CYP2C8 and CYP2C9 alleles (1)(2)(6)(7). These methods are reliable but have several disadvantages, including the time required for enzymatic digestion, lack of feasible high-throughput applications, potential for incomplete digestion, and increased cost per sample (12). Other methods, using the LightCycler (13) and tetra-primer (14) technologies, offer advantages over RFLP but do not provide direct sequencing data.
Pyrosequencing is a less laborious process that relies on the release of pyrophosphate on incorporation of a nucleotide into a DNA sequence to provide direct sequencing data. It is ideal for the determination of allelic variants, and use of multiplex pyrosequencing enables single-tube analysis of numerous target DNA sequences (15)(16). We previously developed a pyrosequencing method for CYP2C9 genotyping, but the original method detects only the two major variants found in Caucasian and African-American populations, CYP2C9*2 and *3 (12). An improvement to our previous reported method was necessary to evaluate CYP2C9*4 and *5, which are located on the same exon as CYP2C9*3. The purpose of the present study was to develop an analytical method that detects all major CYP2C8 variants; the major CYP2C9 variant, CYP2C9*2; and in a single reaction, CYP2C9*3, *4, and *5.
For DNA extraction and isolation, blood samples were collected from 50 consecutively identified healthy volunteers who signed informed consent. The study was approved by the Institutional Review Board at the University of Florida. Ethnicity was self-reported as African American (n =4; frequency, 0.08), Asian (n = 2; frequency, 0.04), Caucasian (n = 36; frequency, 0.72), and Hispanic (n = 8; frequency, 0.16). Approximately 10 mL of blood was withdrawn from each volunteer, and DNA was extracted with a QIAamp® DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Samples were stored at –20 °C after extraction.
The primers used for the PCR reactions and pyrosequencing are shown in Table 1
. PCR reactions (25 µL) consisted of 12.5 µL of HotStarTaqTM Master Mix (Qiagen), 1.5 µL of dimethyl sulfoxide, PCR primers (10 pmol), 7 µL of H2O, and 40 ng of DNA. PCR conditions were as follows: 95 °C for 15 min; 40 cycles consisting of denaturation at 94 °C for 30 s, annealing at 56 °C (55 °C for CYP2C9; 58 °C for CYP2C8 exon 8) for 30 s, and extension at 72 °C for 45 s; and final extension at 72 °C for 7 min.
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Genotyping was performed according to the previously described protocol (12). Briefly, PCR products (10 µL) were immobilized on streptavidin-coated Sepharose beads. Beads were isolated and treated with 700 mL/L ethanol, denaturation buffer, and wash buffer and released into a mixture of annealing buffer containing 10 pmol of sequencing primer (Table 1
). This solution was heated at 80 °C for 2 min and cooled to room temperature. Pyrosequencing was performed for sequence determination and allele designation in a Biotage PSQ HS 96 System, and data were captured with PSQ HS 96 SNP software. The sequences for 10 random individuals were determined in triplicate to verify the robustness of the assay.
Shown in Fig. 1A
are the predicted histograms and resulting pyrograms for CYP2C8 exon 5 in individuals presenting with the following genotypes: CYP2C8*1/*1 (i), CYP2C8*1/*2 (ii), CYP2C8*2/*2 (iii), CYP2C8*1/*4 (iv), and CYP2C8*4/*4 (v). The predicted histogram and resulting pyrogram for an individual presenting as CYP2C9*1/*3 are shown in Fig. 1B
. The allelic frequencies of the sample population are listed in Table 1
. The assay detected no allelic variants in the Asian individuals, and CYP2C9*4 and *5 were not detected in the population studied. Linkage dysequilibrium was apparent between CYP2C8*3 and CYP2C9*2; seven of the eight (87.5%) volunteers with the CYP2C8*3 allele also had the CYP2C9*2 allele. Reanalysis of 10 randomly selected samples in triplicate produced the same genetic sequence information (data not shown).
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The pyrosequencing method presented here is directly applicable to studies involving genetic determination of CYP2C8 and CYP2C9 polymorphisms in a large sample population; our laboratory has used this assay to genotype several hundred samples (unpublished data) from various sources, including whole blood, urine, buccal swabs, and mouthwash rinses. A key advantage of pyrosequencing is that direct sequence data for each DNA are obtained, which means that little interpretation is necessary compared with other methods, such as RFLP analysis, where incomplete digestion may complicate data analysis. Furthermore, the time and cost per sample for pyrosequencing are less than for RFLP analysis (12). A method using real-time PCR to determine CYP2C8 allelic variants was recently reported (17). The PCR method offers an advantage over RFLP in that it can be modified for high-throughput applications, but it does not provide direct sequencing of DNA and therefore still requires some data interpretation. Furthermore, this reverse transcription-PCR method does not detect the CYP2C9 genotype, which is important because a linkage between the allelic variants CYP2C8*3 and CYP2C9*2 exists (18). The data gathered in our investigation support the previous report; we observed similar frequencies of CYP2C8*3 and CYP2C9*2 linkage. Further investigations with a larger sample are necessary, however. This novel method also allows the genotyping of three separate allelic variants in one PCR reaction, but we were unable to detect CYP2C9*4 and *5 carriers because the respective Asian and African-American population sample sizes were small. A larger sample of these populations would be required to identify individuals carrying these alleles. Additionally, with the recent discovery of other CYP2C9 allelic variants (10)(11), adaptations of this method could allow detection of these variants.
Acknowledgments
This project was supported in part by NIH Research Grants R01 HL68834 and MH63458, funded by the National Institute of Mental Health and the Office of Dietary Supplements, and NIH M01 RR00082.
References
-hydroxylase activity in human liver microsomes. Biochem Pharmacol 2002;64:1579-1589.[CrossRef][Web of Science][Medline]
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The following articles in journals at HighWire Press have cited this article:
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  A. Misra, J.-Y. Hong, and S. Kim Multiplex Genotyping of Cytochrome P450 Single-Nucleotide Polymorphisms by Use of MALDI-TOF Mass Spectrometry Clin. Chem., May 1, 2007; 53(5): 933 - 939. [Abstract] [Full Text] [PDF]
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