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Letters to the Editor |
1 Department of Pharmacy, Faculty of Medicine, University of Tokyo Hospital, Tokyo, Japan, 2 Center for Molecular Biology, and Cytogenetics, SRL Inc. Tokyo, Japan
aAddress correspondence to this author at: Department of Pharmacy, Faculty of Medicine, University of Tokyo Hospital, 7-3-1 Hongoh, Bunkyo-ku, Tokyo 113-8655, Japan. Fax 81-3-3816-6159; e-mail tkubota-tky{at}umin.ac.jp.
To the Editor:
Cytochrome P450 2B6 (CYP2B6) metabolizes several clinically important drugs, such as cyclophosphamide (1). Eight variants, CYP2B6*2 through *9 have been reported to date, and assays combining PCR and restriction fragment length polymorphism (RFLP) analysis have been developed for genotyping of CYP2B6*1 through *7 (2)(3). We previously performed PCR-RFLP assays for genotyping of CYP2B6*1 through *7 in 147 Japanese volunteers and found an individual heterozygous for the G516T, A785G, and C1459T mutations in the CYP2B6 gene. The individual was classified as CYP2B6*1/*7 or *5/*6, but we could not determine the CYP2B6 genotype of this individual because the PCR-RFLP assays failed to distinguish the two genotypes. An individual classified as CYP2B6*1/*7 or *5/*6 has also been reported by other authors (4). It is necessary to distinguish CYP2B6*1/*7 and *5/*6 because CYP2B6*5, *6, and *7 have been associated with changes in the production and function of CYP2B6, respectively (2)(4). In haplotype determination, a cloning method is usually performed, but this is time-consuming. We therefore developed a method for rapid haplotype determination based on PCR to distinguish CYP2B6*1/*7 and *5/*6.
The local Institutional Review Board approved this study, and informed consent for participation in this study was obtained from all volunteers.
Primers CYP2B6-A516G (5'-ACC CCA CCT TCC TCT TCC AG-3') and CYP2B6-9R-II (5'-GGA TGA GAA TTT ATT CTG GAC CTC-3') were designed on the basis of the published sequence (5). A GeneAmp® PCR System 9700 (Applied Biosystems) was used for PCR amplifications. In step 1 (Fig. 1
), the following mixture was prepared for the long PCR: 15.75 µL of water, 2.5 µL of 10x LA PCRTM Buffer II, 2.5 µL of the deoxynucleotide triphosphate mixture (2 mM each), 2.0 µL of MgCl2 (25 mM), 0.5 µL of primer CYP2B6-4F (10 µM) (2), 0.5 µL of primer CYP2B6-9R-II (10 µM), 0.25 µL of TaKaRa LA TaqTM (5 U/µL), and 1 µL of genomic DNA (100 ng/µL). The following cycling conditions were used: 94 °C for 1 min, 30 cycles of 94 °C for 20 s and 64 °C for 11 min, and 72 °C for 10 min.
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In step 2, the following mixture was prepared for the allele-specific PCR: 16.55 µL of water, 2.5 µL of 10x LA PCR Buffer II, 2.5 µL of deoxynucleotide triphosphate mixture, 1.7 µL of MgCl2, 0.25 µL of primer CYP2B6-A516G (10 µM), 0.25 µL of primer CYP2B6-9R (10 µM) (2), 0.25 µL of TaKaRa LA Taq, and 1 µL of the fivefold-diluted long-PCR product. The following cycling conditions were used: 94 °C for 1 min, 20 cycles of 94 °C for 20 s and 70 °C for 7 min, and 72 °C for 10 min.
In step 3, the following mixture was prepared for the AlwI RFLP analysis: 7.0 µL of water, 2.0 µL of 10x NEBuffer 4, 1.0 µL of AlwI (5 U/µL; New England Biolabs), and 10 µL of the allele-specific PCR product. The AlwI RFLP mixture was incubated at 37 °C for 2 h. The resulting fragments were identified on a 2% agarose gel stained with ethidium bromide.
In the AlwI RFLP analysis, the allele-specific PCR products derived from CYP2B6*1 and *5 yielded 1067- and 1286-bp fragments, respectively. Our results showed that the 1286-bp fragment was from the individual heterozygous for the G516T, A785G, and C1459T mutations of the CYP2B6 gene, which indicated that the CYP2B6 genotype of this individual was CYP2B6*5/*6.
In conclusion, we have established a method for haplotype determination based on PCR that enables CYP2B6*1/*7 and *5/*6 to be distinguished. This method, which is suitable for routine assay of CYP2B6 genotype, takes 
10 h and is faster than a cloning method.
References
The following articles in journals at HighWire Press have cited this article:
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  J. K. Blievernicht, E. Schaeffeler, K. Klein, M. Eichelbaum, M. Schwab, and U. M. Zanger MALDI-TOF Mass Spectrometry for Multiplex Genotyping of CYP2B6 Single-Nucleotide Polymorphisms Clin. Chem., January 1, 2007; 53(1): 24 - 33. [Abstract] [Full Text] [PDF]
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