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Long PCR-based Genotyping for a Deleted CYP2D6 Gene without DNA Extraction
Biochemistry and molecular biology (Bmb368)
Pondicherry University
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Long PCR-based Genotyping for a Deleted CYP2D6 Gene
without DNA Extraction
Tomoko OTA, Mariko HAYASHIDA, Minori ISHII, Kyoko IWAO-KOIZUMI,
Shigenori MURATA and Kenji KINOSHITA*
School of Pharmaceutical Sciences, Mukogawa Women’s University, Nishinomiya, Japan
Full text and Supplementary materials of this paper are available at jstage.jst.go/browse/dmpk
Summary: In the post-genome era, a simple and inexpensive method for diagnostic analysis is in high demand. Cytochrome P450 (CYP) 2D6 is one of the most widely investigated CYPs in relation to genetic polymorphism. Detection of CYP2D6* 5 is difficult since long PCR is used. Especially for samples without DNA extraction, the detection is not sensitive enough for population analysis. Therefore, we developed a CYP2D6* 5 genotyping method that involves nested long PCR, directly using human whole saliva as a template without DNA extraction. This method will be very useful for genetic diagnoses and can be an efficient tool for individualization of drug therapy in clinical studies.
Keywords: cytochrome P450 2D6; CYP2D6* 5 ; nested PCR; dried saliva
Introduction In general, it is well known that genetic polymorphisms are involved in inter-individual variations in the metabolism of numer- ous drugs in humans) Mutations in a gene coding for a drug- metabolizing enzyme cause enzyme variants with high, low or no activity. Polymorphisms can be classified into three main pheno- types: poor metabolizers (PM), intermediate metabolizers (IM), and rapid metabolizers (RM).2) The PM condition may lead to an excessive or prolonged therapeutic effect or drug-related toxicity after a normal dose, conferring a genetic predisposition to drug- induced adverse effects. In the case of compounds that need to be activated by drug metabolizing enzymes, however, the PM condition may result in decreased response. On the other hand, RMs may not achieve therapeutic levels of the drug given at a standard dose and this might account for a lack of therapeutic effect. CYP2D6 has received significant attention since the beginning of the 1970s) One of the reasons for the significant research interest in this enzyme is the wide inter-individual variation in the enzyme activity of CYP2D, which led to the discovery of deletion and duplication of the CYP2D6 gene. CYP2D6 plays an important role in the metabolism of at least 25% of current therapeutic drugs;3) for example, typical substrates for CYP2D6 are largely lipophilic in nature and include tamoxifen, 4 – 6) dextromethorphan, 7 – 9) co- deine10,11) and numerous other drugs. CYP2D6* 5 causes a defect in enzyme activity, which is one of the most important CYP2D polymorphisms in clinical studies on Japanese subjects) In the former long PCR assay for CYP2D6* 5 ,12) misinter- pretation could occur if the long PCR failed or if insufficient
genomic DNA was added. These failures are avoided in the long PCR by inclusion of the simultaneous amplification of the cloned CYP2D6 as an internal control for the reliability of the PCR. It can prevent misinterpretation without the need for internal control to be performed by using the multiplex primers. Presented here is a specific example of the nested long PCR which is based on the deletion of CYP2D6 for genotyping per- formed directly using dried saliva on filter paper without DNA extraction. In previous reports, CYP2D6* 5 could be detected using an extract sample from whole blood,12,13) The aim of the present study is to demonstrate an inex- pensive and high-throughput genotyping method that detects CYP2D6* 5 by nested long PCR assay using dried whole saliva without DNA extraction. Direct use of dried saliva on filter paper considerably decreases the possibility of contamination between samples. Materials and Methods Human genomic DNA samples: CYP2D6 genotyping was based on long PCR using dried whole saliva disks. Samples for genotype distribution were taken from 46 healthy Japanese volun- teers whose saliva had been analyzed by the long PCR method. A few drops of saliva were applied to the filter paper and dried for one hour at room temperature. The local ethics committee approved this study, and informed consent was obtained from each participating volunteer. Detection of the CYP2D6* 5 genotyping by nested long PCR assay: The first-round long PCR assay was performed with a 50 μL reaction volume containing 5 μL of distilled water, 25 μL
Received October 21, 2013; Accepted December 14, 2013 J-STAGE Advance Published Date: December 31, 2013, doi:10.2133/dmpk-13-NT- *To whom correspondence should be addressed: Kenji KINOSHITA, Ph., School of Pharmaceutical Sciences, Mukogawa Women’s University, 11-68 Koshien Kyuban-cho, Nishinomiya 663-8179, Japan. Tel. ©81-798-45-9982, Fax. ©81-798-41-2792, E-mail: kenji_k@mukogawa-u.ac
Drug Metab. Pharmacokinet. 29 (3): 283–285 (2014). Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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of 2© PCR buffer for KOD FX Neo, 10 μL of 2 mM dNTPs, 15 pmol each of CYP2D6_3 and CYP2D6_4 primers, 30 pmol each of CYP2D6_1 and CYP2D6_2 primers, and 1 U of KOD FX Neo DNA polymerase (1 U/μL, KFX-201, Toyobo, Osaka, Japan). The dried saliva was punched with a 2 mm diameter disk and put into the reaction mixture directly without DNA extraction. PCR primers, shown in Supplementary Table 1, were arranged from the previous report,14) The amplification conditions for the first- round PCR reaction were as follows: 95°C for 5 min, followed by 5 cycles of 98°C for 10 s and 74°C for 10 min, followed by 5 cycles of 98°C for 10 s and 72°C for 10 min, followed by 5 cycles of 98°C for 10 s and 70°C for 10 min, followed by 5 cycles of 98°C for 10 s and 68°C for 10 min, followed by 20 cycles of 98°C for 10 s, 65°C for 30 s and 68°C for 10 min and a final elongation step of 68°C for 10 min. The amplification conditions were arranged from the step-down long PCR assay conditions in the Toyobo protocol. Subsequently, 1 μL of first-round PCR product was subjected to nested PCR in a 50 μL mixture, containing 4 μL of distilled water, 25 μL of 2© PCR buffer for KOD FX Neo, 10 μL of 2 mM dNTPs, 15 pmol each of CYP2D6_3 and CYP2D6_7 primers, 30 pmol each of CYP2D6_5 and CYP2D6_6 primers, and 1 U of KOD FX Neo DNA polymerase. The amplification conditions for the nested PCR assay were the same as for detection of the first-round PCR reaction. The resulting PCR products were separated and detected by electrophoresis in 1% agarose gel. Direct sequencing analysis: After confirmation of the PCR products by the nested long PCR assay, we cut out the gel and puri- fied it using a Takara SUPRECμ-EZ kit (Takara Bio, Otsu, Japan). Sequencing was performed using a BigDye Terminator Cycle Sequencing Kit ver.1 (Applied Biosystems, Foster City, CA). ABI PRISM310 Genetic Analyzer (Applied Biosystems) was used for sequencing.
Results and Discussion
The gene controlling the cytochrome P450 2D6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudo-gene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. For the sequence specificity, the extended primers of several nucleotides to 3A downstream from the previous report13,14) were used for the first-round PCR. These specimens in Figure 1 were used to optimize the experimental conditions of CYP2D6* 5 genotyping. As expected, two bands of 5 kb and 3 kb were amplified using primers CYP2D6_1, CYP2D6_2, CYP2D6_3 and CYP2D6_4, as shown in Figure 1A. After the first-round PCR, it was possible to amplify and view three of the seven specimens in the gel in Figure 1A. For the nested PCR assay, we designed nested primer sets inside of the first-round PCR primers. Since there are intricate repetitive sequences that include 72 bp, 184 bp and 2,526 bp, shown as a combination of ¡, ¢ and £ in Figure 1B, respectively, it was necessary to avoid these sequences. Compared to the previous report,13) it was possible to shorten products even more with primers within 1 kb and 150 bp for wild-type and deleted genes, respectively. As shown in Figure 1B, 4 kb and 3 kb products were amplified using CYP2D6_3, CYP2D6_5, CYP2D6_6 and CYP2D6_7 primers. All genotypes of CYP2D6* 5 could be determined after the nested PCR. The result of the experiment demonstrated that direct PCR amplification using dried saliva as a template could be successfully performed in long PCR. As in our previous report,15) the dried
saliva samples were stable for several weeks at least. Table 1 shows the performance of our long PCR assay in deter- mining the CYP2D6* 5 genotypes of 46 healthy Japanese subjects, and the distribution of genotypes compared with the previous report in 162 Japanese subjects. This result gave us a similar distribution to the previous report) Direct sequence analyses with the primer walking strategy were carried out to confirm the accuracy of the newly developed method. The sequences obtained from the two samples were in agreement with expected sequences. The CYP2D6 gene is extremely polymorphic. To date, more than 100 allelic variants have been described (cypalleles. ki.se/cyp2d6). Many of the drugs today are designed to share
Table 1. Genotype distribution of CYP2D6* 5 in 46 healthy Japanese volunteers (%) Genotype This study Japanese8) 1/ 1 82 87. 1/ 5 17 12. 5/ 5 0 0
Allele frequency This study Japanese8) * 1 91 93. * 5 8 6.
Fig. 1. Scheme of long PCR analyses for CYP2D6* 5 (A) The first-round PCR was performed with four primers, i–iv, as indicated in the upper panel. The dotted line indicates deletion of CYP2D6* 5. Samples were electrophoresed in 1% agarose gels in 0© Tris-acetate-EDTA buffer. Two bands, 5 kb and 3 kb in the gel, were amplified as the result of the first-round long PCR. (B) The nested PCR was performed with four primers, iii and v–vii. There are intricate repetitive sequences around the CYP2D6 gene which are indicated by ¡, ¢ and £. Two bands of 4 kb and 3 kb products were amplified. The detection of CYP2D6* 5 is as follows. Lane a, b, d and f: 1/ 1 , Lane c: 5/ 5 , Lane e and g: 1/ 5 , Lane h: negative control. M is the size standard marker of - HindIII digest.
284 Tomoko OTA, et al.
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Long PCR-based Genotyping for a Deleted CYP2D6 Gene without DNA Extraction
Course: Biochemistry and molecular biology (Bmb368)
University: Pondicherry University
