Vol. 42, No. 10

Biol. Pharm. Bull. 42, 1761–1765 (2019)

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NoteDevelopment of an Inexpensive and Rapid Operation Device forHigh-Throughput Real-Time Quantitative PCR-Based CYP2D6
CNV Genotyping
Miho Imai,a Madoka Kisoi,a Miwako Moritsugu,a Shigenori Murata,a,b Atsushi Ichikawa,b,c andKenji Kinoshita*,a,b,c
aSchool of Pharmaceutical Sciences, Mukogawa Women’s University; Kyuban-cho, Koshien, Nishinomiya, Hyogo
663–8179, Japan: b Institute of Biosciences, Mukogawa Women’s University; Kyuban-cho, Koshien, Nishinomiya,
Hyogo 663–8179, Japan: and c Bio Education Laboratory; Higashiyodogawa-ku, Osaka 533–0033, Japan.
Received February 11, 2019; accepted June 4, 2019
The CYP2D6 gene is the most well characterized gene involved in drug metabolism and is known to haveboth gene duplication and deletion variants. We report an optimized method for the determination of copy
number variation (CNV) in the CYP2D6 gene by a novel puriﬁcation process for a real-time quantitative
PCR. This high-throughput low-cost method accurately determines CNV in the CYP2D6 gene enabling reliable estimates of disease prediction in large epidemiological samples.
Key words copy number variation (CNV); CYP2D6; pharmacogenomics (PGx); real-time quantitative PCR;
water-soluble paper; saliva

INTRODUCTION
The developments of genotyping methods to analyze DNAvariations have contributed enormously to medicine, especially for understanding genes responsible or susceptible tovarious diseases. Genetic variations can be classiﬁed into ﬁve
major classes: variable number of tandem repeat (VNTR),
short tandem repeat or microsatellite (STR), insertion/deletion(I/D) polymorphism, single nucleotide polymorphism (SNP),and copy number variation (CNV). CNV is known to be theprimary source of variation in the human genome, since it
involves deletion or multiplication of DNA segments, and is
the important factor for understanding susceptibility against
diseases. CNV is similar to a type of genomic structuralvariation, which refers to DNA segments with sizes ranging
from one-kilo base to several mega bases in which deletion,
insertion, inversion, duplication, and complex recombination
occur.1–4) Knowing the combination of variants within a given
haplotype, and the diploid content in an individual, plays akey and important role when studying drug metabolism, response, and adverse reactions.
Among CYP enzymes, CYP1, 2, and 3 families are primary
drug metabolizing enzymes and responsible for the metabolism of more than 70% of the drugs in clinical use.5–7) Mostof them are highly polymorphic, allowing clinically signiﬁcantmodiﬁcations of drug metabolizing capacities. Understanding
the combinations of genetic variations is crucial for studying
drug metabolism, response, and adverse reactions. Metabolism
by the CYP2D6 is known to be one of the major genetic polymorphisms inﬂuencing drug-metabolizing activity. Variousdrugs playing an important role in current medicine, such as
antidepressants, anticancer drugs, beta-blockers, and antipsychotics, are metabolized by CYP2D6.8,9)
The CNV of the CYP2D6 gene is the determinant to cause
decreased or increased drug metabolism. Genetically, CYP2D6
is highly polymorphic, and the variants are SNPs, as well as

CNV resulting from CYP2D6 gene deletion or multiplication.
Understanding the characteristics of the CYP2D6 gene locus,genotypes and phenotypes, has an enormous potential in pharmacogenomics (PGx).10) However, the current CNV genotyping method requires DNA puriﬁcation for the use of real-timequantitative PCR, which can put constraints on research, since
it is time-consuming and expensive. Further improvement
or simpliﬁcation of the methods currently available will accelerate and broaden research to be conducted using CNV
genotyping. Namely, genotyping methodologies that are fast,accurate, cost-effective, and that also allow broad marker coverage are the key factors to support and stimulate the growing
number of PGx studies being conducted.
The objective of this study is to introduce a new PGx
method that allows quick, accurate, and inexpensive testing.
We introduce a more comprehensive and robust workﬂow for
high-throughput PGx genotyping experiments, from sample
preparation to data analysis, using a real-time quantitative
PCR system, which is better than previous methods that require extraction and puriﬁcation procedures. The new method
described here is the ﬁrst report to use unprocessed biological
specimens directly as templates for TaqMan® CNV assay. We
believe our new technology will reduce the difﬁculties to conduct large-scale association studies in various ﬁelds.
Samples of either whole blood or saliva are collected for
epidemiological studies involving the analysis of DNA using
high-throughput genotyping methods. Generally, blood collection is not always possible or feasible, particularly in casecontrol studies when requesting blood can potentially reduce
control subjects to participate. A smaller sample size can also
threaten the validity of statistical robustness. Inevitably, extracting oral mucosal cells from the saliva using a swab is lessinvasive than blood sampling. Therefore, dried saliva samples
are often preferred as a source of genomic analysis, since
the sampling process is usually painless and non-invasive.11)
Moreover, its simplicity to sample will extend the capacity by* To whom correspondence should be addressed. e-mail: kenji_k@mukogawa-u.ac.jp© 2019 The Pharmaceutical Society of Japan

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allowing self-sampling and sending the specimen to the laboratory for analysis.

MATERIALS AND METHODS
Materials An oral care sponge swab was purchased fromOsaki Medical Co. (Nagoya, Japan). Biopsy punch (BPP-40F)
was purchased from Kai Industries Co., Ltd. (Tokyo, Japan).
TaqMan® Genotyping PCR Master Mix and TaqMan® Copy
Number Assay (assay id: Hs00010001_cn [Ex9]) with an internal control–ribonuclease (RNase) TaqMan® Copy Number
Reference Assay (assay id: 4403326) were purchased from
Thermo Fisher Scientiﬁc Inc. (Waltham, MA, U.S.A.).
Homemade Sampling Kit Water-soluble paper (60MDP)was purchased from Nippon Paper Papylia Co., Ltd. (Tokyo,Japan), and homemade sampling kits were prepared fromSUN-OAK Co., Ltd. (Tokyo, Japan). Considering the difﬁculties in collecting blood samples, we developed a new sampling
method to collect dried saliva samples using water-soluble
paper.11–13) This sampling kit allows efﬁcient sample collection
for DNA ampliﬁcation and stable preservation for long term

Fig. 1.

Vol. 42, No. 10 (2019)at room temperature. It also simpliﬁed the sampling process.
Thus, an individual can easily learn how to self-sample using
this homemade sampling kit with instruction (Fig. 1).
Subjects CYP2D6 genotyping was based on a real-timequantitative PCR for the TaqMan® Copy Number Assays Protocol (Thermo Fisher Scientiﬁc). A total of 22 healthy female
subjects participated as volunteers in this study. The MedicalEthics Committee of Mukogawa Women’s University approved the study protocol (No. 13–30). Informed consent wasobtained from all the subjects.
Sample Preparation For the validation of this method,
we further quantiﬁed the performance of high-throughput
real-time quantitative PCR-based CNV genotyping directlyfrom oral mucosal cells, excluding the process of a general
DNA puriﬁcation. The results of this study will not only be
informative for genotyping, but also will contribute to transition from low-throughput to high-throughput PCR-based
experiments in other genotyping ﬁelds. With quantiﬁcationof robustness of our new method, we introduce a new device,
which has sufﬁciently overcome the shortcomings of previous methods, by employing direct collection of dried saliva

Development of Homemade Sampling Kit Design(A) A homemade sampling kit, in which water-soluble paper (60MDP) was ﬁxed on a plastic plate (polypropylene: PP, 3 × 3 cm) with an adhesive except for the diameterof about 2 cm in the center part. (B) The picture of the sampling plate. (C) The homemade sampling kit design. (D) After sampling, the kit was folded in half to allow
spontaneous drying of the saliva on the water-soluble paper. Therefore, the folded kit has a hollow structure.

Fig. 2.

Sampling Process and Genetic Test Flow

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samples. The dried saliva samples were prepared as follows:(1) the subjects were asked to rub their inner cheek surfaces
with a swab to collect oral mucosal cells for genotyping, then(2) the oral mucosal cells included in saliva specimens wereapplied to the water-soluble paper, and (3) dried thoroughly
for around 1 h at room temperature. A small piece of a driedsaliva sample for genotyping was used by cutting off andpunching with a 4 mm diameter Biopsy Punch (BPP-40F),
and was directly suspended in 100 µL of distilled water, andheated at 95°C for 5 min. This supernatant of genome DNA
template was added directly to a PCR tube and real-time PCR
based on TaqMan® assay was carried out excepting the usual
extraction and puriﬁcation procedure for genome DNA fromthe saliva (Fig. 2).
Genotyping of CYP2D6 Gene Copy Number Variation
To assess the CYP2D6 gene copy number, we used a com-Fig. 3.

Ampliﬁcation Plots of CYP2D6 and RNase P Genes

The ∆Rn (reﬂecting the quantity of ﬂuorescent probe degraded) is plotted versus
cycle number. (A) Samples with two copies of CYP2D6. In (B) samples with duplication, the Ct value of CYP2D6 decreased compared to RNase P whereas in (C)samples with deletion, the Ct value of CYP2D6 increased compared to RNase P.

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mercial quantitative TaqMan® Copy Number Assay. (assayid: Hs00010001_cn [Ex9]) with an internal control-RNase
TaqMan® Copy Number Reference Assay (assay id: 4403326).
All assays were performed in triplicate in a QuantStudio™
12K Flex Real-Time PCR System. The assays were performedwith TaqMan® Genotyping PCR Master Mix using 4 µL of genomic DNA in a 10 µL reaction. The cycling conditions were:
95°C for 10 min for initial denaturation and enzyme activation, followed by 40 cycles each of 95°C for 15 s and 60°C for
1 min. Relative quantiﬁcation was performed using CopyCaller Software, following the comparative ΔΔCt method (Fig. 3).

RESULTS AND DISCUSSION
To conﬁrm the CYP2D6*5 allele (whole gene deletion),
we performed long-range PCR, which has been establishedpreviously by Ota et al.14) (data not shown). The copy number
analysis for the 22 subjects were tested by a TaqMan® CopyNumber Assay for CYP2D6. All samples performed very wellwith all assays. The data is reported as the estimated numberof CYP2D6 copies, with the error bars representing the estimated copy numbers range. As a quality control measure,
we repeated all three assays within 10% of the samples. All
repeated assays yielded concordant results (Fig. 4). This result
showed that the copy number variation noted with this sample
set was diverse, showing CNV of at least one gene. This genomic DNA had sufﬁcient purity and concentration for usagein TaqMan® Copy Number Assays. The average Ct value ofRNase P of all dried saliva samples were observed at 29.9
cycle ±1.2 (Fig. 5). The genomic DNA copy number in thereaction was calculated from 104 to 105 copies by a calibration
curve (data not shown).
It should be noted, this method of using a new device leads
to a low-cost and a reduction of operation time compared tomethods that are popular. Actually, it takes an average of 2.5 h
and approximately 100 U.S. dollars to measure the CNV ofthe 22 saliva samples. However, experience shows that it took
an average of 15 h and 500 U.S. dollars to measure the same
22 samples by conventional methods using a real-time quantitative PCR, because a saliva sample needs the high-quality
DNA solution to be adjusted to similar concentrations of DNA
in PCR for CNV analysis.
We have developed an optimized CNV genotyping protocol
using the water-soluble paper for a real-time quantitative PCRsystem excluding a general genomic DNA puriﬁcation process. The results of this study have demonstrated that using
saliva (oral mucosal cells) directly to perform real-time quantitative PCR-based genotyping is possible.
The sample-to-CNV procedure with CNV genotyping ona real-time quantitative PCR-based high-throughput platform
yielded an accurate process applicable to large-scale genotyping operations. Moreover, even after six months, our method
gave us exactly the same copy number variation results, andsamples of oral mucosal cells were only stored at room temperature.
This study has shown that we have developed a novel DNA
preparation procedure for accurate real-time quantitative PCRbased genotyping, and the direct use of dried saliva samples
can therefore be used to further expedite the entire genotyping
process, especially when combined with a high-throughput
liquid handling platform. We believe that using our proposedBiol. Pharm. Bull.

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Fig. 4.

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Comparison of Estimated CYP2D6 Gene Copy Numbers

Each sample was assayed in triplicate for the CYP2D6 exon 9. The data presented are the mean CYP2D6 copy numbers with the error bars representing the range ofcopy numbers estimated for each assay.

Fig. 5.

Ampliﬁcation Curves of RNase PThe ∆Rn (reﬂecting the quantity of ﬂuorescent probe degraded) is plotted versus cycle number.

method simpliﬁes current genotyping methods, allowing further usage in diagnostic settings where a large number of samples are analyzed daily using real-time quantitative PCR byTaqMan® copy number assay. For further improvement, usageof samples directly for high-throughput genotyping with theexclusion of a general DNA puriﬁcation will continue to beinvestigated by our lab. Nevertheless, our results demonstrate
this technique is highly feasible as a low-cost alternative of
CNV genotyping. We hope our new methodology will be applied to perform routine genotyping in a truly high-throughputsystem.
Acknowledgments Research reported in this publication was supported by the JSPS KAKENHI, Grant number
JP16K08895. We thank the anonymous volunteers who participated in the blood and saliva sample collection.
Conﬂict of Interest
interest.

The authors declare no conﬂict of

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