Rapid
Automated Genotyping of CYP2C19 and Helicobacter
pylori 23S rRNA Gene in Gastric Juice
Takahisa Furuta,
Yoshihiro Soya, Mitsushige Sugimoto, Masafumi Nishino, Mihoko Yamade, Takahiro
Uotani, Chise Kodaira, Shu Sahara, Hitomi Ichikawa, Takanori Yamada, Satoshi
Osawa, Ken Sugimoto, Masato Maekawa, Hiroshi Watanabe, Kazuo Umemura
Takahisa Furuta, Center for Clinical Research, Hamamatsu
University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192, Japan
Yoshihiro Soya, Tsuruga Institute of Biotechnoogy,
Toyobo Co., Ltd., Osaka, Japan
Mitsushige
Sugimoto, Masafumi Nishino, Mihoko Yamade, Takahiro Uotani, Chise Kodaira, Shu
Sahara, Hitomi Ichikawa, Takanori Yamada, Satoshi Osawa, Ken Sugimoto, First Department of Medicine, Hamamatsu
University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192, Japan
Masahito
Maekawa, Department of Laboratory Medicine,
Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192,
Japan
Hiroshi Watanabe, Department of Clinical Pharmacology and
Therapeutics, Hamamatsu University School of Medicine, Hamamatsu, Japan
Kazuo Umemura, Department of Pharmacology, Hamamatsu
University School of Medicine, Hamamatsu, Japan
Correspondence to: Takahisa Furuta, MD, PhD, Center for Clinical Research, Hamamatsu
University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192, Japan.
furuta@hama-med.ac.jp
Telephone: +081-53-435-2950 Fax: +081-53-435-2951
Received: October 14, 2012
Revised: December 17, 2012
Accepted: December 20, 2012
Published online: April 21, 2013
ABSTRACT
AIM: Genotypes of Helicobacter
pylori (H. pylori) 23S rRNA gene and human CYP2C19 are
predictive determinants of efficacy of triple therapy with proton pump
inhibitor, clarithromycin, and amoxicillin for eradicating H. pylori. We
intended to develop a rapid assay system to determine these genotypes.
METHODS: We designed
primer sets and specific probes for determining SNPs from adenine to guanine at
positions 2142 or 2143 of H. pylori 23S rRNA gene and *2 and *3 alleles
of CYP2C19, using the novel full-automated rapid genetic analyzer (GENECUBE®).
The results were compared with those acquired by direct nucleotide sequencing
of 50 gastric tissue samples. We also determined the presence of SNPs in 132
additional samples of patients¡¯ gastric juices.
RESULTS: The GENECUBE®
genotyping results of the 23S rRNA gene and CYP2C19 from gastric tissue
samples were in complete agreement with those for direct sequencing. The
sensitivity, specificity, and validity of GENECUBE® results were all
100% when compared with clinical data. GENECUBE® genotyping required
approximately 30 min for a single test.
CONCLUSIONS: The GENECUBE®-based
system can simultaneously measure genotypes of H. pylori 23S rRNA gene
and human CYP2C19 from gastric tissues and juices rapidly, which can
greatly aid the tailored H. pylori eradication therapies.
© 2013 ACT. All rights reserved.
Key words:
Helicobacter pylori (H. pylori); 23S rRNA;
CYP2C19; PCR; SNP
Furuta T, Soya Y, Sugimoto M, Nishino M, Yamade M, Uotani
T, Kodaira C, Sahara S, Ichikawa H, Yamada T, Osawa S, Sugimoto K, Maekawa M,
Watanabe H, Umemura K. Rapid Automated Genotyping of CYP2C19 and the Helicobacter Pylori
23S rRNA Gene in Gastric Juice. Journal of Gastroenterology and Hepatology Research 2013; 2(4):
508-512 Available from: URL: http://www.ghrnet.org/index./joghr/
INTRODUCTION
Eradication of H. pylori infection is employed as a method of
treating disorders of the upper gastrointestinal tract, such as peptic ulcer,
gastric mucosa associated lymphoid tissue (MALT) lymphoma, and idiopathic
thrombocytopenic purpura, as well as to prevent gastric cancer[1,2].
Eradication therapy includes administration of a proton pump inhibitor (PPI)
and two antibiotics. Triple therapy with a PPI (either omeprazole,
lansoprazole, rabeprazole, or esomeprazole), clarithromycin, and amoxicillin is
now the first-line regimen among modalities currently in use in Japan[3].
The initial eradication rate of this therapy was approximately 90%. This feat
attributed to the pathogen¡¯s susceptibility to clarithromycin[4-7].
Thus, as the frequency of clarithromycin-resistant strains of H. pylori
has increased[8], the efficacy of triple therapy regimens has
decreased[9], falling to rates as low as 70%[10].
Culture tests
are typically employed to test the susceptibility of H. pylori strains
to antimicrobial agents, such as clarithromycin; however, they require 10-14
days and are of little value in daily clinical practice. Susceptibility of most
H. pylori strains is caused by a point mutation from adenine to guanine
at nucleotide positions 2142 or 2143 in the 23S rRNA gene and are recognized as
single nucleotide polymorphisms (SNPs), designated A2142G or A2143G,
respectively[11-15]. In this manner, drug susceptibility can be determined
by genetic testing.
Another factor
associated with the ability to eradicate H. pylori by the standard
triple therapy is the genotype of CYP2C19[16]. We previously
reported that patients with the rapid metabolizer genotype of CYP2C19
(designated *1/*1) have a lower chance of being successfully treated with
PPI-based therapy at the standard dose[5]. The CYP2C19
genotype is now well known to influence the eradication rates of H. pylori by
PPI-based regimens[17]. Based on the results of CYP2C19 and 23S
rRNA genotyping studies, we planned a tailored regimen based on the genotypes
of CYP2C19 in our patient population and H. pylori 23S rRNA gene,
which resulted in higher eradication rates than those achieved with the
standard regimen[10].
Because human gastric epithelial cells are often present in gastric
juice, DNA extracted from gastric juice samples will contain both the patient
and H. pylori DNA. Therefore, using the DNA sample extracted from
gastric juice samples, genotypes of CYP2C19 in patient population and H.
pylori 23S rRNA gene can theoretically measured.
Here, in order
to make it easy to perform the individualized eradication therapy of H.
pylori based on genotype of CYP2C19 and 23S rRNA genes of H.
pylori in all patients, we developed a high-throughput system for reliably
determining CYP2C19 and 23S rRNA gene genotypes using a automated DNA
analyzer (GENECUBE®) with gastric juice and tissue samples.
MATERIALS
AND METHODS
Study 1
Development of a system that rapidly determines the genotypes of CYP2C19
and the H. pylori 23S rRNA gene using the GENECUBE® automated
DNA analyzer
This study enrolled 50 patients with gastric ulcers (GU, n=22),
duodenal ulcers (DU, n=12), or gastritis (n=19). The subjects
were 36 men and 14 women whose median age (range) was 53 (31-78) years. All had
endoscopically and histologically proven ulcers or active chronic gastritis and
were H. pylori-positive based on the rapid urease test (RUT) (see
below). Written informed consent for participation was obtained from all
patients before the study, and the Human Institutional Review Board of
Hamamatsu University School of Medicine approved the protocol
Biopsies for
the RUT obtained from both the antrum and corpus of the greater curvature by
gastroduodenoscopy were analyzed using a commercial kit (HelicoCheck; Otsuka
Pharmaceutical Co., Ltd., Tokyo, Japan). A color change from yellow to pink
within 24 hours was judged as a positive result.
The GENECUBE®
used in the present study was a novel, fully automated rapid genetic analyzer
capable of extracting nucleic acids from biological material, preparing
reaction mixtures, and amplifying the target gene all within 30 min. Analysis
with this instrument is based on the hybridization of an allele-specific
fluorescence-conjugated probe and PCR amplification within 20 min followed by
melting point analysis. A total of 16 SNPs analyses can be handled
simultaneously. In this study, 4 SNPs, including *2 and *3 mutations of CYP2C19
and A2142G and A2143G mutations of 23S rRNA of H. pylori were analyzed
per one sample. Therefore, a total of 4 samples can be handled simultaneously.
We designed
primers to amplify the 23S rRNA gene based on the H. pylori sequence
deposited in the GenBank: U27270 as follows: forward primer: 5¡¯- GTGGAGGTGAAAATTCCTCCTACCC-3¡¯,
reverse primer 5¡¯- GGCTCCATAAGAGCCAAAGCCCTTAC-3¡¯), probe for detecting the
product (5¡¯- CAAGACGGAAAGACCC-3¡¯-BODIPYFL). The length of the expected
polymerase chain reaction (PCR) product was 141 bp.
PCR was
carried out using approximately 0.1¡Öµg of DNA, and TEST BASIC KIT contains KOD
mix, PPDmix( TOYOBO, Ltd., Osaka, Japan), primers and probe. PCR conditions
were as follows: denaturation at 95¡ãC for 30 s, 50 cycles of denaturation at
97¡ãC for 1 s, annealing at 58¡ãC for 3 s, and extension at 63¡ãC for 5 s. The PCR
products were automatically subjected to melting point analysis, which
consisted of denaturing at 98¡ãC for 30 s, annealing at 40¡ãC for 30 s, and then
heating to 75¡ãC in increments of 0.09¡ãC/s. The display of the data on the
GENECUBE® monitor is shown in Figure 1.
For the
measurement of CYP2C19 genotypes, we designed the oligonucleotide
primers (forward primer: 5¡¯-ACCAGAGCTTGGCATATTGTATCTATACC-3¡¯ and reverse primer
5¡¯-CCAAAATATCACTTTCCATAAAAGCAAG-3¡¯) and probe (5¡¯- TTGATTATTTCCCAGGAACC-3¡¯-BODIPYFL)
for CYP2C19*2 and the oligonucleotide primers (forward primer:
5¡¯-GATGGAAAAATTGAATGAAAACATCAGGATTGTA-3¡¯ and reverse primer
5¡¯-AAAATGTACTTCAGGGCTTGGTCAATA-3¡¯) and probe
(5¡¯-TAAGCACCCCCTGAATCC-3¡¯-BODIPYFL) for CYP2C19*3 based on CYP2C19 sequence
deposited in the GenBank (NG_008384.1). To enable simultaneous measurements of
both genotypes, the PCR conditions were the same as described above for
detecting 23S rRNA gene mutations. To confirm the GENECUBE® SNP
analysis results, we determined the genotypes of CYP2C19 and 23S rRNA gene
using a published direct sequencing method[18,19], with minor
modifications.
Study 2: Analysis of gastric juice sample
Gastric juice samples were collected by aspiration during
gastroduodenoscopy in 132 patients not included in Study 1. These patients
suffered from GU (n=31), DU (n=20), or gastritis (n=81).
This group included 52 men and 80 women, whose median age (range) was 52
(21-80) years. H. pylori infections in 20 of these patients had been
eradicated before the present study. Immediately after the endoscope reached
the stomach, 1-10 mL of gastric juice was aspirated through the suction channel
and collected in a trap placed in the suction line. When gastric juice was
difficult to aspirate due to achlorhydria, 20 mL of water was sprinkled
vigorously on the gastric mucosa using a syringe attached to the suction
channel and collected the water by aspiration. A 20-µL aliquot of
gastric juice was then added to 180 µL of sample solution buffer (Toyobo) (the
final volume was 200 µL) and heated at 95¡ãC for 5 min to extract the DNA. 2-µL
aliquot of 200 µL was subjected to GENECUBE® analysis to test
whether GENECUBE® system could detect the presence or absence of H.
pylori infection using juice samples. The presence of H. pylori
infection was also assessed by RUT and 13C-urea breath test, and
tests for serum anti-H. pylori IgG antibody. When any one of these tests
(RUT, 13C-urea breath test and serum anti-H. pylori IgG
antibody test) was positive, the patient was diagnosed with an H. pylori
infection. The sensitivity and specificity of the GENECUBE® analysis was
evaluated based on the three conventional test
RESULTS
Validation of
GENECUBE® genotyping
Figure 1 shows that GENECUBE® analysis unambiguously
demonstrated wild type or mutant sequences in the 23S rRNA gene at nucleotide
positions 2142 or 2143. Of note is the fact that the analysis took only 30 min.
In contrast, direct sequence analysis of these same 50 samples took
approximately 8 hours.
Table 1
presents the results of the analysis of 23S rRNA gene mutations associated with
susceptibility to clarithromycin by GENECUBE® and direct sequence
analyses. Both methods detected the same mutant sequences in the same 34
samples. Further, sequencing of CYP2C19 by both techniques yielded
identical results and were able to detect the CYP2C19*2 and CYP2C19*3
mutations (Table 2).
Analysis of
gastric juice samples
In Study 2, 26 of the 132 samples were H. pylori-negative based
on analysis of serum anti-H. pylori-IgG, urease and the 13C-urea
breath test, and the remaining 106 were H. pylori-positive based on RUT,
serum IgG, or 13C-urea breath test findings. The results of GENECUBE®
analysis of 23S rRNA gene mutations in subjects based on H. pylori
infection status are displayed in table 3. The GENECUBE® could detect H.
pylori infection in all patients infected with H. pylori based on
analysis of serum anti-H. pylori-IgG, urease and the 13C-urea
breath test. Moreover, there were no false positive results. Thus, the
sensitivity, specificity and validity of the GENECUBE® assay were all 100% in
the present study results. Forty-two of the 106 H. pylori-positive
patients harbored clarithromycin-resistant strains of H. pylori based on
23S rRNA gene sequences. CYP2C19 genotyping was successfully performed for each
sample (Table 4), and we found that the CYP2C19 allele frequency was
almost the same as those reported for samples analyzed in Japan[20].
DISCUSSION
We report here the development of a rapid (30 min per sample) detection
system for determining CYP2C19 genotypes and mutations in the H. pylori
23S rRNA gene associated with resistance to clarithromycin. We were able to
successfully analyze gastric tissue and fluid samples and validated the assay
system by comparing its results to those acquired by direct nucleotide
sequencing. The sensitivity of detection of H. pylori was equal to that
of standard immunological, biochemical, and biological tests. In addition, the
time required to complete the assay makes it suitable for use in daily clinical
practice.
The samples
usually used for the determination of H. pylori infection are taken from
gastric tissue collected by mucosal biopsy during endoscopy. These samples can
be used to detect H. pylori by culture tests, RUT, histology, and PCR.
Genotyping of human DNA can also be performed using these samples[21].
Unfortunately, not all patients can tolerate this procedure, which is
contraindicated in patients with bleeding tendency and those being treated with
either or both anti-coagulants or anti-platelet agents, such as warfarin or
clopidogrel. In contrast, collection of gastric juice samples during endoscopy
can be easily and safely performed in all patients.
In the present
study, we analyzed gastric juice samples acquired during endoscopy to determine
the genotypes of CYP2C19 and the H. pylori 23S rRNA gene, the latter of
which is associated with susceptibility to clarithromycin[13,15,22].
H. pylori and human DNA can be detected by PCR using gastric juice
samples[23], as gastric juice often contains gastric epithelial
cells. Therefore, once DNA is extracted from the gastric juice, we can test for
human and H. pylori DNA. This method of sample collection enabled us to
perform simultaneous analyses of CYP2C19 mutations and 23S rRNA mutation
of H. pylori.
We previously
reported that bacterial susceptibility to clarithromycin and the patient¡¯s CYP2C19
genotype were each associated with the success or failure to eradicate H.
pylori infection5 and that tailored regimens based on CYP2C19 and
23S rRNA gene mutations yielded higher eradication rates than with the
empirical standard therapy[10]. However, standard genotyping
methods, such as PCR-RFLP and direct nucleotide sequencing, require at least
5-10 hours, starting with DNA extraction sequence analysis, a length which has
rendered such methods virtually useless in daily clinical practice. In the
present study, we used the GENECUBE® system, which enabled us to complete
simultaneous analysis of CYP2C19 and 23S rRNA gene mutations within 30 min from
start to finish. In our hospital, the genotyping of CYP2C19 and 23S rRNA is now
completed while patients take a rest for a while after endoscopy. Therefore,
the individualized eradication therapy can be given to patients on the same of
the endoscopy.
Our goal here was to develop a
GENECUBE®-based system for the simultaneous analyses of SNPs in CYP2C19 and in
the H. pylori 23S rRNA gene and to compare the results with those
acquired by direct DNA sequencing to determine the validity of the new system. This
was accomplished successfully, and we were able to show that the sensitivity,
specificity and validity of the system correlated perfectly with non-genetic
tests
Determining a patient¡¯s CYP2C19 genotype and clarithromycin sensitivity
is useful for devising optimal eradication therapy. In our hospital, we have
used this system for advanced treatment in patients infected with H. pylori.
We confess that performing a genotyping test in advance may seem costly (i.e.,
around 150 US dollars), but that this cost can be offset by achieving higher
eradication rates after the first round of individualized therapy. Given that
one of the limitations of the present study is its relatively small sample
size, large-scale prospective studies must be performed to assess the full
clinical potential of the GENECUBE® system and its economic feasibility.
CONCLUSION
The GENECUBE®-based system can simultaneously measure genotypes of H.
pylori 23S rRNA gene and human CYP2C19 from gastric tissues and juices
rapidly, which can greatly aid the tailored H. pylori eradication
therapies.
ACKNOWLEDGMENTS
This work was supported in part by a grant-in-aid from the Ministry of
Education, Culture, Sports, Science and Technology of Japan (20590913). We
thank the staff of the endoscopy unit (Tomoko Tsurumi, Kinuko Maruyama, Chieko
Matsumoto, Akiko Ogi, Urara Kurosawa, Nobue Takahashi, Masayo Serizawa, Keiko
Kikuchi, Satoko Takebayashi, Chikako Sasagase, and Akiko Tomita) for their
help. The contributions of Etsuko Hamada, Ayano Fujiwara, and Katsumasa
Yamanaka in the clinical laboratory unit are also greatly appreciated.
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Peer reviewer: Aydin Seref Koksal, Associate Professor, Department of
Gastroenterology, T¨¹rkiye Y¨¹ksek İhtisas Education and Research Hospital,
06010, Ankara, Turkey.
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