Journal of Pharmacy and Pharmacology 2 (2014) 322-326 D DAVID PUBLISHING

CYP2C19*2, CYP2C19*3, CYP2C19*17 Allele and

Genotype Frequencies in Clopidogrel-Treated Patients
with Coronary Heart Disease in Russian

Karin Badavievich Mirzaev1, Dmitriy Alekseyevich Sychev2, Alla Sergeyevna Yugay1, Andrey Vladimirovich
Grachev3 and Ruslan Yevgenyevich Kazakov4
1. Department of Clinical Pharmacology, I.M. Sechenov First Moscow State Medical University, Moscow 119991, Russian
Federation
2. Department of Therapeutics and Clinical Pharmacology, Russian Medical Academy of Postgraduate Education, Moscow 123995,
Russian Federation
3. Department of Cardiology, “SM-clinic” Medical Center, Moscow 134544, Russian Federation
4. Center for Clinical Pharmacology, Scientific Center on Expertise of Medical Application Products, Moscow 109240, Russian
Federation

Abstract: Individual variation in the response to drug therapy has been mainly attributed to the genetic polymorphism of cytochrome P
450 isoenzymes. Mutation in the gene CYP2C19 (cytochrome P450 2C19) has been shown to influence the clinical efficiency of
clopidogrel. The aim is to investigate the frequencies of CYP2C19*2 (c.G681A; rs4244285), CYP2C19*3 (c.G636A; rs4986893),
CYP2C19*17 (c.C806T; rs12248560) and to compare the allele and genotype frequencies of CYP2C19*2 in patients with CHD
(coronary heart disease) to healthy volunteers. We examined 53 patients with CHD received clopidogrel and 146 healthy volunteers.
CYP2C19*2, CYP2C19*3, CYP2C19*17 carriages were determined by a polymerase-chain reaction. The observed genotype
distribution did not deviate from Hardy-Weinberg equilibrium, it was determined by a Chi-square test with Yates correction. The
frequency of CYP2C19*2 allele reported in patients with CHD and in the healthy volunteers was 16.6% and 13.3%, respectively (P =
0.584). The results of the present study may be helpful in developing current and future directions for its management.

Key words: CYP2C19*2, CYP2C19*17, CYP2C19 polymorphism, pharmacogenetics, clopidogrel resistance, Russian population.

1. Introduction activity. Approximately 85% of absorbed clopidogrel

is hydrolyzed by hepatic carboxylesterase-1 into an
Individual variation in the response to drug therapy
inactive carboxylic acid metabolite—SR 26334 and
has been primarily attributed to the genetic
15% is metabolized in a two-step oxidative process into
polymorphism of cytochrome P 450 isoenzymes.
an active metabolite by the hepatic CYP (cytochrome
CYP2C19 is an isoenzyme that has been shown to
P450). The first step leads to the formation of
metabolize diverse pharmacologically important
2-oxo-clopidogrel. During the second step, the
therapeutic agents including barbiturates, diazepam,
2-oxo-clopidogrel converts into the active
phenytoin, clopidogrel, omeprazole, proguanil and
metabolite—R 130964. It has been suggested that
propranolol [1]. Clopidogrel is a prodrug that requires
CYP2C19 is the major enzyme involved in the
conversion into an active metabolite for biological
generation of clopidogrel active metabolite. There are
28 polymorphic variants of the CYP2C19 gene coding
Corresponding author: Karin Badavievich Mirzaev,
internal medicine resident, research field: pharmacogenomics.
for the CYP2C19 isoenzyme [2]. Patients carrying the
E-mail: karin05doc@yandex.ru.
 CYP2C19*2, CYP2C19*3, CYP2C19*17 Allele and Genotype Frequencies in Clopidogrel-Treated Patients 323
with Coronary Heart Disease in Russian
genetic variant CYP2C19*1 have fully functioned
clopidogrel metabolites and consequently have normal
metabolism of clopidogrel. CYP2C19 G681A (*2) and
CYP2C19 G636A (*3) alleles are associated with CYP
function reduction, impaired clopidogrel
responsiveness and increased subsequent post-PCI
(percutaneous coronary intervention) ischaemic
outcomes. CYP2C19 G681A (*2) allele frequencies
vary across the ethnic groups—50% in people of
Chinese descent, 34% in African Americans, 18% in
Europeans and 19% in Mexican Americans [3-6].
The CYP2C19 C806T (*17) allele is associated with
increased enzymatic activity [7]. Such increased
activity of CYP2C19 may confer a rapid
metabolization of CYP2C19 substrates, which may
lead to an enhanced response to anti-platelet treatment
with clopidogrel. Although this may enhance the
prevention of thrombotic events, it may also increase
the risk of bleeding. Numerous research reports show
that the CYP2C19*1, CYP2C19*2 and CYP2C19*17
influence the efficacy and safety of clopidogrel [8-11].
A class II brecommendation has been given in the ESC
Guidelines for the management of acute coronary
syndromes in patients without persistent ST-segment
elevation 2011 to perform genotyping in high-risk PCI
patients if a change in the anti-platelet therapy will
ensue based on the test results [12]. According to the
American College of Cardiology
Foundation/American Heart Association, a class III c
recommendation has been given to perform
pharmacogenetic testing in patients at a high risk of
stent thrombosis [13]. Despite the proven clinical
efficacy of clopidogrel, high rates of impaired
anti-platelet response to clopidogrel in certain ethnic
groups, which leads to such clinical outcomes as
recurrent thrombotic events, myocardial infarction and
stroke, and such adverse events as bleeding were
demonstrated in many clinical trials [14].
The aim of this study was to investigate the
frequencies of CYP2C19 G681A (*2; rs4244285),
CYP2C19 G636A (*3; rs4986893), CYP2C19 C806T
(*17; rs12248560) and to compare the allele and
genotype frequencies of CYP2C19 G681A (*2;
rs4244285) in patients with CHD (coronary heart
disease) to those of healthy volunteers from the
Russian population.
2. Materials and Methods
2.1 Subjects
This study included 53 patients (20 females and 33
males, with a mean age of 66 ± 10.2 yrs) with coronary
heart disease, receiving treatment with clopidogrel and
146 healthy Caucasian subjects (38 females and 108
males, with a mean age of 38.1 ± 10.1 yrs) from the
Moscow region. A written and informed consent was
obtained from each participant.
2.2 CYP2C19 Genotyping
Four milliliters of venous blood were collected in a
tube containing EDTA (ethylenediaminetetraacetic
acid). Peripheral blood leucocytes were separated by
centrifugation. Genotyping for CYP2C19 was
performed with the use of an oligonucleotide ligation
assay after initial specific amplification by means of a
polymerase chain reaction involving three primers for
the major variant alleles CYP2C19*2 (c.G681A;
rs4244285), CYP2C19*3 (c.G636A; rs4986893),
CYP2C19*17 (c.C806T; rs12248560). Primers:
5’-GATATGCAATAATTTTCCCACTATCATTG-3’
and
5’-GGTGTTCTTTTACTTTCTCCAAAATATCAC-3’
were used to amplify sequence of the CYP2C19*2
(c.G681A; rs4244285) in exon five; for CYP2C19*3
(c.G636A; rs4986893), we used
5’-CACCCTGTGATCCCACTTTC-3’ and
5’-ACTTCAGGGCTTGGTCAATA-3’; for
CYP2C19*17 (c.C806T;
rs12248560)—5’-AAATTTGTGTCTTCTGTTCTCA
AA-3’ and 5’-AATCCCAGTTCTGCCAGCTA-3’.
The sequence of the G allele-specific was
5’-FAM-TTATTTCCCGGGAACC-3’ and the
sequence of the A allele-specific probe was
324 CYP2C19*2, CYP2C19*3, CYP2C19*17 Allele and Genotype Frequencies in Clopidogrel-Treated Patients
with Coronary Heart Disease in Russian

5’-VIC-ATTATTTCCCAGGAACC-3’. “extensive metabolizers”, CYP2C19*2 or CYP2C19*3

Base numbering and allele definitions follow the carriers (*2/*2, *2/*3, *3/*3) as “PM” (poor
nomenclature of the Human CYP (Cytochrome P450) metabolizers), and CYP2C19 (*1/*2, *1/*3, *2/*17,
Allele Nomenclature Committee [2]. The genotypes *3/*17) carriers as “IM” (intermediate metabolizers).
were determined with a TaqMan® Single Nucleotide CYP2C19*17 (*1/*17, *17/*17) carriers are
Polymorphism Genotyping Assay kit and a TaqMan® designated as “UM” (ultra-rapid metabolizers)
Universal PCR Master Mix (Applied Biosystems, (Table 1).
Foster City, CA, USA), according to the
2.3 Statistical Methods
manufacturer’s instructions, using an ABI PRISM®
Sequence Detector 7000 (Applied Biosystems). All the Chi-square test with Yates correction was used to
PCR reactions were carried out in a 10 µL reaction compare the distribution of genotypes and alleles
volume containing genomic DNA 10 ng, between patients and healthy individuals. The
oligonucleotide primers 0.5 pM, 1 µL 10 PCR buffer, adequacy of the fit between the observed and the
dNTPs 250 µM, magnesium chloride 3 mM and DNA estimated genotype frequencies according to the
polymerase 0.25 U. The cycling programme involved Hardy-Weinberg equilibrium was determined by a
preliminary denaturation at 95 °C for 10 min, followed Chi-square test with Yates correction. For all statistical
by 30 cycles of denaturation at 95 °C for 30 s, analyses, a P-value < 0.05 was considered to indicate
annealing at 60 °C for 60 s and elongation at 72 °C for statistical significance. The processing of results was
60 s, followed by a final elongation step at 72 °C for 7 performed in “SPSS (statistical package for the social
min. CYP2C19 *1/*1 carriers are designated as sciences) Statistic 20”.
Table 1 Frequency of CYP2C19*2, CYP2C19*3 and CYP2C19*17 alleles and genotypes in patients and healthy volunteers.
Alleles and genotypes frequencies Predicted phenotype Subject groups P-value
CYP2C19*2 (G681A, rs4244285) Patient Healthy
n 51 146
GG-n (%) Extensive metabolizers 34 (66.7) 111 (76.1)
0.262a
GA-n (%) Intermediate metabolizers 17 (33.3) 31 (21.2)
AA-n (%) Poor metabolizers 0.0 4 (2.7)
G (%) - 83.3 86.6
0.584
A (%) - 16.7 13.4
CYP2C19*3 (G636A, rs4986893)
n 45 -
GG-n (%) Extensive metabolizers 42 (93.3) ndb
GA-n (%) Intermediate metabolizers 3 (6.7) nd
AA-n (%) Poor metabolizers 0 nd
G (%) - 96.7 nd
-
A (%) - 3.3 nd
CYP2C19*17 (C806T, rs12248560)
n 21 -
CC-n (%) Extensive metabolizers 13 (61.9) nd
CT-n (%) Rapid metabolizers 5 (23.8) nd
TT-n (%) Ultra rapid metabolizers 3 (14.3) nd
C (%) - 73.8 nd
-
T (%) - 26.2 nd
a
: differences between patients and control group are not significantly in genotypes and alleles (Chi-square test results were 1.25 (P =
0.262) and 0.84 (P = 0.584), respectively);
b
: nd—no data.
 CYP2C19*2, CYP2C19*3, CYP2C19*17 Allele and Genotype Frequencies in Clopidogrel-Treated Patients 325
with Coronary Heart Disease in Russian
3. Results
The observed genotype distributions did not deviate
from Hardy-Weinberg equilibrium and matched those
reported for white populations (in the patients:
CYP2C19 G681A—X2 = 1.30, P = 0.26, CYP2C19
G636A—X2 = 0.04, P = 0.94 and CYP2C19
C806T—X2 = 1.84, P = 0.18; in the healthy individuals
CYP2C19*2—X2 = 0.28, P = 0.6).
The alleles and genotypes frequencies in the patients
and healthy subjects were considered and presented in
Table 1.
4. Discussion
By providing new data on the pattern of CYP2C19
polymorphism and the relationship between phenotype
and genotype in the Russian population, this study
contributes significantly towards a better
understanding of the prevalence of CYP2C19 in the
Russian population and consequently of the
pharmacological management of a large number of
patients with such ethnic background. The results of
our study were similar to those found in
Gaikovitch’s [15] study of CYP2C19 polymorphisms
in healthy volunteers from the Russian population. The
frequencies of CYP2C19 G681A (*2) in healthy
volunteers reported in this study and in the previous
study were 13.3% and 11.4 % respectively (P = 0.153).
The frequency of CYP2C19 G681A (*2) in patients
with CHD was in line with that documented by
Komarov et al. [16], CYP2C19*2—14.5% (P = 0.147)
and reported by Galiavich et al. [17],
CYP2C19*2—12.5% in patients with CHD from the
Russian population (P = 0.297). Our data demonstrate
that one-third of the clopidogrel-treated patients are
expected to have a lower anti-platelet response. We did
not find CYP2C19 *2/*2 (AA) and CYP2C19 *3/*3 (AA)
genotypes, as they are rare in several ethnic groups
(about 3% of Caucasians, 4%-7% of Afro-Americans
[1]), but they are more prevalent in Korean (12%-16%) [18],
Japanese (18%-23%) [19], Chinese (15%-17%) [20].
To the best of our knowledge, this is the first study
reporting on CYP2C19*17 frequency in the Russian
population.
The CPIC (Clinical Pharmacogenetics
Implementation Consortium) for cytochrome
P450-2C19 (CYP2C19) genotype and clopidogrel
therapy: (1) standard dosing of clopidogrel is
warranted among patients with a predicted CYP2C19
EM or UM phenotype (i.e., *1/*1, *1/*17, and
*17/*17); (2) if the patient is a carrier CYP2C19 PM
(i.e., *2/*2, *2/*3, *3/*3), current literature supports
the use of an alternative antiplatelet agent (e.g.,
prasugrel, ticagrelor) [21].
5. Conclusions
The results of the present study may be helpful for
development of guidelines for CYPC19
genotype-directed antiplatelet therapy based on
CYP2C19 status.
Acknowledgments
The authors wish to thank to “UMA Foundation” for
financial support for publication of this article. The
“UMA Foundation” is one of the leading non-profit
organizations in Russia that supports young scientists
and promotes scientific research.
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