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Abstract
Background: ytochrome P450 CYP2D6 activity affects
antipsychotic therapy safety. 1846G>A (CYP2D6*4) polymorphism frequency varies among different ethnic
groups.
Methods: We studied 1846G>A polymorphism in Tatar
and Russian schizophrenic patients taking different antipsychotics and association of 1846G>A polymorphism and
extrapyramidal disorders (EPD) frequency in schizophrenic patients on haloperidol monotherapy in daily
doses up to 20mg.
Results: Heterozygous 1846GA genotype frequency
among Tatars was lower (23.8% vs. 32.4% in Russians),
but the differences did not reach statistical significance.
The 1846A allele frequency among Tatars was also lower
(11.9% vs. 24.3% in Russians), but the difference was not
quite significant (p=0.0592). Average daily haloperidol
dose in the group without EPD was significantly higher
than in the group with EPD (11.354.6 vs. 13.873.3mg,
p=0.0252), but average daily haloperidol dose/weight
ratios in the compared groups had no significant differences. A statistically significant association between
EPD development and heterozygous 1846GA genotype
and 1846A allele carrier frequency was revealed among
all schizophrenic patients and among those of Tatars.
Conclusions: Further well-designed pharmacogenetic
studies in different Russian regions are needed to
improve psychotropic therapy safety and to establish
*Corresponding author: Irina S. Burashnikova, Department of
Clinical Pharmacology and Pharmacotherapy, Kazan State Medical
Academy, Moushtari, 11, Kazan 420012, Russian Federation,
Phone: +79047604240, Fax: +7 843 2730802,
E-mail: irinabis25@mail.ru
Dmitriy A. Sychev: Department of Clinical Pharmacology, Russian
Medical Academy of Postgraduate Education, Moscow, Russian
Federation
Ruslan E. Kazakov: Federal State Budgetary Institution Scientific
Centre for Expert Evaluation of Medicinal Products of the Ministry
of Health of the Russian Federation, Moscow, Russian Federation
Introduction
ytochrome P450 (CYP P450) 2D6 is one of the major CYP
P450 cytochrome isoenzymes, metabolizing most psychotropic drugs and is involved in the metabolism of many
antidepressants and antipsychotics (e.g. haloperidol,
thioridazine, zuclopenthixol, risperidone, aripiprazole
and others). It is also well known that side effects of antipsychotics, especially those affecting the extrapyramidal
system, may reduce patient compliance [1] or require prescribing of correctors (trihexyphenidyl, biperiden, amantadine) and medicines of other anatomical therapeutic
chemical groups, thus increasing the drug load and the
cost of a schizophrenic patients treatment [2]. In this
regard, the search for methods to prevent extrapyramidal side effects, including the possibility of predicting
them with the help of a personalized medicine approach,
remains actual [3].
The activity of CYP P450 cytochrome isoenzymes is
influenced by endogenous factors such as age, gender and
concomitant diseases, as well as external factors, including nutrition and habits (e.g. smoking, alcohol intake),
concomitant therapy and others. Furthermore, biotransformation is under the control of the appropriate genes
and the individuals genetic characteristics manifest
themselves in variations of drug metabolism.
It was established that CYP P450 2D6 functional activity varies considerably among different races and ethnic
groups, which can be explained by its common diversity
with more than 150 genetic polymorphisms [4, 5]. Despite
the fact that a great number of CYP2D6 polymorphisms
have been described to the moment, the frequency of
many of them is relatively low (1%). The most common
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206Sychev etal.: 1846G>A polymorphism and haloperidol extrapyramidal ADRs in Russians and Tatars
variant of the CYP2D6 gene causing an absence of the
enzymatic activity is 1846A (CYP2D6*4) allele (rs3892097)
[6]. It was formed due to single nucleotide replacement
1846G>A in the genomic DNA sequence (NCBI Reference
Sequence: NG_008376.3).
Taking into account a huge number of CYP2D6 gene
polymorphisms with different functional activity, it is
increasingly challenging to translate diplotypes into phenotype [79].
According to recent data by Gaedigk etal. [7], the ratio
of CYP2D6 phenotypes predicted from genotypes in European populations is about 5% poor metabolizer (gPM), 5%
intermediate metabolizer (gIM), 88% normal (or extensive) metabolizer (gNM) and 2% ultrarapid metabolizer
(gUM).
The CYP2D6*4 allele is present in 70%90% of PM
patients [10] with a maximum rate of up to 20%25%
among Caucasians [11]. A minimal rate of the CYP2D6*4
allele and very rare occurrence rate of PMs (0%1%) is
described in Asians [12], for example, among Koreans [13].
To date, a number of studies evaluating the association of CYP2D6 gene polymorphisms and antipsychotic
therapy safety have been conducted. In some of them, the
researchers found no association between the presence of
functionally defective CYP2D6 alleles and extrapyramidal
side effects [14, 15]. However, in other studies, such relationships were found [16, 17].
It was also shown in a study on a Russian population
that CYP2D6*4 allele frequency was similar among schizophrenic and mentally healthy individuals, thus indicating a lack of association between this polymorphism
and schizophrenia [18]. Thus, results of a study assessing
CYP2D6*4 allele frequency conducted on a schizophrenic
population could be extrapolated to the general regional
population and vice versa.
The Tatarstan Republic is a federal region of Russia
located in the Volga Federal District with a population
>3.8 million, including 2 million ethnic Tatars and
1.5 million ethnic Russians [19]. There is currently no
data on 1846G>A polymorphism prevalence among the
healthy population and schizophrenic patients in the
Tatarstan Republic. Furthermore, to date, the potential
association between 1846G>A polymorphism and antipsychotic-induced extrapyramidal side effects has not been
studied in schizophrenic ethnic Tatars.
The objective of this study was to investigate the frequency of 1846G>A polymorphism among Tatars and
Russians (on a sample of schizophrenic patients from
Tatarstan taking different antipsychotics), as well as to
evaluate the association between 1846G>A polymorphism
Study population
To evaluate possible interethnic differences in 1846A allele frequency, 79schizophrenic patients taking different antipsychotics (37
patients of Russian and 42 patients of Tatar ethnicity) were recruited
in accordance with the inclusion criteria (age18years, diagnosed
F20 (Schizophrenia) according to ICD-10 [20] and exclusion criteria (age<18years, pregnancy, inability of blood sampling because of
patients mental state). A flow-chart of the patient cohort is presented
in Figure 1.
To evaluate the association between 1846A allele carriage
and EPD development, two groups of patients were selected from
the original sample: 1) patients with EPD (EPD+) classified on the
extrapyramidal symptom rating scale [21] and 2) patients without
EPD (EPD). The criteria of EPD diagnosis were the presence of acute
motor disorders: akathisia, dystonia, pseudoparkinsonism, dyskinesia or its combinations. Also, because of the data indicating an
important role of CYP2D6 in the metabolism of haloperidol at low
concentrations/doses [22], only patients receiving haloperidol monotherapy in doses 20mg per day were included. Additional exclusion criteria were the concomitant use of drugs affecting CYP P450
2D6 activity (e.g. selective serotonin reuptake inhibitors, carbamazepine), clinical and/or laboratory signs of liver and/or kidney disease
which could independently influence on the risk of EPD development. We compared two groups of patients (n=27) in each with and
without extrapyramidal side effects.
To evaluate the association between 1846A allele carriage and
extrapyramidal side effects of haloperidol in schizophrenic patients
of Tatar ethnicity, we chose among them two groups with extrapyramidal side effects (n=14) and without extrapyramidal side effects
(n=13).
Sychev etal.: 1846G>A polymorphism and haloperidol extrapyramidal ADRs in Russians and Tatars207
All schizophrenic
patients (n = 79)
II
Russians (n = 37)
Tatars (n = 42)
Schizophrenic
patients taking
other antipsychotic
therapy (n = 25)
III
Patients taking
haloperidol in
monotherapy (n = 27)
Patients taking
haloperidol in
monotherapy (n = 54)
Schizophrenic
patients taking
other antipsychotic
therapy (n =15)
The 1846G allele was identified by the presence of 116 bp, 221 bp
and 22 bp size fragments. Allele 1846 does not have the restriction
site for endonuclease PspN4I and produces only 116 bp and 243 bp
size fragments. Thereafter, the fragments were separated via electrophoresis on an 8% polyacrylamide gel, stained with ethidium bromide and viewed with an ultraviolet transilluminator TFX-20M Gibco
BRL UV Transilluminator (Life Technologies, USA).
Statistical analysis
Statistical analysis of the results was done by nonparametric methods using the Statsoft Statistica V10.0 program (Dell Statistica,
Tulsa, OK, USA). The normality of distributions of samples, which
was evaluated using a W-Shapiro-Wilk test, was taken into account
when choosing a method. Comparisons of the alleles and genotypes
frequencies between Russians and Tatars and between patients
with and without EPD were performed using a 2-test with Yates
208Sychev etal.: 1846G>A polymorphism and haloperidol extrapyramidal ADRs in Russians and Tatars
Table 1:Baseline characteristics of the study population.
Variable
Number of patients
Gender (nSD)
Male, n (%)
Female, n (%)
Ethnicity (nSD)
Tatars, n (%)
Russians, n (%)
1846GA
1846AA
Hardy-Weinberg equilibrium
All schizophrenic
patients
Schizophrenic patients on
haloperidol monotherapy
n=79
41.6 (13.46)
n=54
42.98 (13.5)
n=27
42.7 (13.25)
39 (49.4)
40 (50.6)
26 (48.2)
28 (51.8)
13 (48.1)
14 (51.9)
42 (53.2)
37 (46.8)
27 (50.0)
27 (50.0)
27 (100)
34 (62.96)
18 (33.33)
2 (3.7)
2=0.04 (p>0.05)
19 (70.37)
8 (29.63)
2=0.82 (p>0.05)
54 (68.4)
22 (27.8)
3 (3.8)
2=0.16 (p>0.05)
Table 2:Frequency of genotypes and alleles of the CYP2D6 gene by polymorphic marker 1846G>A in Tatar and Russian schizophrenic
patients.
Genotype CYP2D6*4
Tatars (n=42)
n
1846GG
1846GA
1846AA
Russians (n=37)
n
p-Value
32
10
0
76.2
23.8
22
12
3
59.5
32.4
8.1
0.147
0.456
0.098
Hardy-Weinberg equilibrium
1846G
1846A
74
10
2=0.77 (p>0.05)
88.1
11.9
56
18
2=0.52 (p>0.05)
75.7
24.3
0.059
0.059
correction for continuity or two-tailed Fishers exact test. Comparisons of qualitative characteristics were performed using Fishers
two-tailed 2-test. The differences were considered statistically significant at p<0.05 (with a statistical power >80%). Hardy-Weinberg
equilibrium analysis was performed using online calculator [23].
Results
Of a total of 79 schizophrenic patients, 39 (49.4%) were
male and 40 (50.6%) female. Of these, 42 (53.2%) patients
classified themselves as being of Tatar and 37 (46.8%)
patients of Russian ethnicity. Baseline characteristics of
the study sub-populations are shown in Table 1.
The 1846GG, 1846GA and 1846AA genotypes frequency ratio in all groups of patients corresponded to
the Hardy-Weinberg distribution, because the difference
between numbers of observed and expected individuals
with these genotypes is not significant (Tables 1 and 2).
That allowed us to conclude that the samples were representative and the results reliable.
Sychev etal.: 1846G>A polymorphism and haloperidol extrapyramidal ADRs in Russians and Tatars209
Table 3:Baseline characteristics of the schizophrenic patients with and without extrapyramidal side effects on haloperidol monotherapy.
Variable
EPD+
EPD
p-Value
Number of patients
Age, years, average (SD)
Weight, kg, average (SD)
Daily haloperidol dose, mg, average (SD)
Daily haloperidol dose/weight, mg/kg, average (SD)
Gender
Male, n (%)
Female, n (%)
Ethnicity
Tatars, n (%)
Russians, n (%)
n=27
43.6 (13.5)
74.1 (18.1)
11.35 (4.6)
0.16 (0.08)
n=27
42.33 (13.42)
73.3 (16.2)
13.87 (3.3)
0.19 (0.05)
0.72
0.88
0.025
0.16
14 (51.9)
13 (48.1)
14 (51.9)
13 (48.1)
1.000
1.000
14 (51.9)
13 (48.1)
13 (48.1)
14 (51.9)
1.000
1.000
Table 4:Frequency of genotypes and alleles of the CYP2D6 gene by polymorphic marker 1846G>A in schizophrenic patients receiving haloperidol monotherapy with and without extrapyramidal side effects.
CYP2D6 genotype
1846GG
1846GA
1846AA
Hardy-Weinberg equilibrium
1846G
1846A
EPD+(n=27)
12
14
1
44.4
51.9
3.7
38
16
2=1.6 (p>0.05)
70.4
29.6
p-Value
EPD(n=27)
22
4
1
81.5
14.8
3.7
0.01
0.008
1.509
2=1.69 (p>0.05)
48
88.9
6
11.1
0.03
0.03
Discussion
The patients in our study were on different antipsychotics such as haloperidol, risperidone, paliperidone, quetiapine and others. Despite the CYP P450 2D6 involvement in
the biotransformation of many antipsychotics, there is a
difference in the pharmacological activity of the metabolites of these various drugs. For example, risperidone is
hydroxylated by CYP P450 2D6 to an active metabolite
9-OH-risperidone [24]. CYP P450 2D6 inhibitors reduce
9-OH-risperidone concentration and increase the blood
concentration of risperidone [25], but the total active
concentration of the metabolites remains stable [26].
Therefore, the influence of 1846A allele carriage on EPD
development during risperidone therapy is less predictable. Thus, in the study [27], EPD frequency during risperidone therapy was lower in poor metabolizers (50%)
than in the other phenotypic groups (78%). Haloperidol
210Sychev etal.: 1846G>A polymorphism and haloperidol extrapyramidal ADRs in Russians and Tatars
Table 5:Baseline characteristics of the schizophrenic patients of Tatar ethnicity on haloperidol monotherapy with and without extrapyramidal side effects.
Variable
EPD+
Number of patients
Age, years, average (SD)
Gender
Male, n (%)
Female, n (%)
EPD
p-Value
n=14
39.3 (14.15)
n=13
41.6 (12.49)
0.65
9 (64.3)
5 (35.7)
69.36 (11.55)
11.68 (6.85)
0.174 (0.07)
4 (30.8)
9 (69.2)
69.23 (16.55)
12.27 (7.0)
0.179 (0.05)
0.13
0.13
0.98
0.695
0.85
Table 6:Frequency of genotypes and alleles of the CYP2D6 gene by polymorphic marker 1846G>A in schizophrenic patients of Tatar ethnicity receiving haloperidol monotherapy with and without extrapyramidal side effects.
CYP2D6 genotype
1846GG
1846GA
1846AA
Hardy-Weinberg equilibrium
1846G
1846A
EPD+
EPD
n
p-Value
6
8
42.9
57.1
13
0
100
0
0.002
0.002
20
8
2=2.24 (p>0.05)
71.4
28.6
26
0
100
0
0.005
0.005
Sychev etal.: 1846G>A polymorphism and haloperidol extrapyramidal ADRs in Russians and Tatars211
Conclusions
The 1846A allele frequency in Tatars was lower (11.9%)
than in Russians (24.3%), but the difference was not statistically significant. However, a statistically significant
association between the development of EPD during haloperidol monotherapy and heterozygous 1846GA genotype
and 1846A allele frequency was revealed among all schizophrenic patients and among those of Tatar ethnicity.
More pharmacogenetic studies are needed to assess
the association of different polymorphisms, in particular of 1846G>A, and their effect on drug therapy. This
will provide a better understanding of these links and
hopefully lead to improved therapeutic approaches to
eliminate, or at least ameliorate them, e.g. via personalized therapy. Considering ethnic genetic peculiarities, it
is necessary to initiate well-designed research projects in
different regions of Russia to exclude the influence of nongenetic factors in an attempt to clarify the role of genotyping indications in routine clinical practice.
Acknowledgments: This project forms part of the PhD
thesis in Clinical Pharmacology at Kazan State Medical
Academy, of I.S.B. The authors would like to thank the
following: The Republican Clinical Psychiatric Hospital
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