POLYMORPHISMS OF DRUG-METABOLIZING ENZYMES CYP 1 A 2 , CYP 2 D 6 , GST , NAT 2 AND TRANSPORTER MDR 1 IN POPULATION OF BELARUS : COMPARISON WITH SELECTED EUROPEAN AND ASIAN POPULATIONS

Drug therapeutic efficiency and development of unfavorable pharmacologic responses as well as the disease predisposition are caused first of all by patient’s genetic features. Genetic variations in genes encoding drug-metabolizing enzymes and transporter proteins are essential to understand the ethnic differences in disease occurrence, development, prognosis, therapeutic response and toxicity of drugs. For that reason, it is necessary to establish the normative frequency distribution of genotypes and alleles of these


Introduction
One of the important problems of drug therapy is variability of patients' responses to the drug used.Heterogeneity of organism reactions is also observed when taking vitamins and other bracing products used for correcting a functional state of a healthy human, improving physical efficiency and for recovering organism after endured diseases.As a result of applying the same pharmacologic preparation, the absence of a therapeutic effect is observed in same people and various complications (from slight fatigue to serious health disorders) emerge in others.At present many causes, which underlie interindividual differences in a pharmacologic response (sex, age, weight, pattern of a disease course, concomitant pathology, etc.), are known [1].However, genetic distinctions of a patient are the most important stable factor determining kinetics of metabolic transformations of drugs in organism and heterogeneity of individual's reaction to a drug [2,3].Such distinctions are realized via polymorphic sites of protein genes involved in drug pharmacokinetics and pharmacodynamics.According to an original conception of pharmacologic metrology of academician L. A. Piruzian, an individual human response to drug application is determined by a "starting state" of enzyme systems of chemical compound (xenobiotics) metabolism [4].Genetic polymorphism of xenobiotic biotransformation enzymes results in differences in their activity from several times to scores of times and hundred times.At present regular search for and identification of functionally important polymorphisms of drug metabolism genes are under way for conducting pharmacogenetic testing (PT) since it is considered, at a given moment, one of the most important technologies in personalized medicine [5].The PT results will allow a physician to select a drug and conditions of its dosage individually for every patient providing maximum efficiency and safety of drug therapy.
However, it should be taken into consideration that the occurrence frequency of allelic variants in drug metabolism genes varies greatly in different ethnic groups particularly belonging to diverse races (from 0 to 50 %) [6][7][8].That's why it is necessary to determine frequencies of allelic variants in the given genes in a particular population as far as it is obvious that PT introduction for certain genes will be advantageous only in that case when allelic variants of this genes occur often enough in the population (above 1 %).

Aim
The goal of our investigation was to analyze the frequency distribution of genotypes and alleles of genes encoding drug-metabolizing enzymes (CYP1А2, CYP2D6 -I phase; GSTs, NAT2 -II phase) and transporter protein MDR1 in the population of Belarus and comparisons with other ethnic populations.

Materials and methods
The study population consisted of 538 persons without oncopathology (282 men and 256 women).All participants were Caucasian from Belarus.Written informed consent was obtained from all individuals before enrolment in the study.
As for the age of the examined persons, the sample was divided into two groups: "under 50 years" -322 persons and "above 50 years" -216 persons; smoking status was known in 302 persons (109 smokers and 193 non-smokers).
The Hardy-Weinberg equilibrium for SNPs was tested with the Pearson's χ2 test.The analysis comparing genetic and allelic frequencies among ethnic groups was performed using χ2 tests.An odds ratio (OR) with a 95 % confidence interval (CI) was applied to assess the genetic associations by sex, age, smoking status.All statistical analyses were two-sided, and P<0.05 was considered as statistically significant.Statistical analysis of the material was carried out using SNPStats program (http://bioinfo.iconcologia.net/SNPstats_web)and Statistica 6.0.

Results
Genotype distributions of the polymorphisms were in the Hardy-Weinberg equilibrium, only for rs762551 HWE p-values was < 0,05.
Table 2 presents the data on distribution of alleles and genotypes rs762551 polymorphism of CYP1A2 gene depending on sex, age and smoking status in the population of Belarus.On the population studied, the genotype СС occurs in 6,5 %, СА -48,8 % and АА -44,7 %.Analysis of the relationship between the frequency CYP1A2 genotype and allele distribution and sex, age and smoking status has not revealed any significant difference.
Table 3 presents distribution of CYP1A2 genotype and allele frequencies in various populations.The frequency of the mutant allele А in Belarus did not differ from that in the European and was significantly higher than in the Japanese population.

Note: a -p-value for comparison between the population of Belarus and selected populations
The frequency of the genotype АА rs3892097, determining complete absence of the CYP2D6 enzyme activity, was 6,7 % in the population of Belarus (Table 4).The genotype GA occurred in 35,5 % of the examined persons and 57,8 % were the carriers of the genotype GG.Analysis of the relationship between genotype and allele frequency distributions of rs3892097 polymorphism and sex and smoking status has not revealed any significant difference.There was a difference in the allelic and genotypic distribution between the group of persons under 50 years of age and persons above 50 years.The frequency of GG genotype rs3892097 in persons above 50 years was significantly higher and the combined AG+AA genotype frequency was lower than in persons under 50 years.OR for dominant model GG vs GA+AA was 0,62 CI95 %:0,43-0,91; p=0,012, p log-additive=0,026.

Agricultural and Biological Scienсes
The data on GSTs-gene genotyping in the population of Belarus are given in Table 5.The frequency of the "null genotype" for genes GSTT1 and GSTM1 was 14,9 % and 43,7 % respectively, that is consistent with the occurrence frequency of these genotypes in the Europeoids.No significant differences were revealed in the occurrence frequency of null-genotyping depending on age, sex and smoking status.Analysis of rs1695 polymorphism in the population of Belarus has shown that distribution of genotype frequencies was as follows: the genotype АА -48,0 %; the genotype GA -45,0 % and the genotype GG -7,0 %.No significant differences were also revealed for distribution of GSTP1 genotypes depending on age, sex and smoking status.On the studied group, the frequency of the mutant allele was comparable with frequencies in representatives of the European population and was lower than in the Afro-Americans and higher than in the Chinese population (Table 6).

Note: a -p-value for comparison between the population of Belarus and selected populations
We have carried out NAT2 genotyping for three polymorphic sites: rs1799929, rs1799930 and rs1799931.The frequency of homozygous mutation (ТТ) in the position C481T (rs1799929) was 19,7 % in the studied group, homozygous substitution (АА) in the position G590A (rs1799930) occurred in 8,0 %.It should be noted that no individuals with the genotype АА were detected during studying rs1799931 polymorphism.Analysis of the relationship between the frequency NAT2 genotype and allele distribution and sex and age has not revealed any significant difference.Significant differences were found in the distribution of polymorphic variants of rs1799929 and rs1799930 polymorphisms between smokers and nonsmokers (Table 7).The results of our study showed an association between the carriers of CC genotype rs1799929 polymorphism and GG genotype rs1799930 polymorphism (dominant model) and cigarette smoking (p-value=0,019 and 0,022 respectively).The examined individuals were divided into "fast" and "slow" acetylators according to accepted approaches [19].The holders of three dominant alleles in the homozygous state (481СС, 590GG и 857GG) as well as those in whom only one of the three studied genotypes was in the heterozygous state (481СТ, 590GG, 857GG; 481СС, 590GА, 857GG и 481СС, 590GG, 857GА) were attributed to "fast" acetylators.All the rest of the genotype combinations formed the group of "slow" acetylators.The frequency of "fast" acetylators was 38,5 % in the population of Belarus and that of "slow" acetylators -61,5 % (Table 8).The observed ratio of "fast" and "slow" acetylators differed significantly from distribution of acetylators in the populations of Germany and Italy.

Table 8
The frequency (%) of "fast" and "slow" acetylators in various populations

Table 9
Genotype and allele frequencies of rs1045642 polymorphism in the population of Belarus Agricultural and Biological Scienсes
The CYP1A2 enzyme, a member of the cytochrome P450 superfamily of proteins is a key component of monooxygenases.CYP1A2 catalyzes the metabolic activation of a variety of aryland heterocyclic amines such as 2-aminoanthracene and 2-acetylaminofluoren. Above 40 genetic polymorphisms of the CYP1А2 enzyme were indentified but few of them have been reported to affect the activity of CYP1A2 [12].The CYP1A2*1F polymorphism (rs762551, -163С<A) in the first intron CYP1A2 gene is one of the most common studied variants.For this polymorphism was shown association with the enzyme activity: the allele А determines higher activity of the CYP1A2 enzyme and the allele С -low activity.
CYP2D6 is an important polymorphic phase-I drug-metabolism enzyme and plays an important role in the metabolism of a variety of drugs and environmental compounds.CYP2D6 is involved in metabolism of more than 20 % drugs (for instance, debrisoquine, antidepressants, beta-blockers et al [5,7,10,24].The CYP2D6 gene located at chromosome 22q 13.2 is one of the most polymorphic CYP450 genes.At present above 70 alleles of this gene are known, although multiple allele identified no function [25].CYP2D6*3, CYP2D6*4, CYP2D6*5, and CYP2D6*6 are reported to be associated with enzymatic activity and varies from complete absence ("poor" metabolizer) to ultra-fast metabolism ("fast" metabolizer).However, it was shown that 75 % of "poor" metabolizers were carriers of CYP2D6*4 (rs3892097,1846G>A) polymorphism [24].
Glutathione-S-transferase (GST) and N-acetyltransferase (NAT) are key enzymes of the second phase in biotransformation.Out of these enzymes glutathione transferases of μ class (GSTM1), θ class (GSTТ1) and π class (GSTP1) are the most studied.
GSTМ1 and GSTТ1 polymorphisms are caused by the presence of extensive deletion in the coding region ("null genotype").It is important to note that about 50 % of the Europeans have homozygous deletion of GSTM1 gene and 10-20 % -homozygous deletion of GSTТ1 gene [28].Synthesis of the corresponding protein product does not occur at the given mutations.The presence of the "null genotype", even if for one of these genes (GSTM1 or GSTТ1), is related to an increase in risk of multifactorial disease development.In recent years the data on association of GSTM1 and GSTТ1 with development of unfavorable reactions after chemotherapy came to light [28].
The enzyme glutathione S-transferase of π class (GSTP1) is involved in metabolism of wide spectrum xenobiotics including drugs.The rs1695 polymorphism in the 5 th exon of GSTP1 gene, manifesting itself in substitution of isoleucine 105 by valine (Ile105Val), is associated with development of drug resistance in oncologic disease treatment.Watson et al. showed that allele G (Ile/Val and Val/Val) were characterized by a reduced conjugation activity of the enzyme as compared to individuals with the genotype АА (Ile/Ile) [16].
N-acetyltransferase 2 (NAT2) is a major enzyme of acetylating xenobiotics with the primary aromatic and hydrazine structure.NAT2 metabolizes and detoxifies xenobiotics such as caffeine, tobacco smoke, pesticides, and drugs [29,30] Metabolic acetylation polymorphism, which manifests itself by the presence of "fast" and "slow" acetylators in the population of phenotypes, is known.Thirteen point mutations, in the coding region NAT2, which form 36 alleles in different combinations is its bases [31].Pronounced ethnic differences are a distinction of NAT2 polymorphism.Among populations of Europe and North America, 40-70 % are "slow" acetylators whereas among populations of the Pacific shore of Asia (the Japanese, the Chinese, Koreans) only 10-30 % of representatives are "slow" acetylators [19,31].Individual and ethnic differences in the acetylation rate exert an effect on development of unfavorable reactions when taking some drugs.
Our study showed an association between the carriers of CC genotype rs1799929 polymorphism and GG genotype rs1799930 polymorphism and cigarette smoking.It is possible that the Agricultural and Biological Scienсes carrier of CC genotype rs1799929 and GG genotype rs1799930 ("fast" acetylator status) reduces a detrimental effect of smoking.
In recent years the influence of Р-glycoprotein, encoded by MDR1 gene, on drug pharmokinetics was studied.P-glycoprotein is involved in elimination of a lot of drugs into extracellular space [6,32].The level of enzyme expression causes drug concentration in cell: the higher is expression of an active transporter, the faster substrate or its metabolites are removed from cell and the quicker their concentration is reduced.The rs1045642 polymorphism (3435С<Т) was revealed to be associated with the level of Р-glicoprotein expression: СС-homozygotes are characterized with a high level of Р-glicoprotein expression, ТT-homozygotes -with a low level and heterozygotes СT -with an intermediate level [33].The frequency of С3435Т substitution differed greatly among ethnics groups: the frequency of the ТT genotype in the European populations -17-28 % [34][35][36], in the Iranians -19 % [37], in the Japanese -12 % [38], in the Afro-Americans -1 % [35].The genotype frequency of rs1045642 in Belarus corresponded to that in the European population and was significantly higher than in the Japanese and in the Afro-Americans.
No significant differences in sex were revealed for distribution of MDR1 genotypes.However, it is known from the literature data that Р-glycoprotein expression was higher by a factor of 2,4 in men than in women.Therefore pharmacogenetic investigations on metabolism of drugs, being Р-glycoprotein substrate, should be performed taking into account sex differences [39,40].Application of such an approach allows differentiated prescription of drug therapeutic dose for men and women.

Conclusion
Study on genetic polymorphism of biotransformation enzymes involved in drug metabolism was first carried out in the population of Belarus.Our results indicate that clinically important genes are genetically highly variable and differ considerably between populations.These investigations can underlie an individual genetic passport which enables correction of treatment (drug selection and conditions of its dosage) in view of individual features of patients that will make pharmacotherapy more effective and safe.It should be emphasized that a genetic component of drug efficiency and/or toxicity can be determined before starting treatment.

Table 1
Characteristic of the polymorphisms genotyped in this study

Table 2
Distribution of CYP1A2 genotype and allele frequencies in the population of Belarus

Table 3
Genotype and allele frequencies (%) of rs762551 polymorphism CYP1A2 gene in some populations

Table 4
Genotype and allele frequencies of rs3892097 polymorphism in the population of BelarusNote: a -significant difference between the group of persons under 50 years of age and persons above 50 years

Table 5
Distribution of GSTs genotype and allele frequencies in the population of Belarus

Table 6
Genotype and allele frequencies (%) of rs1695 polymorphism in different ethnic groups

Table 7
Genotype frequencies of rs1799929 and rs1799930 polymorphisms in smokers and nonsmokers No significant differences in age, sex and smoking status were revealed for distribution of MDR1 genotypes.