DISTRIBUTION OF THE MOST COMMON GENETIC VARIANTS
ASSOCIATED WITH A VARIABLE DRUG RESPONSE IN THE
POPULATION OF THE REPUBLIC OF MACEDONIA
Kapedanovska Nestorovska A1, Jakovski K2, Naumovska Z1, Hiljadnikova Bajro M1,
Sterjev Z1, Eftimov A1, Matevska Geskovska N1, Suturkova L1, Dimitrovski K3,
Labacevski N3, Dimovski AJ
*Corresponding Author: Aleksandar J. Dimovski, MD., Ph.D., Center for Biomolecular and Pharmaceutical Analysis,
Faculty of Pharmacy, University Ss Cyril and Methodius, Mother Theresa 47, Skopje 1000, Republic of Macedonia.
Tel: +389-2-3217-580; +389-2-3119-694. Fax: +389-2-3290-830; +389-2-3123 054. E-mail: adimovski@ff.ukim.edu.mk
page: 5
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GENETIC VARIANTS ASSOCIATED WITH A VARIABLE DRUG RESPONSE
Phase I: Oxidation, CYP450 Family (CYP2D6,
CYP2C19, CYP2C9, CYP3A5). The CYP450 isoform
activity has emerged as a major determinant of the
variability in drug metabolism and response as these
enzymes are responsible for about 80.0% of all Phase
I DMEs. Despite the large number of CYP genes and
enzymes, 57 individual CYP genes organized in 18
gene families comprising 44 subfamilies, only the
members of the CYP2 and CYP3 gene families have a
significant importance since they contribute to the metabolism
of the majority of clinically important drugs
[5,6]. The CYP2D6 enzyme, al-though expressed at
low levels, constituting only 4.0-6.0% of the CYP
content in the liver, metabolizes 25.0 to 30.0% of all
clinically used drugs including anti-arithmics, antidepressants,
anti-psychotics, beta blockers and codeine.
The enzyme is the only one among the CYPs
DMEs that is not inducible, and therefore, the genetic
variation contributes largely to the inter-individual
variation in the enzyme activity [7].
The CYP2C19 enzyme plays a critical role in the
oxidative biotransformation of approximately 10.0%
of the commonly used drugs belonging to drug classes
such as proton pump inhibitors, tricyclic anti-depressants,
selective serotonin reuptake inhibitors, benzodiazepines,
barbiturates, phenytoin, voriconazole,
nelfinavir and proguanil [7,8], whereas, the CYP2C9
enzyme contributes to the metabolism of 15.0% of
clinically important drug classes, such as nonsteroidal
anti-inflamatory drugs, angiotensin II receptor
antagonist, anti-diabetic drugs, diuretic torsemide as
well as the narrow therapeutic index drugs such as
phenytoin and warfarin [7,9]. The CYP3A enzymes
are responsible for the metabolism of approximately
50.0% of the currently marketed drugs, including steroids,
anti-depressants, immunosuppressive agents
and anti-biotics. The CYP3A5 is the primary extrahepatic
CYP3A isoform, and typically, displays decreased
catalytic activity compared to CYP3A4 [5,9].
The CYP450 allelic variant and numerous subvariants reported to date are summarized at the Human CYP
Allele Nomenclature Committee website (http:// www.
cypalleles.ki.se/).
Currently, over 100 allelic variants and numerous
subvariants have been identified within the CYP2D6
gene. The most important non functional alleles that
are predominantly responsible for poor metabolizers
(PMs) and account for 90.0-95.0% of PMs in
indigenous Europeans are CYP2D6*3, CYP2D6*4
and CYP2D6*6 [10]. The CYP2D6*4 with an allele
frequency of 12.0-21.0%, is the most common allele
associated with PMs. Other detrimental alleles,
CYP2D6*3 and CYP2D6*6, are frequently found in
indigenous Europeans (1.0-2.0%), but are very rare
or absent in other populations. The most common
alleles with severely reduced activity are represented
by CYP2D6*9, CYP2D6*10, CYP2D6*17 and
CYP2D6*41. The frequency of CYP2D6 deletions
ranges from 2.0-7.0% in indigenous Europeans, 6.0%
in Blacks and less than 1.0% in Asians and 2.0-10.0%
of the populations carry multiple copies of the functional
CYP2D6 allele [7,11].
The majority of PMs of CYP2C19 are due to the
CYP2C19*2 (681G>A) and CYP2C19*3 (636G>A)
allelic variants that are non functional alleles, resulting
in a complete loss of enzyme activity. They account
for 95.0% of PMs. The frequency of the CYP2C19*2
allele ranges between 15.0-17.0% in indigenous Europeans
and Blacks and up to 30.0% in the Asian
population. The most common allele associated with
ultrarapid enzyme activity, CYP2C19*17 (–806C>T),
has a similar allelic frequency of 15.0-25.0% in both
the indigenous European and Black populations, and
much reduced frequency (4.0%) in Asians [7,12].
The most prevalent CYP2C9 alleles in the European
population are CYP2C9*2 (430C>T) and
CYP2C9*3 (1075A>T). Both are decreased-function
alleles, lead to significant reduction in the enzymatic
activity of the CYP2C9 enzyme and account for 85.0%
of PM phenotypes. The reported frequencies of CYP2C9*
2 and CYP2C9*3, ranges between 13.0-22.0%
and 4.0-6.0%, respectively, in indigenous Europeans,
but are much lower in Blacks and Asians [7,9,12].
CYP3A5 is a highly polymorphic enzyme as a result
of mutations that severely diminish the synthesis
of the functional CYP3A5 protein. The CYP3A5*3C
(6986A>G) mutant is the major defective allele, leading
to alternative splicing and protein truncation,
which in turn results in the absence of the enzyme
activity. It is widely detectable in the indigenous European
population (85.0-98.0%) with a lower frequency
in the Black (27.0-48.0%) and Asian (60.0-75.0%)
populations [7,12,13].
Distribution of the CYP450 (CYP2D6, CYP2C19,
CYP2C9, CYP3A5) Genetic Variants in
the Population of the Republic of Macedonia. The
allelic and genotype frequency distributions in our
population and the correlated CYP450 phenotype status
for the investigated DMEs are presented in Table
1. The CYP2D6 gene is considered a challenge for
genotyping because its genetic variants include not
only single nucleotide polymorphisms (SNPs), but
copy number variations (CNVs) as well. The optimum
amount of genotyping required to accurately
predict the CYP2D6 phenotype remains debatable.
A total of 11 different CYP2D6 variant alleles have
been identified in our population. The prevalence of
both inactive (*3,*4 and *6) and decreased activity
)*9, *10 and *41) alleles was estimated to be 0.324.
The most frequently observed variant allele was *4,
accounting for 0.575 of all variant alleles. A total of
33 different genotypes were identified. The geno-typephenotype
association was made according to the
CYP2D6 DPWG Genotype Phenotype Classification
System (http://www.pharmgkb.org). Table 2 summarizes
the resulting CYP2D6 genotypes and corresponding
phenotypes recognized in the population of
the Republic of Macedonia. Our results regarding the
CYP2D6 profile were generally in accordance with the
frequencies reported for other European populations
(Supplementary Table 1A; references 5-18). Statistically
significant differences were found in comparison
to the prevalence of the CYP2D6 gene duplication and
deletion. We report a higher frequency of CYP2D6
duplications/deletions in the Republic of Macedonia
compared to that of Germany, France, Russia and
Croatia [5,14,17]. These findings regarding the distribution
of the CYP2D6 CNV status corresponds to the
north-south gradient theory, suggesting a decreasing
prevalence of CYP2D6 CNV from the Mediterranean
countries towards Northern Europe. The frequency
distribution of CYP2D6 PMs is similar to that of the
other European populations [10,11,15-23].
All six possible CYP2C19*2 and *17 genotypes
with the corresponding phenotypes [*1/*1 and *1/*2,
both specifying extensive metabolizers (EMs); *1/*17
and *17/*17 indicative for ultrarapid metabolizers
(UMs); *2/*2 predicting PM and the *2/*17 combined heterozygous genotype with a difficult to predict
phenotype] have been identified [23] (Table 1). The
detected frequency of the most common CYP2C19
variant alleles in the population of the Republic of
Macedonia is within the range comparable with other
European populations (Supplementary Table 1A; references
7,11-14,18-36). According to the previously
reported data [24], the CYP2C19*3 allele has a very
rare occurrence in the population of the Republic of
Macedonia and therefore, it was not a subject of comparison
in this study.
The allelic frequencies for CYP2C9*2 and CYP2C9*
3 found in our population correspond to the
general frequencies reported for the various ethnic
groups throughout Europe (Supplementary Table 1B;
references 11-14,20,23, 34-47). The frequencies of the CYP2C9 genotypes associated with PM phenotypes
(0.297), *2/*2 and *3/*3 were found to be 0.026 and
0.010 [23], respectively (Table 1).
The CYP3A5*3 allele was abundantly present in
the specimens of our study with a frequency of 0.908.
We found no statistically significant difference compared
to the frequencies reported for other Europeans
(Supplementary Table 1B; references 13,36,48-52).
Since heterozygotes and wild types for CYP3A5*3
may have a CYP3A5 activity, we estimate that 0.82
of the population of the Republic of Macedonia do
not have a CYP3A5 enzymatic activity [25]. This is
in agreement with the finding that 10.0% of the indigenous
European population are high expressers
of CYP3A [13].
Phase II: Conjugation (GSTT1, SULT1A1;
UGT1A1). Extensive polymorphism also occurs in
the majority of Phase II drug metabolizing enzymes.
Sulfo-transferases and UDP-glucuronosyltransferases
are two important phase II enzyme families that
catalyze the sulfate and glucuronide conjugation of
many endogenous and exogenous substances. The
most frequent polymorphism in the SULT1A1 gene
is the SULT1A1*2 (638G>A) that is correlated with a
diminished capacity to sulphate SULT1A1 substrates.
The greatest clinical significance of SULT1A1 is its
role in the metabolism of estrogens and tamoxifen
[26]. UGT1A1 pharmacogenetics has been studied
more extensively than that of other uridine 5’-diphospho-
glucuronosyltransferases (UGTs). The greatest
clinical significance has the UGT1A1*28 variant allele
correlated with an altered level of the enzyme
expression and therefore a low enzymatic activity.
It has been associated with adverse drug reactions
(ADRs) due to reduced clearance, most notably lifethreatening
toxicity of chemotherapy with a high-dose
irinotecan [27].
Ethnic variations in allele frequencies observed
for SULT1A1*2 ranges from 0.332 in indigenous
Europeans, 0.080 in Asians and 0.294 in African-
Americans. The frequency of the UGT1A1*28 allele
varies among ethnicities, being the highest in those
of African (43.0%) or European (39.0%) descent and
the lowest in those of Asian (16.0%) descent [26,27].
Glutathione S-transferases (GSTs), are active in
the detoxification of a wide variety of potentially toxic
and carcinogenic electrophiles in conjugation with
glutathione (GSH). The most common polymorphism
is caused by a gene deletion that results in the absence
of GSTT1 enzyme activity and has been associated with a reduction of the relapse risk in several tumor
types (acute lymphoblastic leukemia, acute myeloblastic
leukemia, breast cancer, ovarian cancer and
lung cancer) treated with chemotherapy. Reported
frequencies for the GSTT1 null genotype from different
ethnic groups show a range of 16.0-60.0% for
Asians, 15.0-26.0% for Africans and 10.0-20.0% for
the European population [26,28].
Distribution of the Phase II Conjugation
(GSTT1, SULT1A1; UGT1A1) Genetic Variants
in the Population of the Republic of Macedonia.
For the SULT1A1 (638G>A) polymorphism, approximately
one-third of the population from the Republic
of Macedonia carry the low-activity SULT1A1*2 allele
and 17.0% are predicted to be homozygous for
the SULT1A1*2 genetic variant [29] (Table 3). The
distributions of the UDP-glucuronosyltransferase
(UGT)1A1*28 allele and genotype frequencies in our
population are summarized in Table 3 [30]. Regarding
the GSTT1 genotype distribution, 16 (0.13) out
of 128 study subjects were predicted to carry the null
genotype.
We found no significant difference in SULT1A1*2
allelic and genotype distribution in our population
compared to the one reported for other Europeans
(Supplementary Table 2; references 53-57). Our findings
regarding allelic and genotypic frequencies for
UGT1A1*28 genetic variant are comparable to those
observed in the Framingham Heart Study that comprises
subjects of almost entirely European descent
from England, Ireland, France and Italy, and to the
variant allele frequency reported for the Turkish population
(Supplementary Table 2; references 58-63). The
results concerning the population frequency of GSTT1
null genotypes corroborate the regional frequency
gradients of the GSTT1 null allele that exist within
the European populations (Supplementary Table 2;
references 18, 64-70).
Drug Target Genetic Variants (MTHFR,
TYMS-TSER, VKORC1). Polymorphisms in drug
targets are also an important area for pharmacogenetic
studies, since an over-expression or under-expression
of drug targets could also lead to resistance or toxicity
in standard chemotherapy regimens. Two polymorphisms
of folate cycle enzymes, methylenetetrahydrofolate
reductase (MTHFR) C677T and thymidylate
synthase (TYMS) enhancer region (TSER) 28 bp tandem
repeat, are the two most important intracellular
targets for several chemotherapeutic drugs, including
antifolates (metotrexate) and fluoropirimidines (5FU,
capecitabine), respectively [31]. The most common
MTHFR 677C>T variant is associated with a decreased
enzyme activity and increased metotrexate
intolerance. The polymorphism of a 28 bp sequence in
the 5’ promoter enhancer region of the TS gene (TSER)
varies from two (TSER*2) to nine (TSER*9) copies of
the tandem repeat, with TSER*2 and TSER*3 being
the most common alleles. The second SNP within
the second repeat of TSER*3 (TSER*3RG>C), upregulates
the TS expression and is associated with
resistance to TS-targeted chemotherapy. The MTHFR
C677T polymorphism shows wide regional and ethnic
variations among the European population (23.0-
41.0%), Asian countries (40.0-44.0%) and Africans
(12.0, 6.0%) [31,32]. Inter-population differences
have also been documented for TYMS-TSER genetic
variants; the frequencies reported for TSER*3 allele
within the Asian, indigenous European and African
populations were 0.81, 0.60 and 0.52, respectively
[32,33].
The VKORC1 gene encodes for the enzyme
vitamin K epoxide reductase that is the pharmacodynamic
target of coumarinic oral anticoagulants.
The VKORC1 (–1639 G>A) variant, the one SNP
included in the International Warfarin Pharmacogenetics
Consortium (IWPC) dosing algorithm, has been
shown to confer a high risk of bleeding, predisposing
the individuals carrying these polymorphisms to
hemorrhagic incidents. In general, the A allele has an
increasing frequency from Western Europe (40.0%)
towards Eastern Asia (90.0%) [34].
Distribution of the Drug Target Genetic Variants
in the Population of the Republic of Macedonia.
The allelic frequencies and the genotype distributions
of the MTHFR 677C>T [35], TYMS-TSER
*2/*3 (2R/3R; 3C/3G) [36] and VKORC1 –1639G>A
genetic variants in our population are presented in Table
4. Both MTHFR C677T and TYMS-TSER genetic
variants show wide regional and ethnic variations
among the European population (Supplementary Table
2; references 71-90). Significantly higher frequencies
of the MTHFR 677T allele was observed when
compared to the frequencies reported in the populations
of the Nordic countries, Turkey and Croatia.
There are few population studies of the TYMS-TSER
locus in the literature and the frequencies reported
for the TSER*3 allele correspond to that found in
our population (Supplementary Table 2; references
80-90). The prevalence of VKORC1 –1639G>A in
the population of the Republic of Macedonia is in
accordance with the values reported for other European
populations (Supplementary Table 2; references
42,91-94).
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