FREQUENCIES OF SINGLE-NUCLEOTIDE POLYMORPHISMS
AND HAPLOTYPES OF THE SLCO1B1 GENE IN SELECTED
POPULATIONS OF THE WESTERN BALKANS
Daka Grapci A1, Dimovski AJ2, Kapedanovska A2, Vavlukis M3, Eftimov A2, Matevska
Geshkovska N2, Labachevski N4, Jakjovski K4, Gorani D5, Kedev S3, Mladenovska K2,*
*Corresponding Author: Professor Kristina Mladenovska, Faculty of Pharmacy, Center for Biomolecular Pharmaceutical
Analyses, University “Ss Cyril and Methodius” in Skopje, Blv. “Mother Theresa” 47, 1000 Skopje,
Republic of Macedonia. Tel: +389-2-3126-032. Fax: +389-2-3132-015. E-mail: krml@ff.ukim.edu.mk
page: 5
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MATERIALS AND METHODS
Subjects and Study Protocol. For the aim of this
study, a total of 233 Caucasian patients (age 18-72
years, average body mass index (BMI) 26.20 kg/m2,
109 women and 124 men) with hypercholesterolemia
type IIa or IIb, were selected randomly from the outpatients
evaluated for coronary heart disease at the
University Clinic of Cardiology in Skopje (RoM) and
the University Clinical Center in Prishtina, Clinic for
Internal Diseases (RoK). Of these, 156 (66.95%) were
Macedonians, 64 (27.47 %) Albanians, four (1.72%)
Turks and nine (3.86%) Gypsies. Due to the low number
of patients, the data for the groups of Turks and
Gypsies are not presented in this paper. Therefore,
the evaluated group of patients (220 individuals, 105
female and 115 male patients) consisted of 70.91%
Macedonians (n = 156, 73 women and 83 men) and
29.09% Albanians (n = 64, 32 women and 32 men).
Initially, the study protocol was approved by
the Ethics Committee of the Faculty of Pharmacy
and Committee for Clinical Studies of the Faculty
of Medicine, University “Ss. Cyril and Methodius”
(UKIM), Skopje, RoM, and the Ethics Committee
and Committee for Clinical Studies of the Faculty of
Medicine, University in Prishtina, RoK. All participants
received oral and written information and gave
a written informed consent before entering the study.
Exclusion criteria (note: not relevant for the results
present in this study, but important for the overall
aim of the research) included cancer in remission
for period shorter than 5 years, Cushing syndrome,
hyperthyroidism, positive hepatitis B surface antigen,
hepatitis C virus antibody, fibromyalgia, myopathy,
rhabdomyolysis, malabsorption syndrome, renal failure,
liver disease, McArdle disease, women who are
pregnant, nursing or have planned a pregnancy, drugs
interacting at the level of SLCO1B1. Data for BMI,
cigarette smoking, blood pressure, alcohol consumption,
physical activity and pharmacotherapy were also
collected and recorded. To evaluate the frequency of
genetic variations in genes encoding SLCO1B1, one
blood sample was obtained from each participant for
DNA extraction on the first day of the hospital visit.
In this study, 140 DNA samples obtained from
the DNA bank of the Center for Biomolecular Analysis
at the UKIM-Faculty of Pharmacy, Skopje,
RoM, were also analyzed for the diversity of the
SLCO1B1 gene. These samples were obtained from
healthy individuals (of Caucasian ethnicity, 78.57%
Macedonians, 21.43% Albanians, 79 males, average
age 48.0 ± 12.9, BMI 26.16 kg/m2) selected by
medical history, physical examination and routine
laboratory tests before entering the study. Considering
that there was no significant difference (p >0.05)
in the allelic frequencies of SLCO1B1 variants and
genotype distributions between healthy subjects and
patient groups, statistical analysis was also performed
on the total population consisted of 360 subjects,
of which 73.89% were Macedonians (n = 266, 129 women and 137 men) and 26.11% Albanians (n = 94,
42 women and 52 men).
Genomic DNA Extraction and Genotyping
Procedures. Three mL venous blood samples
drawn with EDTA as anticoagulant were collected
and stored at 4 °C prior to DNA isolation. DNA isolation
was performed at the Center for Biomolecular
Pharmaceutical Analyses, UKIM-Faculty of Pharmacy,
Skopje, RoM, using the Qiamp DNA Blood kit
(Qiagen GmbH, Hilden, Germany) according to the
manufacturer’s protocol. The samples were kept at
–20 °C until further analysis. The SLCO1B1 SNPs to
be genotyped were selected on the basis of literature
data [6,13,20,28,29] and a previous study in which
151 subjects were included [30]. The following variants
in the SLCO1B1 gene were analyzed: c.388A>G
(Asn130Asp, rs2306283), c.521T>C (Val174Ala,
rs4149056), c.571T>C (Leu191Leu, rs414 9057),
c.597C>T (Phe199Phe, rs2291075), c.1086C>T
(Tyr362Tyr, rs57040246), c.1463G>C (Gly488Ala,
rs5950 2379), c.*439T>G (rs4149087, the position
is given with the first nucleotide 3’ of the stop codon
(TAA) set to *1) using TaqMan allelic discrimination
assay (Applied Bio-systems, Foster City, CA, USA).
Polymerase chain reaction was performed on the
quantitative real-time PCR (q-PCR) system Mx3005P
(Strata gene, La Jolla, CA, USA) using TaqMan genotyping
protocols (TaqMan®Drug Metabolizing
assay; Applied Bio-systems) in total volume of 12.5
μL under following conditions: one cycle of 2 min.
at 50 °C, one cycle of 10 min. at 95 °C, and 50 cycles
of 15 seconds at 92 °C and 1 min. at 60 °C.
Population Genetics and Statistical Analysis.
The study sample alleles and genotype frequencies
were estimated with a gene counting method.
The agreement with Hardy-Weinberg equilibrium
(HWE) of the observed genotypic distribution for
the SLCO1B1 gene was tested with the χ2 test. The
statistical analysis was carried out using the Statistical
Package for the Social Sciences (SPSS Inc., Chicago,
IL, USA) software (v. 19.0).
Genetic diversity was quantified between the
members of the same ethnic population, between
the ethnic populations, and between different ethnic
populations and the global population. Population
comparisons were also performed with the χ2 test of
population differentiation. Odds ratios (ORs) were
calculated with 95% confidence interval (95% CI).
For multiple comparisons, Bonferroni’s post hoc
test was used. Statistically significant differences
were those where the p value was less 0.05. Linkage
disequilibrium for each pair of SNPs within each
population was quantified (correlation r2 and coefficient
of linkage disequilibrium D’ values) to find
the haplotypes in the study groups. The statistical
analyses were carried out using the SHEsis software
platform for the analysis of LD, haplotype construction
and genetic association at polymorphism loci
(http://analysis2.bio-x.cn/myAnalysis.php) [31]. The
haplotypes were presented with their previously assigned
names, as cited in the study of Pasanen et
al. [13] in which allelic frequencies at 11 variant
sites were determined (g.11187G>A, g.11110T>G,
g.10499A>C, c.388A>G, c.411G>A, c.463C>A,
c.521T>C, c.571T>C, c.597C>T, c.1929A>C and
c.*439T>G). Considering that five of these SNPs
and two other SNPs have been analyzed in the present
investigation, one haplotype has several names
and there are haplotypes that we designated as new.
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