THE 3’ END PROTHROMBIN GENE VARIANTS IN SERBIAN
PATIENTS WITH IDIOPATHIC THROMBOPHILIA Aradjanski M1, Djordjevic V1, Pruner I1,*, Tomic B1, Gvozdenov M1, Kovac M2,3, Radojkovic D1 *Corresponding Author: Dr. Iva Pruner, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe
444A, PO Box 23, 11010 Belgrade, Serbia. Tel: +381-11-397-6658. Fax: +381-11-397-5808. E-mail: iva.pruner@
gmail.com page: 43
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MATERIALS AND METHODS
the database of over 4000 patients who were referred
to the Institute of Molecular Genetics and Genetic
Engineering, University of Belgrade, Belgrade,
Serbia for genetic testing of thrombophilia during
the period from 2000 to 2013. Anamnestic data were
gathered from all the participants and the following
subjects were excluded from further investigation: the
patients who developed the first thrombotic event at
an age above 50 years, the patients with malignancies,
diabetes, antiphospholipid antibodies, deficiency of
natural inhibitors (antithrombin, protein C, protein
S), and the carriers of FVL and FII G20210A gene
mutations [26]. Finally, the study group included 100
patients (age range 16-63, median 38 years; male/
female: 45/55) with a clinical picture of recurrent
thrombotic events (DVT, PE, FL) or the combination
of two or three thrombotic events (DVT, PE, FL, MI
and stroke).
The control group comprised 100 healthy subjects
(age range: 20-66 years, median 39; male/female:
82/18) with no history of thrombotic events.
Informed consent was obtained from all participants
and the study protocol was approved by the local
research ethics committee.
Laboratory Methods. Peripheral blood was
taken on 3.8% sodium citrate as anticoagulant. Genomic
DNA was purified from 200 μL of human
whole blood using the QIAamp DNA blood mini
kit (Qiagen GmbH, Hilden, Germany) according to
the manufacturer’s protocol. The blood and DNA
samples were stored at –20 °C until further use.
The 715 bp fragment that includes the last intron
and exon, 3’UTR and the flanking region of the FII
gene (primers: 5’-GGA AAC GAG GGG ATG CCT
GT-3’ and 5’-CCT GCC ATC TTT CCT CTC AC-3’),
was amplified by polymerase chain reaction (PCR).
The PCR reactions were performed in a 25 μL final
volume: 1 × Kapa Buffer B; 2.5 mM MgCl2; 200 μM
dNTPs; 1U Kapa Taq polymerase (Kapa Biosystems,
Boston, MA, USA), 10 pmol of forward and reverse
primers and 200 ng of DNA. The thermal cycle profile
was: initial denaturation at 95 °C for 5 min. and 37
cycles consisting of denaturation at 95 °C for 1 min.,
annealing at 61 °C for 1 min. and polymerization at
72 °C for 1 min. were applied. Final extension of the
PCR products was at 72 °C for 10 min.
Sequencing of the amplified 715 bp fragments
was performed according to the manufacturer’s protocol,
using the BigDye™ Terminator Version 3.1
Ready Reaction Kit (Applied Biosystems, Foster
City, CA, USA) on a 3130 Genetic Analyzer (Applied
Biosystems). Two sequencing reactions for the
fragment of interest were performed for each sample
using the forward primer (5’-TCT AGA AAC AGT
TGC CTG GC-3’) or reverse primer (5’-GAA TAG
CAC TGG GAG CAT TGA-3’).
Statistical Analysis. Statistical analysis was performed
using MedCalc 12.2.1.0 statistical software
(MedCalc Software bvba, Ostend, Belgium). The prevalence of detected gene variants was compared
between patients and controls with the use of Fisher’s
exact test. The odds ratio (OR) and 95% CI (95%
confidence interval) were also estimated. A p value
of <0.05 was considered to be statistically significant.
Deviations of genotype distributions from Hardy-
Weinberg equilibrium were assessed by the c2-test.
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