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

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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