Deep vein thrombosis (DVT) is the most common clinical manifestation of venous thromboembolism with multi factorial pathogenesis. Venous thromboembolism (VTE) occurs as a result of interaction of series of different risk factors including acquired and hereditary conditions. Generally, a tendency toward venous thrombosis can arise from hyperactive anticoagulant pathways, hypoactive anticoagulant mechanisms or hypoactive fibrinolysis [1]. Venous thromboembolism is highly heritable and multiple coinherited genetics risk factors increase the incidence risk [2]. The most common identified mutations interfering with VTE are: 1691 (G>A) factor V Leiden (FVL), 20210 (G>A) prothrombin (PT), deficiency of protein C, protein S and antithrombin III.
Blood coagulation factor V is coded by a gene located on the long arm of chromosome 1 (1q23) and consists of 25 exons and 24 introns [3]. Transition in exon 10 of gene F5 causes a substitution (R506Q) known as factor V Leiden that has been recognized as the most prevalent genetic risk factor for VTE. Heterozygotes and homozygotes of the 1691 mutation are associated with a 7- and 80-fold increased risk of venous thrombosis, respectively [4]. It has been estimated that the prevalence of 1691 mutation in the patients with DVT varies between 20.0 and 30.0% [5]. Estimated frequencies of 1691 mutation among Caucasians range from 2.0 to 9.5%, moderate prevalence is observed in Hispanic Americans and Indians (1.0-5.0%), whereas it is rare in Asians and Africans (<1.0%) [6,7].
A G to A transition at position 20210 within the 3’ untranslated region (3’UTR) of the
The C to T transition at position 677 in exon 4 of the
Although 1691, 20210 and 677 mutations have been recognized as the most common risk factor for VTE, there are only a few reports of prevalence of these mutations in the healthy Bosnian population [12,13]. Our study was conducted in order to investigate the frequency of these mutations and their association with DVT in the Bosnian population.
The DNA was isolated from EDTA anti-coagulated whole blood (Vacutainer Becton Dickinson, Meylan Cedex, France) using the commercial FlexiGene DNA Isolaton Kit (250) (Qiagen GmbH, Hilden, Ger- many). The 1691, 20210 and 677 mutations were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques that were in accordance with previously described protocols [8,14,15]. The genotypes were determined by electrophoresis in 4.0% agarose gel (Sigma Aldrich Chemie GmbH, Münich, Germany) stained with ethidium bromide (Sigma Aldrich).
Statistical analyses were performed by using MedCalc 12.4.0.0 (MedCalc Software, Ostend, Belgium). Deviation of allele and genotype distribution from the Hardy-Weinberg equilibrium was assessed by the χ2 test. For all mutations, the odds ratio (OR) and their 95% confidence intervals (CI) were calculated according to McHugh [16] to estimate the risk for DVT. Statistical significance was set at a
In this study, we investigated the frequency of the most common prothrombotic factors and their association with DVT in patients with DVT and healthy controls in Bosnia and Herzegovina. The frequencies of allele and genotypes of 1691, 20210 and 677 mutations are summarized in Table 1. Of the three tested mutations, only the 1691 mutation was shown to be significantly associated with DVT with an OR (95% CI) = 6.0 (2.62-14.14);
Allele and genotype frequencies of the 1691 (G>A) FVL, 20210 (G>A) PT and 677 (C>T) MTHFR mutations.
Mutations | Alleles | Cases | Controls | OR For wild-type | |
---|---|---|---|---|---|
1691 (G>A) FVL | wild type G/G | 88 (79.30) | 199 (96.14) | 6.0 (2.62-14.4) | 0.0001 |
allele G | 196 (89.50) | 406 (98.06) | 5.6 (2.4-12.7) | 0.0001 | |
20210 (G>A) PT | wild type G/G | 108 (97.30) | 207 (100.00) | 13.5 (0.6-263.9) | 0.087 |
allele G | 219 (98.64) | 414 (100.00) | 13.5 (0.6-263.9) | 0.087 | |
677 (C>T) MTHFR | wild type C/C | 43 (38.74) | 91 (43.96) | 1.24 (0.77-1.98) | 0.368 |
allele C | 133 (66.16) | 274 (70.26) | 0.82 (0.57-1.19) | 0.309 |
FVL: factor V Leiden; PT: prothrombin; MTHFR: methylene tetrahydrofolate reductase; OR: odds ratio; 95% CI: 95% confidence interval.
Table 2 shows the allele and genotypes frequencies of the 1691, 20210 and 677 mutations according to gender. Allele and genotype frequencies of the 1691 mutation did not differ significantly between genders in compared groups (χ2 = 0.359;
Allele and genotype frequencies of factor 1691 (G>A) FVL, 20210 (G>A) PT and 677 (C>T) MTHFR mutations according to gender.
Mutations | Alleles | Patients | Controls | ||||||
---|---|---|---|---|---|---|---|---|---|
Men | Women | χ2 (df = 1) For wild-type | Men | Women | χ2 (df = 1) For wild-type | ||||
1691 (G>A) FVL | wild type G/G | 38 (73.08) | 46 (77.96) | 0.359 | 0.549 | 97 (95.10) | 102 (97.14) | 0.582 | 0.445 |
allele G | 89 (86.40) | 103 (88.80) | 0.287 | 0.592 | 199 (97.54) | 226 (98.26) | 0.771 | 0.387 | |
20210 (G>A) PT | wild type G/G | 51 (98.07) | 57 (96.32) | 0.226 | 0.634 | 102 (100.00) | 105 (100.00) | – | – |
allele G | 103 (99.03) | 116 (98.30) | 0.223 | 0.636 | 204 (100.00) | 210 (100.00) | – | – | |
677 (C>T) MTHFR | wild type C/C | 18 (34.61) | 25 (42.37) | 0.701 | 0.402 | 42 (41.18) | 49 (46.67) | 0.873 | 0.350 |
allele C | 61 (64.21) | 72 (67.92) | 0.165 | 0.680 | 133 (68.91) | 141 (71.94) | 0.428 | 0.512 |
FVL: factor V Leiden; PT: prothrombin; MTHFR: methylene tetrahydrofolate reductase.
The frequencies of combined genotypes of the 1691, 20210 and 677 mutations are presented in Table 3. Two individuals (1.16%) with combined heterozygous genotypes of 1691 and 20210 were part of the patients’ group. The highest frequency (14.89%) of combined genotypes was detected for compound heterozygotes for 1691 and 677 mutations in the patients’ group. This combination of genotypes was observed in 6.19% controls. In the patients’ group we observed 6.39% individuals who were heterozygous for 1691 (GA) and homozygous (TT) for 677. Based on that, we did not identify any homozygote (AA) for 1691 in the control group, while its combination with the other two mutations was absent.
The Frequencies of combined genotypes of 1691 (G>A) FVL, 20210 (G>A) PT and 677 (C>T) MTHFR.
Combined Genotypes | Patients | Controls |
---|---|---|
1691 (G>A) FVL (GG) and 20210 (G>A) PT (GG) | 85 (98.84) | 199 (100.00) |
1691 (G>A) FVL (GG) and 677 (C>T) MTHFR (CC) | 34 (72.34) | 90 (92.78) |
1691 (G>A) FVL (AA) and 677 (C>T) MTHFR (CT) | 2 (4.25) | 0 (0.00) |
20210 (G>A) PT (GG) and 677 (C>T) MTHFR (CC) | 43 (97.72) | 91 (100.00) |
FVL: factor V Leiden; PT: prothrombin; MTHFR: methylene tetrahydrofolate reductase.
The observed prevalence of the 1691mutation in this study corresponds to the reported prevalence in the healthy Caucasian population [2,17-20]. The relationship between the 1691 mutation and VTE is commonly recognized [21]. According to the data, about 18.0% of patients with first occurrence of DVT and about 40.0% of thrombophilic families carry 1691 [5,14]. In this study, the 1691 mutation was shown to be significantly associated with DVT with an OR (95%CI) = 6.0 (2.62-14.14);
The second genetic risk factor for vein thrombosis, the 20210 mutation, was present in the Caucasian population with a frequency of 0.7 to 4.0%, with an average close to 2.0%. The prevalence of this mutation was higher in the Southern European countries than in Northern countries, in spite of presence of overlapping between north and south. The 20210 mutation was found to be very rare or even absent in Asian and African populations and American Indians and Australian Aborigines [25]. The greatest frequency of 20210 is reported in Hispanics and Mexican Mestizos. The prevalence of this mutation in patients with VTE was 8.0% and an even higher prevalence (18.0%) was found in selected families with thrombosis. In our investigated group, three out of 111 patients were heterozygotes for the 20210 mutation. In the control group, the 20210 mutation was absent. Our results are in contrast with the results of Adler
Two common polymorphisms of the
Researching the distribution of the 1691, 20210 and 677 mutations according to gender is especially important in women because they undergo hemostatic changes during the pregnancy. We did not observe the difference in the frequencies of these mutations between men and women in the patients’ group or in the control group. However, we found statistically significant difference in distribution of the 1691 mutation between women with DVT and women in the control group and between men in compared groups. We can only speculate that men and women who were heterozygotes or homozygotes for the 1691 mutation were more prone to develop DVT. Literature data have shown that the combined effect of more than one genetic variant can double or triple the risk for VTE. The combination of the most frequent genetics risk factor, 1691 and 20210, has been frequently found in patients with VTE. The prevalence of 0.4% of both mutations in individuals with VTE was observed by Ridker
The frequency of the 1691, 20210 and 677 mutations in the healthy population in Bosnia and Herzegovina is similar to previously reported prevalence of these mutations in other populations in the region. Based on our results, we can conclude that only the 1691 mutation was significantly associated with DVT in our population. We did not confirm association of other two studied mutations, 20210 and 677, with DVT in the investigated sample of the Bosnian population. The observed frequency of combined heterozygosity for the 1691 and 677 mutations in our study and their possible impact on developing DVT imply that further studies on a larger number of patients from all of Bosnia and Herzegovina are needed for confirmation of our findings.