Frequency of thrombophilia associated genes. Population – based study.

Background: Thrombophilia is a hypercoagulable state that may have a genetic basis (inherited) or can be acquired. It is a multifactorial condition and only the mutual interactions between the environment and genes may lead to the development of clinical manifestation. This state is the main factor promoting venous (rarely arterial) thromboembolism (VTE). Inherited thrombophilia is mainly associated with two pathogenic variants in the V coagulation factor ( FV ) and the prothrombin ( FII ) genes. The aim of our study was to evaluate the frequency of two pathogenic variants in FII and FV genes as inherited thrombophilia factors in a group within the Polish population in comparison with other described populations. Methods: All studied groups consisted of 633 unrelated patients aged between 18 and 70. Individuals in the research group come from the Podlasie region of Poland. Genotyping of FII and FV variants was performed using the 7900HT Fast Real-Time PCR System and were genotyped by TaqMan assay. Results: The pathogenic allele frequency for A allele was 0.03 (3%) and 0.07 (7%) for FII and FV genes, respectively. The GA/AA genotypes (c.*97G>A variant) were observed in only 33 (5.03%) individuals in the studied group. Additionally, the frequency of GA/AA genotypes was over 17.4% in the coagulation factor V. Co-incidence of heterozygous genotype GA of variants FII and FV genes was observed in only 4 subjects. Conclusions: The FII gene variant shown in our study is less frequent than in other European countries (about 6%). In contrast, the A allele of the FV gene occurs with a frequency similar to that of Northern, Central and South Central Europe (about 5%).

Genotyping of FII and FV variants was performed using the 7900HT Fast Real-Time PCR System and were genotyped by TaqMan assay. Results: The pathogenic allele frequency for A allele was 0.03 (3%) and 0.07 (7%) for FII and FV genes, respectively. The GA/AA genotypes (c.*97G>A variant) were observed in only 33 (5.03%) individuals in the studied group. Additionally, the frequency of GA/AA genotypes was over 17.4% in the coagulation factor V. Co-incidence of heterozygous genotype GA of variants FII and FV genes was observed in only 4 subjects. Conclusions: The FII gene variant shown in our study is less frequent than in other European countries (about 6%). In contrast, the A allele of the FV gene occurs with a frequency similar to that of Northern, Central and South Central Europe (about 5%).

Background
Thrombophilia can be defined as a predisposition to form clots inappropriately and may be inherited or acquired [1,2] and this is the main factor promoting venous (rarely arterial) thromboembolism is responsible for about 30,000 deaths per year [5]. VTE is not only a risk factor for mortality (strokes, heart attacks), but it also causes morbidity post-thrombotic syndrome and chronic thromboembolic pulmonary hypertension (CTEPH) which reduce the quality of life for patients.
The acquired tendency to develop thrombophilia and VTE is connected with a lot of clinical or environmental-hypercoagulable states. The most commonly acquired thrombophilia factors are laparoscopic surgery, arthroscopic knee surgery, increasing age, obesity, smoking, immobility due to sitting and bed rest more than three days [6]. Other thrombophilia and VTE risk factors are malignancy, pregnancy or polycythemia rubra vera. Oral drug and transdermal contraceptives, hormone replacement therapy and chemotherapeutic drug -tamoxifen are a strong hypercoagulable states [6]. The other VTE risk factor is antiphospholipid syndrome (APS) This rare autoimmune condition leads to venous/arterial thrombosis or pregnancy morbidity [7].
Inherited thrombophilia is mainly associated with two pathogenic variants in V coagulation factor (FV) and the prothrombin genes. In the FV gene the presence of the Leiden mutation c.1601G>A (p.Arg534Gln;R506Q; rs6025; NM_000130.4) occurrs in 1-7% of people in Europe and is rare or absent in individuals of African , Japanese or native American origin . The prothrombin gene (coagulation factor II, FII) mutation is connected with thrombophilia c.*97G>A (previously designated as G20210A or 20210G>A; rs1799963; NM_000506. 3) and is present in 0,7 -4% of people in the general population [8,9]. Both pathogenic variants occur most often in the heterozygous form (referred to as carrier state) together or separately. Patients with either the heterozygous FV Leiden or FII mutation are at a mild risk of thrombosis and 3.8 and 4.9 times, respectively more prone to a first blood clot. However, if the patient is the carrier of both heterozygous mutations, then the risk becomes higher and increases by up to 20 times. Homozygotous patients with FII and FV mutations are very rare in the general population [9]. A deficiency of antithrombin, protein C, protein S, plasminogen and dysfibrinogenemias are less in common inherited thrombophilias. All known genetic factors are present in about 25% of unrelated VTE cases and in about 63% of patients with a family history of VTE. These inherited thrombophilias have limited clinical significance in primary care but they may be important for patients with DVT or PE. Deep vein thrombosis is a major risk factor for the development of PE, which is a life-threatening condition. Thrombophilia is a multifactorial condition and only the mutual interactions between the environment and genes may lead to the development of clinical manifestations [4,8,9].
In respect to the above-mentioned, the aim of our study was to evaluate the frequency of two pathogenic variants in FII and FV genes as inherited thrombophilia within a group of Polish population in comparison with other described populations. In the current group we would like to assess the incidence of alleles and genotypes only as a population study without reference to the clinical features of patients. Performing a case-control study is the next step in our research among in Polish population.

Methods
The study group was composed of 633 unrelated patients between the ages of 18 and 70,

Results
The frequency of genotypes and alleles of coagulation factors FII (rs1799963) and FV (rs6025) in our studied population is shown in Table 2. Our study showed that the pathogenic variants in FII and FV genes were observed in 2.7% (A) and 7% (A) of the analyzed group of people, respectively. The pathogenic allele frequency in the studied group for A allele was 0.03 (3%) and 0.07 (7%) for FII and FV genes respectively ( Table 2)

Discussion
The coagulation factor V variant was first described in 1994 by Bertina and et al [10]. This single point mutation, described as a transition of G (guanine) to A (adenine) in 1601 nucleotide position in exon 10, causes changes in the protein chain; the substitution of arginine for glutamine (R506Q) [11]. This pathogenic variant leads to the resistance of coagulation factor V to proteolytic inactivation by the activated protein C (APC), which is consequently related to a predisposition to thrombosis.
Factor V Leiden is a poor risk factor of VTE when we compared it with the impact of natural anticoagulants deficiency, but it may be present in around 20% of patients with venous thrombosis [12]. Notwithstanding the aggregation of their data with the current data (in our study n=633 and in Alder study n=1588-total n=2221) indicates a frequency value for Poland of 4,5% [16]. The distribution of the A allele of the FV gene shown in our study places Poland, according to the study by Clark et al. in the group of European countries with a frequency above 3% , regardless of whether we add our result to the one previously published by Alder [16,17]. These include Northern European countries such as Sweden (4. 5%), Norway (4. 2%), Denmark (3. 9%), U. K. (3. 7%) [17][18][19][20], and Central European countries like Germany (3. 8%), The Czech Republic (5. 1%), Italy (4%), Greece (3. 2%), Hungary (3. 8%) Austria (2. 8%) [17]. The Romanian value (8. 3%) was found to be significantly higher than in all European countries [21]. However, in Slavic countries a lower frequency of this allele was observed (<3%) [a]. In countries such as Slovenia, Croatia, Bosnia and Herzegovina, Serbia and Montenegro, the distribution of c. 1601A ranged from 1. 5 to 2. 5% [22]. Also in the countries of the Eastern European bloc -Finland, Russia, Ukraine and Belarus the frequency of this variant is low (0. 6-2.4%) [22,23]. Interestingly, in Western European countries such as France, Spain and Portugal, the frequency of the A allele is very similar to that of Slavic countries (2%, 0. 9%, 1. 2% respectively) [17]. The result of the frequency of the A allele FV gene differentiates our country in this respect from the group of Slavic countries to which Poland belongs. This can be explained by the influence of geopolitical conditions in with Poland has been entangled over the centuries. This probably influenced the penetration of the c. 1601A allele into the population. Also, the historical migration of the population, which leads to ethnic differences, has influenced the distribution of the allele within the European population. The number of individuals in groups also influences the results obtained. In our study for variant 20210G>A in the FII gene, we observed a frequency of prevalence with the value 0.03. By way of comparison, in the Bosnian population, the A variant of the FII gene occurred at a frequency of 6%, 1% in the Saudi population and 5.4% in the Italian population [24][25][26]. No data on the distribution of this allele in other populations is available. Allele frequency: - † -0.03 (A) ; ‡ -0.07 (A).
Tab. 3. Recommendation of ACMG (American College of Medical Genetics and Genomics).