Molecular analysis of prothrombotic gene variants in venous thrombotic diseases. Different risk factors in different sex and clinical forms. CURRENT STATUS: POSTED

Background The role of prothrombotic gene variants as risk factors for venous thrombotic diseases is controversial and the ordering of tests for thrombophilia in the clinical context is scarcerly respective of evidence-based data. Methods We studied FVL, FVR2, FII G20210A, MTHFR C677T and A1298C, beta-fibrinogen -455 G>A, FXIII V34L, HPA-1 L33P variants and PAI-1 4G/5G alleles in: 343 patients with deep vein thrombosis (DVT), 164 with pulmonary embolism (PE), 126 with superficial vein thrombosis (SVT), 118 with portal vein thrombosis (PVT), 75 with cerebral vein thrombosis (CVT), 119 with retinal vein thrombosis (RVT) in comparison with 430 subjects from the general population (GP) of the same geographical area (Southern Italy). Results About 40% of patients with DVT, PE and SVT have a prothrombotic predisposition represented by FVL, FVR2 and FII G20210A variants (significantly more frequent than in the general population), significantly higher in PE males. While, in patients with PVT and CVT only FII G20210A variant is more frequent particularly in females. Finally, RVT is poorly related to prothrombotic risk factors, confirming that local vascular and other factors have a pivotal role in its pathogenesis. Conclusions Our data indicate that only FVL, FVR2 and FII G20210A are related to vein thrombotic diseases while all the other gene variants, frequently ordered in the clinical context, do not have a role as risk factors. Furthermore, the evidence of a sex difference for some variants, once confirmed in larger populations, may help to promote sex-specific prevention of such diseases.

thrombotic diseases while all the other gene variants, frequently ordered in the clinical context, do not have a role as risk factors. Furthermore, the evidence of a sex difference for some variants, once confirmed in larger populations, may help to promote sex-specific prevention of such diseases.

Background
Venous thromboembolism (VTE) includes deep vein thrombosis (DVT) and pulmonary embolism (PE) that frequently appears as a complication of DVT. More recently, thromboflebitis was classified as superficial vein thrombosis (SVT), and was recognized as a high-risk condition to generate PE in turn [1]. For other rarer entities with unusual thrombosis locations, like cerebral [2], portal [3] and retinal vein [4], it is discussed if the pathogenesis and risk factors are the same of VTE [5]. Finally, VTE is the third more frequent cause of cardiovascular diseases and death and represents a relevant medical and social problem for the high occurrence and for the severity of the phenotype in a percentage of cases [6,7]. Such diseases may be prevented and effectively treated; therefore, the search of risk factors is a major objective of biomedical research [8].
Venous thromboembolism is a multifactorial condition which results from the combination of multiple and synergic predisposing factors, acquired and inherited ones. Among them, hypercoagulability states, now termed "thrombophilia", play a pivotal role in the pathogenesis of VTE. Some variants found in genes involved in haemostasis and its inhibition display a procoagulant effect [5]. The aim of this study was to assess the allele and genotype frequency of nine prothrombotic gene variants in different forms of venous thrombosis in comparison to a large group of subjects from the general population of Campania region (Southern Italy) and to compare the frequency of such variants in patients of different sex and age.

Results
General population. The allele and the genotype frequencies of each gene variant are displayed in Tables 1A and 2 (column GP). No differences were observed between males and female neither in the allele or the genotype frequency (Table 1B, column GP).
Deep vein thrombosis, pulmonary embolism, superficial vein thrombosis. For each of these three diseases, the allele frequency of FVL, FVR2 and FII G20210A variants was significantly higher in DVT, PE and SVT patients with respect to subjects from the GP. For all the other variants we did not find any significant difference (Table 1A). This result was mirrored by a different distribution of the genotype frequency of FVL, FVR2 and FII G20210A variants between the groups of patients with DVT, with PE and with SVT and the general population (Table 2). In particular, each of the three variants determined a significantly increase in the odds of disease in subjects bearing the specific variant (Table   3).
Finally, when assessing sex differences in the variants-diseases association, a significant interaction was observed for the FVL variant that causes almost a six-fold increase in the odds of PE disease in male patients while failed to be a significant predictor in females (Table 3). Moreover, the allele frequencies of FVL and FVR2 were higher in males than in females with DVT, PE or SVT, although significantly only for PE subjects (Table 1B). No significant interaction effect was observed with age (first episode before or after 50 years old) in DVT, PE and in SVT patients for each of the variants (Table 3).
Portal venous thrombosis. The allele (Table 1A) and the genotype (Table 2) frequency of the FVL and of the FII G20210A variants was significantly different between the group of patients with PVT and the GP, leading to a significant increase in the odds of PVT associated to these two variants (Table 4A). For all the other variants no differences were observed between patients with PVT and the GP. The analysis of sex and age differences did not reveal any significant interaction effect (Table 4A), even if a higher (but not significant) frequency of FII G20210A was found in female patients (Table 1B). Table 1A and Table 2, only the allele and the genotype frequency of the FII G20210A variant was significantly different in the patients with CVT respect to GP, leading to a significant increase in the odds of CVT associated to this variant (Table 4B). For all the other variants, no significant differences were observed between patients with CVT and the GP. No significat interaction effects with sex and age were observed for all the variants (Table 4B), only the frequency of FII G20210A variant was higher (although not significantly) in females (Table 1B).

Retinal venous thrombosis.
For none variant the allele (Table 1A) or the genotype (Table 2) frequency was significantly different between patients with RVT and the GP.
Similarly, no significat interaction effects with sex and age were observed for these variants (data not shown).

Discussion And Conclusions
This study firstly compared the allele and the genotype frequency for nine prothrombotic gene variants between patients with various forms of venous thrombosis and a large group of control subjects from the same geographical area.
Pulmonary embolism, DVT and SVT share a genetic predisposition represented by FVL, FVR2 and FII G20210A gene variants, whose frequency is significantly higher as compared to the GP. These data match with the current literature [5,[18][19][20]. Interestingly, the allele frequency of FVL and FVR2 resulted higher in males than in females with PE, DVT and SVT, although significantly only for PE subjects (while in the general population no sex difference was identified for all the variants examined). A sex difference in the allelic frequency of FVL and FII G20210A variants was previously found by our group in females with young AMI and for MTHFR C677T mutation in males with AMI [11]. Differently, most previous studies did not find (or did not assess) a sex difference for prothrombotic variants and PAI-1 4G/5G alleles did not demonstrate a relationship with VTE [25]. While, differently from our study, MTHFR A1298C and C677T variants were found as risk factors for VTE [26]; and another study [27] revealed that only the MTHFR A1298C but not the C677T MTHFR variant was a risk factor for VTE. A significant increase of the PAI-1 4G allele frequency was found only in VTE patients that had also the FVL [28]. We did not confirm such data (data not shown).
In patients with portal vein thrombosis, FVL and FII G20210A variants resulted more frequent as compared to the GP in agreement to a previous study [4], while Parik et al. [29] excluded a major role of prothrombotic gene variants in portal thrombosis. The major limit of our study is that all cases with portal vein thrombosis had chronic liver disease and it is known that inflammation, fibrosis and alterations of local circulation are the main pathogenic basis of portal hypertension in such patients [3]. Interestingly, the frequency of FII G20210A is higher (although not significantly) in our females with PVT.
In patients with cerebral venous thrombosis we found only a significantly higher frequency of the FII G20210A variant respect to the GP, and such data match with two studies [2,30]. While, our data exclude a role of MTHFR and FVL variants that in previous studies had been reported to be associated to a higher risk for the CVT [30,31]. Interestingly, also in CVT (as we observed in PVT) the frequency of FII G20210A is higher in females than in males. Cerebral venous thrombosis is typical of young subjects and is about three times more frequent in females [2], and our results confirm this data supporting a role of hormones in its pathogenesis in addition to local risk factors [32]. However, a role of the FII G20210A, particularly in females with CVT, seems to be considered and confirmed in larger populations.
Finally, prothrombotic gene variants have a scarce role as risk factors for retinal vein thrombosis. It is well known that RVT has peculiar local risk factors different from those that act in other forms of VTE, while the potential role of thrombophilia (including FVL and G20210A) seems to be marginal [33]. Similarly, Janssen [4] found that FVL and G20210A and other prothrombotic gene variants have a minor role in the pathogenesis of RVT, being atherosclerosis the main cause [33,34]. Thus, routine testing for prothrombotic variants must be considered not advisable in patients with RVT [33].
From all these observations, it may be concluded that prothrombotic gene variants have a relevant role in DVT, PE and SVT: about 40% of patients show a prothrombotic predisposition represented by at least one among FVL, FVR2 or FII G20210A variants. Such predisposition is significantly more pronounced in males confirming that sex-related risk factors act in VTE. All the other prothrombotic variants do not have a role, despite the ordering of tests for thrombophilia is strongly heterogeneous and still scarcerly respective of evidence-based data. While, portal and cerebral vein thrombosis seem to be less related to prothrombotic gene variants, and only FII G20210A seems to act as a risk factor, particularly in females, again confirming the existence of sex-related risk factors.

Patients
The study was approved by the Ethical Committee of the University Federico II, Naples, Italy (protocol n. 380/18) and was conducted in accordance with the Helsinki Declaration.
Before the analysis, the data were anonimyzed. Our laboratory acts as the reference lab for molecular diagnostics in Campania Region (Southern Italy, about 5 million of inhabitants). During the last twelve years (i.e., 2007-2018), we received several thousands of requests for molecular analysis of thrombophilia for different diseases. We now retrospectivelly analyzed the results obtained in patients referred as affected by various forms of venous thrombotic diseases in comparison to a group of subjects from the general population (GP) of the same geographical area (Campania region, Southern Italy).
For each subject, we recorded all anagraphical and clinical data and verified that the diagnosis of each disease had been performed according to current guidelines [14][15][16].
Patients with doubtful diagnosis were excluded from the study. In addition, in cases of more patients belonging to the same family, we considered only the first case temporally analyzed. In details, our study population included: i) 430 subjects from the GP that at the anamnesis did not refer episodes of venous thrombosis (median age: 43 years; range: 5-85 years; 265 females); ii) 343 patients that experienced at least an episode of DVT (median age: 53 years; range: 10-92 years; 172 females); iii) 164 patients that experienced at least an episode of PE (median age: 56 years; range: 12-93 years; 101 females); iv) 126 patients that experienced at least an episode of SVT (median age: 53 years; range: 16-79 years; 81 females); v) 118 patients that experienced at least an episode of PVT as a complication of chronic liver disease (median age: 54 years; range: 6-79 years; 52 females); vi) 75 patients that experienced at least an episode of CVT (median age: 40 years; range: 1-76 years; 54 females); vii) 119 patients that experienced at least an episode of RVT (median age: 55 years; range: 5-88 years; 65 females). The informed consent was obtained to anonymously use all samples for research purposes.

DNA extraction
A blood sample was collected by venipuncture using the Vacutainer system in EDTA tubes.
DNA was extracted from leukocytes using a commercial automated procedure (Roche, Italy). The DNA was spectrophotometrically quantified (also to verify the purity) and analyzed by real time PCR (Roche, Italy) for: FVL (R506Q variant); FVR2 (H1299R variant);