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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 44  |  Issue : 1  |  Page : 21-27

Thrombophilic gene polymorphisms are associated with deep venous thrombosis in a cohort of Egyptian patients


1 Department of Clinical and Chemical Pathology, Faculty of Medicine, Beni Suef University, Beni Suef, Egypt
2 Internal Medicine Department, Faculty of Medicine, Cairo University, Giza, Egypt
3 Department of Critical Care, Faculty of Medicine, Beni Suef University, Beni Suef, Egypt

Date of Submission18-Jan-2019
Date of Acceptance30-Jan-2019
Date of Web Publication27-Sep-2019

Correspondence Address:
Dina H El-Dahshan
Clinical and Chemical Pathology Department, Faculty of Medicine, Beni Suef University, Beni Suef, 19773
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_3_19

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  Abstract 


Background There is several evidence suggesting, that inherited thrombophilia increases the susceptibility to venous thromboembolism (VTE).
Aim is to detect prothrombotic genes polymorphisms possible association with the risk of occurrence of venous thrombosis.
Methods A group of eleven gene polymorphisms related to the risk of thromboembolic events were studied; including Factor V Leiden(FVL) 1691G>A, Methylene tetrahydrofolate reductase gene (MTHFR) C677>T and others. Genotyping was done using PCR, and identification of corresponding bands using the StripAssay in 493 patients with clinical symptoms of deep venous thrombosis (DVT). Five hundred age and sex matched healthy individuals were included as a control group.
Results Genotyping of DVT patients showed significantly higher prevalence of FVL 1691G>A, MTHFR C677>T, MTHFR 1298A>C, Prothrombin 20210G>A, PAI 4G, FVIII V34L, B Fibrinogen 455G>A, HPA-1, ACE and APO E e4 gene polymorphisms (p<0.001). Seven and 11 fold increased susceptibility to DVT among patients with MTHFR C and MTHFR A mutations (OR 7.318& 11.23) respectively. Moreover a strong association was detected between DVT and mutations in genes: PAI 4G (OR 46.987), B Fibrinogen (OR 3.331), Ace (OR 187.663) and Apo E (OR 386.361).
Conclusion Individuals carrying thrombophilic genes polymorphisms are at a greater risk for venous thrombosis.

Keywords: DVT, factor V leiden, gene polymorphism, MTHFR, PAI, prothrombin gene, thrombophilic genes


How to cite this article:
Essa HH, Gobran H, Sabry SM, El-Dahshan DH. Thrombophilic gene polymorphisms are associated with deep venous thrombosis in a cohort of Egyptian patients. Egypt J Haematol 2019;44:21-7

How to cite this URL:
Essa HH, Gobran H, Sabry SM, El-Dahshan DH. Thrombophilic gene polymorphisms are associated with deep venous thrombosis in a cohort of Egyptian patients. Egypt J Haematol [serial online] 2019 [cited 2019 Oct 23];44:21-7. Available from: http://www.ehj.eg.net/text.asp?2019/44/1/21/268000




  Introduction Top


An understanding of the mechanisms that promote thrombosis has improved significantly. Deep venous thrombosis (DVT) has become a health burden with about 0.1–0.2% annual incidence [1]. Normal circulation and thus hemostasis entails a delicate balance between the main clotting processes: procoagulant, anticoagulant, and fibrinolytic systems.

Procoagulant clotting proteins initiate and propagate normal clot formation. Once the clot has initiated, naturally occurring anticoagulant proteins regulate the clotting cascade. These anticoagulant proteins confine the clot to the injured site by downregulating the procoagulant system.

After clot formation and bleeding control, the fibrinolytic system is activated leading to clot removal and restoring of normal vascular architecture. In thrombophilic diseases, thrombosis may be due to increased levels of procoagulants, decreased levels of natural anticoagulants, or defects in the fibrinolytic system. In addition, vascular wall abnormalities may promote clot formation [2].

Venous thrombosis is related to three pathological factors commonly known as ‘Virchow’s triad.’ The components of Virchow’s triad include vessel damage, blood hypercoagulability, and blood stasis [3]. The term thrombophilia is used to describe a group of diseases in which there is a predisposition to, may be repeated and extended over a period of time, excessive clotting [4]. These conditions include and are not limited to: (a) family history of increased clotting or a diagnosis of a demonstrated genetic mutation such as factor V Leiden (FVL), protein C and S deficiencies, antithrombin deficiency, and prothrombin (PT) 20210A mutations, methylenetetrahydrofolate reductase (MTHFR) C677T causing hyperhomocysteinemia, plasminogen activator inhibitor-1 (PAI-1), or (b) an acquired condition such as lupus anticoagulant or antiphospholipid antibody syndrome, which can occur alone as a manifestation of an autoimmune disorder, or as part of a syndrome such as systemic lupus erythematosus. Family studies showed that genetic mutations accounts for 60% of DVT cases [5].


  Aim Top


The aim of this study was to understand the prevalence of genetic mutations predisposing to thrombophilia between healthy control and DVT patients. In an attempt to investigate their association with the risk of occurrence of venous thrombosis.


  Patients and methods Top


Study population

In this study, the case group comprised 493 (369 men and 124 women) DVT patients, complaining of clinical symptoms of DVT. All cases group individuals were recruited following referral by a clinician from different centers after confirmed diagnosis with the presence of thrombus using Doppler ultrasound. Cases with a positive history of predisposing factors to DVT such as surgery, hospitalization, and prolonged immobilization were excluded. Five hundred (314 men and 186 women) age-matched and sex-matched healthy patients with negative family and personal history of thromboembolic events were included as the control group.

Laboratory investigations

Using ELIZA, all participants were screened for protein C antigen (REAADS catalog #: K035-001), protein S antigen (catalog #: K036-001), and anticardiolipin immunoglobulin (Ig)G/IgM (REAADS catalog #: K023-001). Also antithrombin III and lupus anticoagulant (stago lot #231775) were tested. Flowcytometry: The presence of CD16 and CD56 was investigated with flowcytometry, Coulter Beckman Elite (catalog # code F7011#M7 304; Dako, Michigan, USA).

Detection of genetic variants

DNA extraction and genotyping

From each patient and control, 5–10 ml of venous blood was collected on EDTA and stored at −20°C until genomic DNA extraction was done. Genomic DNA was extracted from leukocytes using GIAamp DNA Blood Mini kit (Qiagen, Venlo, Netherlands), Genetic variations of 11 mutations in 10 genes associated with venous thromboembolism (VTE) and cardiovascular disease were studied included: FVL 1691G>A, two mutations in MTHFR gene C677>T and 1298A>C mutations, PT 20210G>A mutation, PAI-1 4G, factor VIII (FVIII) V34L, β-fibrinogen (FGB) −455G>A, angiotensin-converting enzyme (ACE), human platelet antigen 1b (HPA-1), apolipoprotein B (Apo B) R3500Q, and apolipoprotein E (Apo E)/E4.

CVD StripAssays (catalog # 4-360, ViennaLab, Austria) were used with the immobilized oligos on a test strip. DNA extraction and amplification with multiplex PCR and simultaneous biotin labeling were done according to the manufacturer’s instructions using a thermocycler, then hybridization was done directly on the StripAssay test strips and finally identification of the labeled product bands was done by the naked eye.

Statistical methods

Data were coded and entered using the statistical package for the social sciences (SPSS), version 24 (IBM corporation, Huoston, TX, USA). Data were summarized using mean and SD for quantitative variables and frequencies (number of cases) and relative frequencies (percentages) for categorical variables. For comparing categorical data, χ2-test was performed. Fisher’s exact test was used instead when the expected frequency is less than 5 [6]. Genotype and allele frequencies were calculated in the disease and the control groups. Odds ratio (OR) with 95% confidence intervals (CIs) was calculated using logistic regression [7]. P values less than 0.05 were considered as statistically significant.


  Results Top


The results of this study showed that the presence of anticardiolipin antibodies (IgG and IgM) was significantly higher in DVT patients (P=0.007 and 0.030, respectively). Also, the prevalence of protein C and protein S deficiency was significantly higher in DVT patients than controls (P=0.007 and 0.030). Moreover, the lupus anticoagulant frequency was significantly higher in patients (P=0.015). Both CD16 and CD56 surface antigens were not detected in any of our patients and controls. The results of different laboratory tests performed to our patients and controls are displayed in [Table 1].
Table 1 Comparing the results of laboratory investigations in deep venous thrombosis patients and control group

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Analyzing the prevalence of different genotypes of each of the thrombophilic genes, and comparing between cases group and healthy control group showed statistically significant higher prevalence of these mutations in patients than controls (P<0.001) in: FVL 1691G>A, MTHFR C677>T, MTHFR 1298A>C, PT 20210G>A, PAI 4G, FVIII V34L, FGB 455G>A, HPA-1, ACE, and Apo E e4 gene mutations. Genotyping heterozygosis, homozygosis frequencies for all studied polymorphisms are given in [Table 2]. Comparing genotype distribution in male and female patients showed significant difference in FVIII and ACE gene mutations (P=0.033 and 0.007, respectively; [Table 3]), while comparing control men and women, there was no significant difference in gene polymorphism frequency between sexes.
Table 2 Comparing the distribution of 11 gene polymorphisms among patients and control groups

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Table 3 Comparison of genotype of 11 polymorphisms between men and women of deep venous thrombosis patients group

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Finally, when the heterozygote and homozygote genotypes were added to form a new group (carrier group or mutant), there was a statistically significant difference between DVT patients and control in the frequency of nine of the 11 studied genes ([Table 4]). Analyzing the OR in our genotyping results has shown that patients with MTHFR mutations showed significantly increased susceptibility to DVT. Patients carrying the MTHFR T mutation showed seven-fold more susceptibility to DVT than controls: 95% CI: 5.133–10.434 (OR 7.318, P<0.001), while patients with the MTHFR C mutation were 11-fold more susceptible to DVT than controls: 95% CI: 8.056–15.664 (OR 11.23, P<0.001). These results highlighted us to detect the significant difference of prevalence of double heterozygosity of the MTHFR among DVT patients that was present in 18.1% of patients and in 1.4% of normal individuals (P<0.001). In addition to mutations of PAI 4G (OR 46.987), FGB (OR 3.331), ACE (OR 187.663), and Apo E (OR 386.361) showed significant association between gene mutation and the occurrence of DVT ([Table 4]).
Table 4 Association of 11 gene polymorphisms with deep venous thrombosis

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  Discussion Top


DVT is considered the most common clinical manifestation of VTE. Its pathogenesis is multifactorial occurring due to an interaction of many risk factors including both acquired and hereditary conditions. Generally, a tendency toward venous thrombosis can arise from hypoactive anticoagulant mechanisms or defective fibrinolysis [8]. VTE is mainly inherited in addition to multiple coinherited genetic risk factors which raise the incidence risk [9].

Previous studies have discussed the polymorphisms in genes involved in hemostasis and how they could be related to the increased risk of intravascular coagulation. The most common identified mutations involved in VTE are: 1691 (G>A) FVL, 20210 (G>A) PT, deficiency of protein C, protein S, and antithrombin III [10].

Hence, to our best knowledge, investigating the prevalence of 10 thrombophilic gene polymorphisms in DVT patients and their association with the disease are done in the current study for the first time in the northern region of Upper Egypt.

A Lebanese study [11] performed genotyping using the StripAssay used in this study to evaluate its added value in genotyping. They recommended these strips to be used in screening for VTE and cardiovascular disease (CVD) genetic risk factors. Using the CVD strip with 11 gene polymorphisms led to identify new mutations and single nucleotide polymorphisms in nearly two-thirds of patients with a normal genotype when they were negative when typed for FVL, PTH, and MTHFR polymorphisms.

Taken together, our results showed that DVT patients had significantly higher frequency of FV G1691A Leiden, MTHFR C, MTHFR A, PT G20210A, PAI, FVIII, FGB, HPA-1, ACE, and Apo E gene mutations when compared with healthy controls (P<0.001). These findings agree with most of the previously published data. Meyer et al. [12] showed that the prevalence of FV G1691A polymorphism is higher in DVT patients than controls. Moreover, Arsov et al. [13] and Juul et al. [14] have also reported the positive association of FV G1691A polymorphism with DVT.

In the study by Arsov et al. [13], they reported that the prevalence of the FV G1691A polymorphism among patients with VTE disease was 21.1%, compared with that in healthy controls (5.5%). This prevalence of FV G1691A is in accordance with our results as the polymorphism was found in 36.8% of our patients and in 5.8% of controls.

In this study, the results of analysis of PT G20210A polymorphism have shown a significantly higher prevalence of polymorphism among DVT patients than controls (P<0.001). These results were in accordance with De Stefano et al. [15] who found that carriers of FV G1691A and PT G20210A polymorphisms have an increased risk of DVT.

In contrast, Lijfering et al. [16] reported that FV G1691A and PT G20210A polymorphisms did not have any influence on the risk of venous thrombosis. Another study by Bouaziz-Borgi et al. [17] reported that the frequency of PT G20210A polymorphism was higher in DVT patients compared with controls, but the difference was not statistically significant.

A study conducted in the Balcan region by Jusić-Karić et al. [18] studied the frequency and association of 1691 (G>A) FVL, 20210 (G>A) PT, and 677 (C>T) MTHFR with DVT. This study included 111 DVT patients and 207 healthy controls in the Bosnian population. In agreement with this study, their results showed a higher prevalence of polymorphic genotypes among DVT patients. On the other hand, mutation 1691 (G>A) FVL was only significantly associated with DVT with an OR=6.0 (95% CI: 2.62–14.14; P=0.0001), while in this study genotype analysis in the three mutations 1691, 20210, and 677 showed significantly higher prevalence of mutations in DVT patients (P<0.05 and 0.001).

A mega European by Simone et al. [19], study including 11,000 patients and 21,000 volunteer controls, performed pooled analysis of more than 31 studies of different genotypic variants of a group of thrombophilic gene polymorphisms in adults case-control studies, through January 2010. In this study, the common polymorphisms FVL and PT 20210A were confirmed to be intermediate risk factors for VTE. The calculated OR was 4.4 and 2.8, respectively.

In contrast to the results of the two aforementioned studies (18 and 19), our results showed that neither the 1691 (G>A) FVL (OR=0.698) nor the PT 20210 (G>A) had an association with DVT, while the significant association was revealed in MTHFR 677 (C>T) with an OR=7.318 (95% CI: 5.133–10.434; P=0.0001). This may be explained by the different distribution pattern of the studied genotypes in the two studies. Simone et al. [19] reported that the prevalence of FVL, PT 20210A, heterozygous MTHFR C677T, and homozygous MTHFR C677T was 16, 5, 43, and 10%. These data had different patterns among our patients in FVL and PT 20210A 36.7 and 44%, while it was nearly similar in heterozygous and homozygous MTHFR C677T 33.1 and 9.7%.

Analyzing the influence of PAI-1 4G/5G polymorphism on the risk of DVT showed a strong association with venous thrombotic events (OR 46.987; 95% CI: 32.32–68.311). In a recent study by Vuckovic et al. [20], they compared the frequency of polymorphism into two groups of DVT patients and random controls, each group consisted of 100 individuals. Their results were not in agreement with our results as they stated that their analysis of venous thrombotic risk was influenced by PAI-1 genotype and showed no influence of PAI-1 4G/5G gene variant (OR 0.57; 95% CI: 0.27–1.20).Moreover, it is worth noting that the presence of these prothrombotic gene mutations is important especially in women as they are prone to hemostatic changes during pregnancy. In this study, we compared the frequencies of prothrombotic gene mutations in DVT patients of both sexes; in the 10 studied genes we observed a statistically significant difference in the frequencies of FVIII (P=0.033) and ACE (P=0.007) mutations between men and women. Our results are in agreement with Jusić-Karić et al. [18] who did not observe any difference in the frequencies of 1691 (G>A) FVL, 20210 (G>A), and MTHFR 677 (C>T) mutations among men and women. On the other hand, Farajzadeh et al. [21] found that the frequency of PT G20210A and PAI-1 4G/5G polymorphisms was statistically different between men and women in the case group (P<0.05).

To compare the results of this study, with the results of other studies in the region, the distribution of gene polymorphism among different ethnic groups was analyzed. A study in the Northwestern Iran [21] showed that DVT patients (n=193) had significantly higher frequency of FV (G1691A) and PAI-1 4G/5G polymorphisms when compared with healthy controls (n=500) (P<0.05). Moreover, they reported a higher prevalence of PT G20210A polymorphism, but did not show a statistically significant difference, which was again similar to our study results (P<0.001).


  Conclusion Top


This study demonstrated that inherited gene polymorphisms are associated with the risk of DVT. FVL, PTH, and MTHFR mutations are significant risk factors. Moreover, many other polymorphisms are involved in the susceptibility to the disease such as PAI, Apo E, and ACE but more studies are required to emphasize their role in the disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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