Objective
Knowledge about risk factors for venous thromboembolism (VTE) is still limited. A recently found haplotype within the natural anticoagulant protein annexin A5 (ANXA5) exerts an important modulating effect on gene expression.
Study Design
Eighty-three nonanticoagulated patients with a documented pregnancy-related VTE and 195 controls were investigated. The presence of the ANXA5 haplotypes was determined.
Results
Twenty-seven patients (32.5%) carried the M2 haplotype. Among them, 17 (63.0%) had a history of VTE in puerperium and 10 (37.0%) during pregnancy. The prevalence of the M2 haplotype was different as compared with that recorded among controls (odds ratio, 2.7; 95% confidence interval, 1.5–4.9, P < .001). A logistic regression analysis, correcting for potential confounders (age at which the thrombotic event occurred, factor V Leiden, and factor IIA20210 variants) showed a significant increase (odds ratio, 3.4; 95% confidence interval, 1.7–6.7) of the occurrence of VTE in carriers of the M2 haplotype as compared with noncarriers.
Conclusion
The M2 haplotype within the ANXA5 gene may represent a new thrombophilic risk factor for pregnancy-related VTE.
Venous thromboembolism (VTE) is common in whites, affecting 1 of 1000 individuals every year and is strongly associated with life-threatening pulmonary embolism (PE), which is still the leading cause of maternal mortality in developed countries.
Pregnancy itself is a risk factor for VTE, and this tendency can be enhanced by inherited or acquired thrombophilia. Indeed, women with thrombophilia are at increased risk of developing VTE, which is an important cause of morbidity and mortality during pregnancy. The incidence of antenatal deep vein thrombosis (DVT) is about 0.61 per 1000 pregnancies in women younger than 35 years and 1.21 per 1000 in women older than 35 years. The risk of VTE is similar in all 3 trimesters of pregnancy, and the risk of VTE after hospital discharge after delivery is greater. The incidence of postpartum DVT is about 0.30 per 1000 pregnancies in women younger than 35 years, rising to 0.72 per 1000 pregnancies in women older than 35 years.
Interestingly age over 35 years, obesity, multiparity, prolonged bed rest, mode of delivery (cesarean section, especially urgent ones) significantly increase the risk of DVT. The association between thrombophilia and pregnancy-related VTE has been addressed: the relative risks are different and depend on the type of inherited or acquired thrombophilia.
The risk of VTE is 4- to 5-fold greater in pregnant than nonpregnant women and can be 20-fold higher in the postpartum period. Abnormalities within the gene loci encoding for natural anticoagulants (antithrombin, protein C, and protein S) and fibrinogen have been shown to be rather uncommon risk factors for VTE. In patients from European ancestry, a common mutation within the gene of the coagulation factor V (FV; Leiden mutation) and one within the factor II (FII) gene (a G-to-A transition at nucleotide position 20210) have been shown to account for a large number of cases of thromboembolism. Although the knowledge of a series of major risk factors for vein thrombosis has been greatly improved, there exist many thrombotic events whose pathogenesis is unclear.
Annexins are proteins with a high Ca ++ -dependent affinity for anionic phospholipids. Annexin V (ANXA5), the most abundant member of this group of proteins, is found in many tissues and in the bloodstream and plays a potent antithrombotic role. The circulating ANXA5 is probably released from cells present in the vessel wall. After its secretion into plasma, it binds blood cells and probably endothelial cells, accounting for the low levels of free protein in plasma. The activation of platelets and red cells significantly increases the number of binding sites for ANX5 on these cells. Moreover, apoptosis is another process associated with phospholipid exposure and thus with greater binding of ANXA5 to the cell surface. It has been shown that ANXA5 prevents formation of prothrombinase and tenase complexes assemble and can interfere in activation of protein C on the surface of endothelial cells.
Finally, in the venous blood, it inhibits platelet adhesion, and in the arterial blood, it reduces aggregate formation. Recently a common haplotype (M2) within the ANXA5 gene has been demonstrated to diminish the ANXA5 promoter activity and to be a risk factor for recurrent fetal losses. In addition, hypertensive disorders of pregnancy have been also shown to be associated with the carriership of the M2 haplotype.
In a cohort of patients with documented pregnancy-related VTE, we investigated whether the presence of the M2 haplotype is associated with the occurrence of venous thrombosis.
Materials and Methods
Patients and controls
Since 2004-2008, 2745 patients with VTE were referred to the Thrombosis Centre at the Istituto di Ricovero e Cura a Carattere Scientifico “Casa Sollievo della Sofferenza” (San Giovanni Rotondo, Italy). Among them, 105 had suffered from a pregnancy-related VTE. Eight of them did not give the informed consent for the investigation of the ANX A5 haplotype, and deoxyribonucleic acid (DNA) aliquots for 14 were not available.
Thus, 83 nonanticoagulated women (median age, 35 years; range, 18–75 years), with a documented episode of DVT in a leg during pregnancy or in the puerperium (6 weeks after pregnancy), referred at least 3 months after the thrombotic episode for a work-up, were investigated. The median age (range) at the time of the first thrombotic episode was 30 years (17–42 years).
A complete clinical summary with emphasis on personal history for thromboembolic disease was obtained from all subjects by a specially trained staff. DVT was diagnosed by ultrasonography; PE by helical computed tomography (CT), or angiography or ventilation-perfusion lung scan. Overall, 8 women (9.6%) also experienced an objectively confirmed episode of PE, as diagnosed by helical CT, angiography, or high-probability ventilation-perfusion lung scan. A recurrence of VTE was observed in 5 patients (6.0 %). A reported recurrent thromboembolism was accepted as such if it had been confirmed by objective diagnostic testing (ie, ultrasonography showed a newly noncompressible venous segment or perfusion lung scan) or helical CT or angiography revealed defects in previously perfused areas or in the original area if the initial episode was previously demonstrated to be resolved in case of PE.
One hundred ninety-five apparently healthy parous women (median age, 34 years; range, 18–66 years) randomly selected from a southern Italian general population without a history of venous thromboembolism served as controls. Both cases and controls were white and were from the same region. The 2 groups were comparable for sex and social status.
After approval of the local ethics committee, the study was carried out according to the principles of the Declaration of Helsinki; informed consent was obtained from all the subjects.
Blood collection and coagulation tests
Blood samples were collected into vacuum plastic tubes containing 3.8% trisodium citrate and centrifuged at 2000 × g for 15 minutes to obtain platelet-poor plasma. The latter was frozen and stored in small aliquots at –70°C until tested. Antiphospholipid antibodies (lupus anticoagulant [LAC] and immunoglobulin G [IgG] anticardiolipin antibodies [aCL] [enzyme-linked immunosorbent assay (ELISA), Byk Gulden, Milan, Italy]) antithrombin, protein C, amidolytic and immunological (Behring, Marburg, Germany), and total and free protein S antigen (ELISA, Diagnostica Stago, Asnières, France) were determined in all patients, as reported elsewhere. Clotting assays were performed on a KC4 Amelung coagulometer (Amelung, Geesthacht, Germany). Interassay and intraassay coefficients of all the variables never exceed 8.0% and 5.0%, respectively.
DNA extraction and analysis
DNA was extracted from peripheral blood leukocytes according to standard protocols. FV Leiden and FIIA20210 gene polymorphisms were investigated as previously described.
The presence of M2 haplotype (a set of 4 consecutive nucleotide substitutions in the ANXA5 gene promoter –19G/A, +1A/C, 27T/C, and 76G/A) was investigated, as described by Bogdanova et al. When only 2 of the 4 variants (+1A/C and 27T/C) were present, the haplotype was defined as M1. Briefly, a polymerase chain reaction was carried out to amplify the appropriate region of the ANXA5 promoter. The amplification was achieved by priming with a forward (5′-GCGAGGACAAGAGGTCTCC-3′) and a reverse (5′-CATGGCGACTACTCAGGTCA-3′) oligonucleotide.
The contents of each reaction were 0.1 μg of genomic DNA, 10 pmol of each primer, 100 μM of deoxynucleotide triphosphates, 5mM Tris HCl (pH 8.4), 25 mM KCl, 1.5 mM MgCl 2 , dimethylsulfoxide 5% (volume/volume), and 1 U of Platinum Taq DNA polymerase (Invitrogen Corp, Carlsbad, CA) in a volume sample of 50 μL. After a predenaturation at 95°C for 2 minutes, cycling conditions were 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 1 minute in 35 cycles using a Perkin Elmer-Cetus thermal cycler (PerkinElmer, Foster City, CA). Amplified DNA fragments were subjected to direct sequencing analysis using an ABI PRISM 3100 genetic analyzer sequencer (PE; Applied Biosystems, Foster City, CA).
Statistical analysis
All the analyses were performed using SPSS version 11.0 (SPSS Inc, Chicago, IL). Between groups, differences in means were evaluated by the Mann-Whitney U test, after grouping homozygous and heterozygous carriers of the FV Leiden mutation and homozygous and heterozygous carriers of the FIIA20210 allele, whereas differences in frequencies were calculated using χ 2 statistics.
The observed numbers of ANXA5 haplotypes were compared with those expected for a population in Hardy-Weinberg equilibrium using a χ 2 test. Where appropriate, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Adjusted ORs and 95% CIs were calculated by logistic regression models that controlled for potential confounding variables such as age when the thrombotic event occurred, FV Leiden and FII A20210 mutations, and the presence of the M2 haplotype.
In the logistic regression analyses, an enter method with stepwise selection of the variables to set up the system was used, in which the “P to enter” and “removal” values were set at P = .05 and P = .10, respectively. For patients with recurrences, the age at the first thrombotic episode was taken into account.
Additional multinomial logistic regression models were performed to have a measure (adjusted ORs) of the independent association with the ANXA5 M2 haplotype in patients with pregnancy-related conditions as compared with controls. Statistical significance was taken as P < .05.
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