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The G534E-polymorphism of the gene encoding the Factor VII-activating protease is a risk factor for venous thrombosis and recurrent events
Thrombosis Research 130 (2012) 441–444
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Regular Article
The G534E-polymorphism of the gene encoding the Factor VII-activating protease is a risk factor for venous thrombosis and recurrent events Parviz Ahmad-Nejad a, 1, Carl-Erik Dempfle b, 1, Christel Weiss c, Peter Bugert d, Martin Borggrefe b, Michael Neumaier a,⁎ a
Institute for Clinical Chemistry, University Medical Centre Mannheim, Medical Faculty Mannheim, University of Heidelberg, Germany I. Department of Medicine, University Medical Centre Mannheim, Medical Faculty Mannheim, University of Heidelberg, Germany Department for Statistical Analysis, University Medical Centre Mannheim, Medical Faculty Mannheim, University of Heidelberg, Germany d Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessen, Mannheim, Germany b c
a r t i c l e
i n f o
Article history: Received 29 September 2011 Received in revised form 6 February 2012 Accepted 9 February 2012 Available online 14 March 2012 Keywords: Deep venous thrombosis factor VII-activating protease polymorphism predisposition thrombophilia
a b s t r a c t Introduction: A single nucleotide polymorphism of the factor VII activating protease (FSAP), FSAP Marburg I (rs7080536) has been identified as a risk factor for venous thrombosis, but its clinical role has so far been controversial in part due to small cohort sizes. The aim of the present case-control study was to elucidate the impact of the FSAP Marburg I polymorphism (FSAP-MI) on the development of venous thromboembolic disease (VTE) with other known sequence variations, including Factor V Leiden (rs6025) and Factor II G20210A (rs1799963). Materials and Methods: The study included 891 patients (312 male and 579 female) with a history of deep venous thrombosis (DVT) and/or pulmonary embolism (PE) and 1283 healthy blood donors with no history of venous thromboembolic disease. Results: We found that besides to the well-established aforementioned sequence variations of FV and Prothrombin, the FSAP Marburg I (FSAP-MI) polymorphism was significantly associated with the development of DVTs (1.65 (1.16-2.34) OR (95% CI)) and recurrent thromboembolic events (DVT and PE) (2.13 (1.353.36) OR (95% CI)). Comparing patients displaying one or more events FSAP-MI was still associated with the development of recurrent thromboembolic events (1.64 (1- 2.69) OR (95% CI)). Conclusions: We conclude that FSAP Marburg-I genotyping may be used to determine the risk for thromboembolic disorders in patients with suspected thrombophilia and known DVT or PE. © 2012 Elsevier Ltd. All rights reserved.
Introduction Venous thromboembolism (VTE) is a frequent clinical condition, with more than 74.000 patients being hospitalized every year in Germany (www.gbe-bund.de) [1]. The incidence of deep vein thrombosis (DVT) is 1 per 1000 person-years [2] with a 10-year recurrence risk of 30% [3]. Furthermore, deep vein thrombosis can lead to lifethreatening pulmonary embolism [4]. Various genetic risk factors for VTE have been identified. including the Factor V R506Q (Leiden) and Prothrombin G20210A variants [5–8]. Factor V Leiden is found in 12 to 30% of patients suffering from VTE and is associated with a 7-fold increased risk of thrombosis. Heterozygous Factor II G20210A occurs in 7 to 18% of patients with VTE and increases the risk
⁎ Corresponding author at: University Medical Centre Mannheim, Medical Faculty Mannheim, University of Heidelberg, Institute for Clinical Chemistry, Theodor Kutzer Ufer 1-3, D-68167 Mannheim, Germany. Tel.: + 49 621 383 2222; fax: + 49 621 383 3819. E-mail address: [email protected] (M. Neumaier). 1 Contributed equally and share first authorship. 0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.02.009
approximately 3-fold. Nevertheless, both genetic variants can only explain a fraction of VTE events [7] suggesting that additional genetic predisposition factors may exist. The Factor VII activating protease (FSAP, or Hyaluronan binding protein 2; HABP2; OMIM# 603924) is a 70 kDa heterodimeric protein first identified by Choi-Miura et al. [9]. The corresponding gene has 13 exons, spans 35 kb and is located on chromosome 10 (10q25-q26) [10]. Among other functions, FSAP may promote coagulation by activating Factor VII. Additionally it contributes to fibrinolysis by activating single-chain plasminogen activators [11]. The Marburg-I polymorphism of FSAP was first identified and functionally characterized in healthy individuals with normal plasma FSAP levels, but reduced pro-urokinase activation by Roemisch et al. in 2002 [12]. They found heterozygosity for a sequence variation in exon 13 of the FSAP gene (rs7080536). Since the phenotype of this polymorphism potentially leads to an unbalanced protein function favoring coagulation, it has been investigated in several diseases [13–17]. A number of studies have dealt with FSAP Marburg I in the context of DVT, PE, VTE or recurrent VTE [18–22]. Briefly, the FSAP Marburg I sequence variation was reported as a significant risk factor
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for VTE among Germans [19,23] whereas studies in Dutch and Italian subjects (LETS study) [18,20], in a further German population [21], and in Californians of European ancestry could not confirm this SNP as a significant risk factor [24]. The FSAP Marburg I-polymorphism has been suggested to predispose for thromboembolic diseases, but the original results have not been confirmed by further investigations. In a case-control study we therefore determined the individual gene polymorphisms for Factor V, Factor II and FSAP Marburg I in 891 patients with a positive history of DVT and/or PE to analyse their roles in DVT, PE, VTE and recurrent VTE. Material and Methods Subjects The present case-control study included 891 patients (312 male and 579 female; average age 49.61 ± 15.96 years) with a history of DVT and/or PE. The patients were recruited from the local Haemostasis and Thrombosis outpatient clinic (University Medical Centre Mannheim) between August 2006 and January 2010. Consecutive healthy German blood donors from Mannheim (n = 1283) with no history of VTE were recruited from the Institute for Clinical Chemistry and the Institute of Transfusion Medicine and Immunology following informed consent (control group; 279 male and 1004 female; average age 45.65 ± 17.52 years old). DNA-Isolation DNA was isolated from K-EDTA anticoagulated peripheral blood on a PerkinElmer Multiprobe II EX equipped with a magnetic separator (AGOWA Maxisep 7200) using the AGOWA® mag Maxi DNA Isolation Kit (AGOWA GmbH, Berlin) in an automated protocol. PCR and Sequencing All genotyping assays were performed on the pyrosequencing instrument PSQ MA96 (Biotage, Sweden/Qiagen, Germany). PCRs and genotyping for FV-Leiden, FII g21210a were carried out as published previously [25]. The protocol for genotyping the FSAP-Marburg Ipolymorphism is available in the supplemental section of the manuscript. All genotyping assays were performed in a blinded fashion entirely independent to results of the clinical examinations and patient's history. Statistical Analyses Statistical calculations were performed with the SAS System (Release 9.2; SAS Institute Inc., Cary, NC, USA). Differences between two groups were tested using Fisher's exact and Chi-square test to evaluate qualitative data. Differences were considered significant when the p-value were less than 0.05. Results Patient Characteristics The present case-control study included 891 patients and 1283 healthy blood donors. A history of deep venous thrombosis (DVT) in the patient group was reported by 770/891 (86.42%) patients and pulmonary embolism (PE) by 292/891 (32.77%) patients. 272/891 (30.53%) patients had suffered from two or more thromboembolic events and 22 (2.46%) patients reported additionally a history of ischemic stroke. The demographic and clinical characteristics of the study population and the control group are displayed in Table 1.
Table 1 Patient characteristics. Control group (n = 1283) Age Gender Patient group (n = 891) Age Gender Clinical characteristics Patient group (n = 891) Deep venous thrombosis (DVT) Pulmonary embolism (PE) Positive family history for VTE Stroke Recurrent VTE
Mean: 45.65 Male 279 (21.75%) Female 1004 (78.25%)
Std Dev: 17.52
Mean: 49.61 Male 312 (35.02%) Female 579 (64.98%)
Std Dev: 15.96
positive 770 (86.42%) 292 (32.77%) 264 (29.63%) 22 (2.46%) 272 (30.53%)
negative 121 (13.58%) 599 (67.23%) 627 (70.37%) 869 (97.53%) 619 (69.47%)
DVT: deep venous thrombosis; PE: pulmonary embolism; VTE venous thromboembolic events; Std Dev: standard deviation.
Genotype Data Heterozygous or homozygous Factor-V-Leiden mutations were detected in 227 (25.82%) and 23 (2.62%) patients, respectively. The prothrombin G20210A variant was found in 67 (7.78%) patients, 65 being heterozygous and 2 homozygous. The FSAP-Marburg-I polymorphism was present in 68 patients (7.63%), 65 being heterozygous sequence variation and 3 with a homozygous genotype. Frequencies of the three aforementioned nucleotide exchanges for all clinical subcohorts are summarized in Table 2. Frequency and percentage of all genotype combinations are listed in supplemental table 2. Genotype-phenotype Association Since published data concerning the frequency of the FSAP Marburg-I polymorphism are inconsistent we genotyped more than 1200 healthy blood donors. In contrast to previously published investigations we detected the FSAP-MI sequence variation more frequently, with 5.14% heterozygote carriers of the mutation. Additionally, we found two homozygous carriers in the control group. Statistical analysis revealed a significant association between the FSAP-Marburg I and venous thromboembolism. DVT was significantly associated with the FSAP-MI sequence variation (p = 0.0138; Fisher´s Exact Test -supplemental table 1). No significant association could be observed for pulmonary embolism alone (p = 0.155). Patients with a history of thromboembolic events (DVT and/or PE) displayed a significant association between the recurrence of these events and FSAP Marburg-I mutation (p = 0.035 – supplemental table 1). The frequency of the FSAP-MI genotype was significantly higher in patients suffering from recurrent VTE than in controls (p = 0.0029 – supplemental table 1). Further statistical analyses revealed that carriers of the FSAP-MI polymorphism displayed higher odds ratios for DVT (odds ratio 1.65 [95% confidence interval 1.16-2.34], p = 0.007) and for recurrent thromboembolic events (odds ratio 2.13 [95% confidence interval 1.35-3.36], p = 0.0021- Table 3). Comparing patients having one event (DVT/PE) with patients suffering from recurrent venous thromboembolic events demonstrated a significant higher risk for FSAP-MI carriers (odds ratio 1.64 [95% confidence interval 1-2.69], p = 0.0491- Table 3). In our cohort 22/250 (8.8%) and 4/67 (6%) patients were positive for FSAP-MI and FV-Leiden or for FSAP-MI and the Prothrombin mutation at the same time. Among 23 patients that were carriers of FV-Leiden and the protrombin mutation FSAP-MI was additionally present in two cases (2/23; 8.7%). In 574/852 (67.37%) patients with DVT and in 158/260 (60.78%) patients with recurrent VTE no FV-Leiden and no Prothrombin g20210a-Mutation could be detected. 44 (8.3%) of these DVT patients and 18 (11.4%) of patients with recurrent VTE were solely
P. Ahmad-Nejad et al. / Thrombosis Research 130 (2012) 441–444 Table 2 Sequence variations for controls and cohorts.
Table 4 Frequencies of combined sequence variations.
Control group (n = 1283) Male Female FSAP
279 (21.75%) 1004 (78.25%) Negative (WT): 1215 (94.0%)
Patient group (n = 891) FSAP Negative (WT): 820 (92.03%)
DVT (n = 770) FV-Leiden (10 missing) Prothrombin g20210a (28 missing) FSAP MI PE (n = 292) FV-Leiden (3 missing) Prothrombin g20210a (9 missing) FSAP MI DVT + PE (n = 891) FV-Leiden (12 missing) Prothrombin g20210a (20 missing) FSAP MI Rec. VTE (n = 272) FV-Leiden (3 missing) Prothrombin g20210a (9 missing) FSAP MI
443
Heterozygotes: 66 (5.14%) Homozygous: 2 (0.16%) Heterozygotes: 68 (7.63%) Homozygotes: 3 (0.34%)
Negative/wildtype
Heterozygous
Homozygous
526 (69.21%)
211 (27.76%)
23 (3.03%)
680 (91.64%)
60 (8.09%)
2 (0.27%)
705 (91.56%)
62 (8.05%)
3 (0.39%)
229 (79.24%)
53 (18.34%)
7 (2.42%)
263 (92.93%)
20 (7.07%)
0 (0%)
272 (93.15%)
18 (6.16%)
2 (0.68%)
629 (71.56%)
227 (25.82%)
23 (2.62%)
794 (92.22%)
65 (7.55%)
2 (0.23%)
820 (92.03%)
68 (7.63%)
3 (0.34%)
174 (64.68%)
84 (31.23%)
11 (4.09%)
244 (92.78%)
19 (7.22%)
0 (0%)
243 (89.34%)
27 (9.93%)
2 (0.74%)
DVT: deep venous thrombosis; PE: pulmonary embolism; VTE venous thromboembolic events; rec.: recurrent.
positive for the FSAP-MI. A genetic predisposition to DVT or recurrent VTE would not have been recognized, if FSAP-MI had not been determined in these cases (see Table 4). Discussion Despite all preventive measures VTE still represents one of the most frequent clinical condition in our times. The annual incidence is estimated as 1-2 cases per 1000 individuals in the average population [26]. Several genetic predispositions leading to enhanced coagulation activation, impaired coagulation activation, or impaired Table 3 Genotype-phenotype association. FSAP MI
negative
positive
OR (95% CI)
Fisher's Exact Test
DVT PE DVT + PE Rec. VTE Control
705 (91.56%) 272 (93.15%) 820 (92.03%) 243 (89.34%) 1215 (94.7%)
65 (8.44%) 20 (6.85%) 71 (7.97%) 29 (10.66%) 68 (5.3%)
1.65 (1.16-2.34) 1.55 (1.10-2.18) 2.13 (1.35-3.36)
P = 0.0070 P = 0.3223 P = 0.0158 P = 0.0021
FSAP MI Rec. VTE DVT + PE (single events)
negative 243 (89.3%) 577 (93.2%)
positive 29 (10.7%) 42 (6.8%)
OR (95% CI) 1.64 (1- 2.69) 1.30 (0.87.1.93)
Chi-square P = 0.0491 P = 0.1936
DVT: deep venous thrombosis; PE: pulmonary embolism; VTE venous thromboembolic events; rec.: recurrent.
Patients suffering from DVT and/or PE
FSAP-MI Negative/ wildtype
FSAP-MI Heterozygous or homozygous
FV-Leiden (n = 250) FII g20210a (n = 67) FV-Leiden and FII g20210a (n = 23) Patients negative for FV-Leiden and FII g20210a DVT + PE (n = 574) Rec. VTE (n = 158)
228 (91.2%) 63 (94.0%) 21 (91.3%)
22 (8.8%) 4 (6%) 2 (8.7%)
530 (%) 140 (88.6%)
44 (8.3%) 18 (11.4%)
DVT: deep venous thrombosis; PE: pulmonary embolism; VTE venous thromboembolic events; rec.: recurrent.
fibrinolysis have been identified. Molecular biology–based methods for the detection and risk assessment of thrombophilia have gained widespread clinical use [27–31]. In this study, we have focused on the influence of the FSAP MarburgI sequence variation as a genetic thrombophilic risk factor on DVT, PE and recurrent thromboembolic disorders. According to the present results, FSAP Marburg I is a candidate marker for DVT and recurrent thromboembolic events, similar to the Factor V Leiden- and Prothrombin G20210A-variants. The FSAP Marburg I SNP was previously reported as an independent risk factor for VTE among Germans [19], but other studies have not confirmed this observation [18,20,21]. While in the initial investigation heterozygosity for FSAP MI was detected in 8/213 (3.8%) patients with VTE, the frequency was 2.3% within the control group (n = 213) [19]. Studies in Dutch cohort (LETS study) and in another German population also did not confirm the role of FSAP MI as a significant risk factor for DVT [20,21]. Specifically, Weisbach et al. [20] and van Minkelen et al. [21] reported higher frequencies of heterozygous FSAP MI carriers (6.3% in n = 471 and 6.4% in n = 241, respectively) within their control groups leading to non-significant associations between the Marburg-I polymorphism and VTE. Finally, Hoppe et al. determined the frequency of heterozygosity of FSAP MI to 3.2% in a healthy German population (n= 281) [32]. These incongruent observations have prompted us to enlarge our control group to n = 1283 for a more precise determination of the prevalence of FSAP MI in the average population. We found that the frequency of heterozygosity for the FSAP Marburg-I polymorphism is 5.14% in the healthy control group consistent with the data by Weisbach et al. that reported a frequency between 5.2-5.7% in healthy controls [21]. Analyzing our both cohorts statistically a significant association of FSAP MI with DVT (n = 780), DVT/PE (n = 891) and recurrent VTE (n = 272) can be demonstrated. The discrepancies to previously published manuscripts might be caused by specifics of the cohorts analysed on the one hand or by differently defined phenotypes and study designs applied. The study obtains an evaluation of the FSAPMI sequence variation in patients suffering from DVT, PE and recurrent thromboembolic disorders. It cannot be totally excluded that our findings were influenced by biases due to different selection criteria for cases and controls and local demographic factors of the patients included compared to other studies. To further validate the role of FSAP-MI in recurrent venous thromboembolism we compared patients displaying one with patients suffering from more than one thrombembolic event. Again a significant association of FSAP-MI with recurrent event could be demonstrated. Risk estimates for an additive model providing an OR for heterozygotes compared with wildtype carriers (OR 1.52 (1.08-2.17)) and homozygotes compared with wildtype carriers (2.22 (0.37- 13.33)) should only be regarded tentatively due to the small numbers available for calculation. In contrast to our findings, Gulesserian et al. could not demonstrate that FSAP-MI serves as a risk factor for recurrent venous thromboembolism [22]. This discrepancy might be explained by differences
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in the study design applied. The present study retrospectively analyses a patient cohort suffering from different thromboembolic disease entities and does not include a defined period of observation for recurrent VTE. Gulesserian et al. investigated 854 patients with a first unprovoked VTE for an average of 41 months after discontinuation of anticoagulation for the development of a secondary VTE [22]. Nevertheless they found that carriers of FSAP MI had a 1.5-fold higher risk of recurrent VTE than those without the mutation. The 95% confidence interval, however, was ranging widely from 0.6 to 2.8 in their study due to the relatively small number of carriers of the variation investigated [22]. In summary, the FSAP-MI polymorphism predisposes for VTE and recurrent VTE. In our study, 64.4% (574/891) of the DVT-patients and 58.1% (=158/272) of the patients suffering from recurrent VTE displayed no FV-Leiden or prothrombin mutation. In these cases, the determination of FSAP would make an additional contribution. In 7% and 10% of the cases, a genetic predisposition namely the FSAP-MI could be determined. Combination of FSAP-MI with FV-Leiden or prothrombin mutation might impose an additional risk. We suggest including FSAP MI determination next to FV-Leiden and Prothrombin g20210a-genotyping in patients with suspected thrombophilia or known DVT to determine their risk for thromboembolic events. Conflict of Interest Statement None. Acknowledgements We appreciate the excellent technical assistance of Romy Eichner. Appendix A. Supplementary Data Supplementary data to this article can be found online at doi:10. 1016/j.thromres.2012.02.009. References [1] http://www.gbe-bund.de. The Federal Health Monitoring of the Federal Ministry of Health- Federal Republic of Germany. http://www.gbe-bund.de. [2] Naess IA, Christiansen SC, Romundstad P, Cannegieter SC, Rosendaal FR, Hammerstrom J. Incidence and mortality of venous thrombosis: a population-based study. J Thromb Haemost 2007;5:692–9. [3] Heit JA, Silverstein MD, Mohr DN, Petterson TM, Lohse CM, O'Fallon WM, et al. The epidemiology of venous thromboembolism in the community. Thromb Haemost 2001;86:452–63. [4] Stein PD, Patel KC, Kalra NK, Petrina M, Savarapu P, Furlong Jr JW, et al. Estimated incidence of acute pulmonary embolism in a community/teaching general hospital. Chest 2002;121:802–5. [5] Emmerich J, Rosendaal FR, Cattaneo M, Margaglione M, De Stefano V, Cumming T, et al. Combined effect of factor V Leiden and prothrombin 20210A on the risk of venous thromboembolism–pooled analysis of 8 case-control studies including 2310 cases and 3204 controls. Study Group for Pooled-Analysis in Venous Thromboembolism. Thromb Haemost 2001;86:809–16. [6] Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;88:3698–703. [7] Rosendaal FR. Venous thrombosis: a multicausal disease. Lancet 1999;353:1167–73. [8] Rosendaal FR, Doggen CJ, Zivelin A, Arruda VR, Aiach M, Siscovick DS, et al. Geographic distribution of the 20210 G to A prothrombin variant. Thromb Haemost 1998;79:706–8.
[9] Choi-Miura NH, Tobe T, Sumiya J, Nakano Y, Sano Y, Mazda T, et al. Purification and characterization of a novel hyaluronan-binding protein (PHBP) from human plasma: it has three EGF, a kringle and a serine protease domain, similar to hepatocyte growth factor activator. J Biochem 1996;119:1157–65. [10] Sumiya J, Asakawa S, Tobe T, Hashimoto K, Saguchi K, Choi-Miura NH, et al. Isolation and characterization of the plasma hyaluronan-binding protein (PHBP) gene (HABP2). J Biochem 1997;122:983–90. [11] Choi-Miura NH, Yoda M, Saito K, Takahashi K, Tomita M. Identification of the substrates for plasma hyaluronan binding protein. Biol Pharm Bull 2001;24:140–3. [12] Romisch J, Factor VII. activating protease (FSAP): a novel protease in hemostasis. Biol Chem 2002;383:1119–24. [13] Ireland H, Miller GJ, Webb KE, Cooper JA, Humphries SE. The factor VII activating protease G511E (Marburg) variant and cardiovascular risk. Thromb Haemost 2004;92:986–92. [14] Kanse SM, Parahuleva M, Muhl L, Kemkes-Matthes B, Sedding D, Preissner KT. Factor VII-activating protease (FSAP): vascular functions and role in atherosclerosis. Thromb Haemost 2008;99:286–9. [15] Sedding D, Daniel JM, Muhl L, Hersemeyer K, Brunsch H, Kemkes-Matthes B, et al. The G534E polymorphism of the gene encoding the factor VII-activating protease is associated with cardiovascular risk due to increased neointima formation. J Exp Med 2006;203:2801–7. [16] Willeit J, Kiechl S, Weimer T, Mair A, Santer P, Wiedermann CJ, et al. Marburg I polymorphism of factor VII–activating protease: a prominent risk predictor of carotid stenosis. Circulation 2003;107:667–70. [17] Wasmuth HE, Tag CG, Van de Leur E, Hellerbrand C, Mueller T, Berg T, et al. The Marburg I variant (G534E) of the factor VII-activating protease determines liver fibrosis in hepatitis C infection by reduced proteolysis of platelet-derived growth factor BB. Hepatology 2009;49:775–80. [18] Franchi F, Martinelli I, Biguzzi E, Bucciarelli P, Mannucci PM. Marburg I polymorphism of factor VII-activating protease and risk of venous thromboembolism. Blood 2006;107:1731. [19] Hoppe B, Tolou F, Radtke H, Kiesewetter H, Dörner T, Salama A. Marburg I polymorphism of factor VII-activating protease is associated with idiopathic venous thromboembolism. Blood 2005;105:1549–51. [20] van Minkelen R, de Visser MCH, Vos HL, Bertina RM, Rosendaal FR. The Marburg I polymorphism of factor VII-activating protease is not associated with venous thrombosis. Blood 2005;105:4898 author reply 9. [21] Weisbach V, Ruppel R, Eckstein R. The Marburg I polymorphism of factor VII-activating protease and the risk of venous thromboembolism. Thromb Haemost 2007;97:870–2. [22] Gulesserian T, Hron G, Endler G, Eichinger S, Wagner O, Kyrle PA. Marburg I polymorphism of factor VII-activating protease and risk of recurrent venous thromboembolism. Thromb Haemost 2006;95:65–7. [23] Hoppe B, Dörner T, Kiesewetter H, Salama A. Marburg I polymorphism of factor VII-activating protease and risk of recurrent venous thromboembolism. Thromb Haemost 2006;95:907–8 author reply 8. [24] Pecheniuk NM, Elias DJ, Xu X, Griffin JH. Failure to validate association of gene polymorphisms in EPCR, PAR-1, FSAP and protein S Tokushima with venous thromboembolism among Californians of European ancestry. Thromb Haemost 2008;99:453–5. [25] Verri A, Focher F, Tettamanti G, Grazioli V. Two-step genetic screening of thrombophilia by pyrosequencing. Clin Chem 2005;51:1282–4. [26] Bates SM, Ginsberg JS. Clinical practice. Treatment of deep-vein thrombosis. N Engl J Med 2004;351:268–77. [27] Austin H, CDS, Lally C, Bezemer ID, Rosendaal FR, Hooper WC. New gene variants associated with venous thrombosis: a replication study in White and Black Americans. J Thromb Haemost 2011;9:489–95. [28] Bezemer ID, Bare LA, Arellano AR, Reitsma PH, Rosendaal FR. Updated analysis of gene variants associated with deep vein thrombosis. JAMA 2010;303:421–2. [29] Bezemer ID, Bare LA, Doggen CJ, Arellano AR, Tong C, Rowland CM, et al. Gene variants associated with deep vein thrombosis. JAMA 2008;299:1306–14. [30] Bezemer ID, Doggen CJM, Vos HL, Rosendaal FR. No association between the common MTHFR 677C->T polymorphism and venous thrombosis: results from the MEGA study. Arch Intern Med 2007;167:497–501. [31] van Minkelen R, de Visser MC, Houwing-Duistermaat JJ, Vos HL, Bertina RM, Rosendaal FR. Haplotypes of IL1B, IL1RN, IL1R1, and IL1R2 and the risk of venous thrombosis. Arterioscler Thromb Vasc Biol 2007;27:1486–91. [32] Hoppe B, Tolou F, Dörner T, Kiesewetter H, Salama A. Gene polymorphisms implicated in influencing susceptibility to venous and arterial thromboembolism: frequency distribution in a healthy German population. Thromb Haemost 2006;96: 465–70.
Contents lists available at SciVerse ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
The G534E-polymorphism of the gene encoding the Factor VII-activating protease is a risk factor for venous thrombosis and recurrent events Parviz Ahmad-Nejad a, 1, Carl-Erik Dempfle b, 1, Christel Weiss c, Peter Bugert d, Martin Borggrefe b, Michael Neumaier a,⁎ a
Institute for Clinical Chemistry, University Medical Centre Mannheim, Medical Faculty Mannheim, University of Heidelberg, Germany I. Department of Medicine, University Medical Centre Mannheim, Medical Faculty Mannheim, University of Heidelberg, Germany Department for Statistical Analysis, University Medical Centre Mannheim, Medical Faculty Mannheim, University of Heidelberg, Germany d Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessen, Mannheim, Germany b c
a r t i c l e
i n f o
Article history: Received 29 September 2011 Received in revised form 6 February 2012 Accepted 9 February 2012 Available online 14 March 2012 Keywords: Deep venous thrombosis factor VII-activating protease polymorphism predisposition thrombophilia
a b s t r a c t Introduction: A single nucleotide polymorphism of the factor VII activating protease (FSAP), FSAP Marburg I (rs7080536) has been identified as a risk factor for venous thrombosis, but its clinical role has so far been controversial in part due to small cohort sizes. The aim of the present case-control study was to elucidate the impact of the FSAP Marburg I polymorphism (FSAP-MI) on the development of venous thromboembolic disease (VTE) with other known sequence variations, including Factor V Leiden (rs6025) and Factor II G20210A (rs1799963). Materials and Methods: The study included 891 patients (312 male and 579 female) with a history of deep venous thrombosis (DVT) and/or pulmonary embolism (PE) and 1283 healthy blood donors with no history of venous thromboembolic disease. Results: We found that besides to the well-established aforementioned sequence variations of FV and Prothrombin, the FSAP Marburg I (FSAP-MI) polymorphism was significantly associated with the development of DVTs (1.65 (1.16-2.34) OR (95% CI)) and recurrent thromboembolic events (DVT and PE) (2.13 (1.353.36) OR (95% CI)). Comparing patients displaying one or more events FSAP-MI was still associated with the development of recurrent thromboembolic events (1.64 (1- 2.69) OR (95% CI)). Conclusions: We conclude that FSAP Marburg-I genotyping may be used to determine the risk for thromboembolic disorders in patients with suspected thrombophilia and known DVT or PE. © 2012 Elsevier Ltd. All rights reserved.
Introduction Venous thromboembolism (VTE) is a frequent clinical condition, with more than 74.000 patients being hospitalized every year in Germany (www.gbe-bund.de) [1]. The incidence of deep vein thrombosis (DVT) is 1 per 1000 person-years [2] with a 10-year recurrence risk of 30% [3]. Furthermore, deep vein thrombosis can lead to lifethreatening pulmonary embolism [4]. Various genetic risk factors for VTE have been identified. including the Factor V R506Q (Leiden) and Prothrombin G20210A variants [5–8]. Factor V Leiden is found in 12 to 30% of patients suffering from VTE and is associated with a 7-fold increased risk of thrombosis. Heterozygous Factor II G20210A occurs in 7 to 18% of patients with VTE and increases the risk
⁎ Corresponding author at: University Medical Centre Mannheim, Medical Faculty Mannheim, University of Heidelberg, Institute for Clinical Chemistry, Theodor Kutzer Ufer 1-3, D-68167 Mannheim, Germany. Tel.: + 49 621 383 2222; fax: + 49 621 383 3819. E-mail address: [email protected] (M. Neumaier). 1 Contributed equally and share first authorship. 0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.02.009
approximately 3-fold. Nevertheless, both genetic variants can only explain a fraction of VTE events [7] suggesting that additional genetic predisposition factors may exist. The Factor VII activating protease (FSAP, or Hyaluronan binding protein 2; HABP2; OMIM# 603924) is a 70 kDa heterodimeric protein first identified by Choi-Miura et al. [9]. The corresponding gene has 13 exons, spans 35 kb and is located on chromosome 10 (10q25-q26) [10]. Among other functions, FSAP may promote coagulation by activating Factor VII. Additionally it contributes to fibrinolysis by activating single-chain plasminogen activators [11]. The Marburg-I polymorphism of FSAP was first identified and functionally characterized in healthy individuals with normal plasma FSAP levels, but reduced pro-urokinase activation by Roemisch et al. in 2002 [12]. They found heterozygosity for a sequence variation in exon 13 of the FSAP gene (rs7080536). Since the phenotype of this polymorphism potentially leads to an unbalanced protein function favoring coagulation, it has been investigated in several diseases [13–17]. A number of studies have dealt with FSAP Marburg I in the context of DVT, PE, VTE or recurrent VTE [18–22]. Briefly, the FSAP Marburg I sequence variation was reported as a significant risk factor
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for VTE among Germans [19,23] whereas studies in Dutch and Italian subjects (LETS study) [18,20], in a further German population [21], and in Californians of European ancestry could not confirm this SNP as a significant risk factor [24]. The FSAP Marburg I-polymorphism has been suggested to predispose for thromboembolic diseases, but the original results have not been confirmed by further investigations. In a case-control study we therefore determined the individual gene polymorphisms for Factor V, Factor II and FSAP Marburg I in 891 patients with a positive history of DVT and/or PE to analyse their roles in DVT, PE, VTE and recurrent VTE. Material and Methods Subjects The present case-control study included 891 patients (312 male and 579 female; average age 49.61 ± 15.96 years) with a history of DVT and/or PE. The patients were recruited from the local Haemostasis and Thrombosis outpatient clinic (University Medical Centre Mannheim) between August 2006 and January 2010. Consecutive healthy German blood donors from Mannheim (n = 1283) with no history of VTE were recruited from the Institute for Clinical Chemistry and the Institute of Transfusion Medicine and Immunology following informed consent (control group; 279 male and 1004 female; average age 45.65 ± 17.52 years old). DNA-Isolation DNA was isolated from K-EDTA anticoagulated peripheral blood on a PerkinElmer Multiprobe II EX equipped with a magnetic separator (AGOWA Maxisep 7200) using the AGOWA® mag Maxi DNA Isolation Kit (AGOWA GmbH, Berlin) in an automated protocol. PCR and Sequencing All genotyping assays were performed on the pyrosequencing instrument PSQ MA96 (Biotage, Sweden/Qiagen, Germany). PCRs and genotyping for FV-Leiden, FII g21210a were carried out as published previously [25]. The protocol for genotyping the FSAP-Marburg Ipolymorphism is available in the supplemental section of the manuscript. All genotyping assays were performed in a blinded fashion entirely independent to results of the clinical examinations and patient's history. Statistical Analyses Statistical calculations were performed with the SAS System (Release 9.2; SAS Institute Inc., Cary, NC, USA). Differences between two groups were tested using Fisher's exact and Chi-square test to evaluate qualitative data. Differences were considered significant when the p-value were less than 0.05. Results Patient Characteristics The present case-control study included 891 patients and 1283 healthy blood donors. A history of deep venous thrombosis (DVT) in the patient group was reported by 770/891 (86.42%) patients and pulmonary embolism (PE) by 292/891 (32.77%) patients. 272/891 (30.53%) patients had suffered from two or more thromboembolic events and 22 (2.46%) patients reported additionally a history of ischemic stroke. The demographic and clinical characteristics of the study population and the control group are displayed in Table 1.
Table 1 Patient characteristics. Control group (n = 1283) Age Gender Patient group (n = 891) Age Gender Clinical characteristics Patient group (n = 891) Deep venous thrombosis (DVT) Pulmonary embolism (PE) Positive family history for VTE Stroke Recurrent VTE
Mean: 45.65 Male 279 (21.75%) Female 1004 (78.25%)
Std Dev: 17.52
Mean: 49.61 Male 312 (35.02%) Female 579 (64.98%)
Std Dev: 15.96
positive 770 (86.42%) 292 (32.77%) 264 (29.63%) 22 (2.46%) 272 (30.53%)
negative 121 (13.58%) 599 (67.23%) 627 (70.37%) 869 (97.53%) 619 (69.47%)
DVT: deep venous thrombosis; PE: pulmonary embolism; VTE venous thromboembolic events; Std Dev: standard deviation.
Genotype Data Heterozygous or homozygous Factor-V-Leiden mutations were detected in 227 (25.82%) and 23 (2.62%) patients, respectively. The prothrombin G20210A variant was found in 67 (7.78%) patients, 65 being heterozygous and 2 homozygous. The FSAP-Marburg-I polymorphism was present in 68 patients (7.63%), 65 being heterozygous sequence variation and 3 with a homozygous genotype. Frequencies of the three aforementioned nucleotide exchanges for all clinical subcohorts are summarized in Table 2. Frequency and percentage of all genotype combinations are listed in supplemental table 2. Genotype-phenotype Association Since published data concerning the frequency of the FSAP Marburg-I polymorphism are inconsistent we genotyped more than 1200 healthy blood donors. In contrast to previously published investigations we detected the FSAP-MI sequence variation more frequently, with 5.14% heterozygote carriers of the mutation. Additionally, we found two homozygous carriers in the control group. Statistical analysis revealed a significant association between the FSAP-Marburg I and venous thromboembolism. DVT was significantly associated with the FSAP-MI sequence variation (p = 0.0138; Fisher´s Exact Test -supplemental table 1). No significant association could be observed for pulmonary embolism alone (p = 0.155). Patients with a history of thromboembolic events (DVT and/or PE) displayed a significant association between the recurrence of these events and FSAP Marburg-I mutation (p = 0.035 – supplemental table 1). The frequency of the FSAP-MI genotype was significantly higher in patients suffering from recurrent VTE than in controls (p = 0.0029 – supplemental table 1). Further statistical analyses revealed that carriers of the FSAP-MI polymorphism displayed higher odds ratios for DVT (odds ratio 1.65 [95% confidence interval 1.16-2.34], p = 0.007) and for recurrent thromboembolic events (odds ratio 2.13 [95% confidence interval 1.35-3.36], p = 0.0021- Table 3). Comparing patients having one event (DVT/PE) with patients suffering from recurrent venous thromboembolic events demonstrated a significant higher risk for FSAP-MI carriers (odds ratio 1.64 [95% confidence interval 1-2.69], p = 0.0491- Table 3). In our cohort 22/250 (8.8%) and 4/67 (6%) patients were positive for FSAP-MI and FV-Leiden or for FSAP-MI and the Prothrombin mutation at the same time. Among 23 patients that were carriers of FV-Leiden and the protrombin mutation FSAP-MI was additionally present in two cases (2/23; 8.7%). In 574/852 (67.37%) patients with DVT and in 158/260 (60.78%) patients with recurrent VTE no FV-Leiden and no Prothrombin g20210a-Mutation could be detected. 44 (8.3%) of these DVT patients and 18 (11.4%) of patients with recurrent VTE were solely
P. Ahmad-Nejad et al. / Thrombosis Research 130 (2012) 441–444 Table 2 Sequence variations for controls and cohorts.
Table 4 Frequencies of combined sequence variations.
Control group (n = 1283) Male Female FSAP
279 (21.75%) 1004 (78.25%) Negative (WT): 1215 (94.0%)
Patient group (n = 891) FSAP Negative (WT): 820 (92.03%)
DVT (n = 770) FV-Leiden (10 missing) Prothrombin g20210a (28 missing) FSAP MI PE (n = 292) FV-Leiden (3 missing) Prothrombin g20210a (9 missing) FSAP MI DVT + PE (n = 891) FV-Leiden (12 missing) Prothrombin g20210a (20 missing) FSAP MI Rec. VTE (n = 272) FV-Leiden (3 missing) Prothrombin g20210a (9 missing) FSAP MI
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Heterozygotes: 66 (5.14%) Homozygous: 2 (0.16%) Heterozygotes: 68 (7.63%) Homozygotes: 3 (0.34%)
Negative/wildtype
Heterozygous
Homozygous
526 (69.21%)
211 (27.76%)
23 (3.03%)
680 (91.64%)
60 (8.09%)
2 (0.27%)
705 (91.56%)
62 (8.05%)
3 (0.39%)
229 (79.24%)
53 (18.34%)
7 (2.42%)
263 (92.93%)
20 (7.07%)
0 (0%)
272 (93.15%)
18 (6.16%)
2 (0.68%)
629 (71.56%)
227 (25.82%)
23 (2.62%)
794 (92.22%)
65 (7.55%)
2 (0.23%)
820 (92.03%)
68 (7.63%)
3 (0.34%)
174 (64.68%)
84 (31.23%)
11 (4.09%)
244 (92.78%)
19 (7.22%)
0 (0%)
243 (89.34%)
27 (9.93%)
2 (0.74%)
DVT: deep venous thrombosis; PE: pulmonary embolism; VTE venous thromboembolic events; rec.: recurrent.
positive for the FSAP-MI. A genetic predisposition to DVT or recurrent VTE would not have been recognized, if FSAP-MI had not been determined in these cases (see Table 4). Discussion Despite all preventive measures VTE still represents one of the most frequent clinical condition in our times. The annual incidence is estimated as 1-2 cases per 1000 individuals in the average population [26]. Several genetic predispositions leading to enhanced coagulation activation, impaired coagulation activation, or impaired Table 3 Genotype-phenotype association. FSAP MI
negative
positive
OR (95% CI)
Fisher's Exact Test
DVT PE DVT + PE Rec. VTE Control
705 (91.56%) 272 (93.15%) 820 (92.03%) 243 (89.34%) 1215 (94.7%)
65 (8.44%) 20 (6.85%) 71 (7.97%) 29 (10.66%) 68 (5.3%)
1.65 (1.16-2.34) 1.55 (1.10-2.18) 2.13 (1.35-3.36)
P = 0.0070 P = 0.3223 P = 0.0158 P = 0.0021
FSAP MI Rec. VTE DVT + PE (single events)
negative 243 (89.3%) 577 (93.2%)
positive 29 (10.7%) 42 (6.8%)
OR (95% CI) 1.64 (1- 2.69) 1.30 (0.87.1.93)
Chi-square P = 0.0491 P = 0.1936
DVT: deep venous thrombosis; PE: pulmonary embolism; VTE venous thromboembolic events; rec.: recurrent.
Patients suffering from DVT and/or PE
FSAP-MI Negative/ wildtype
FSAP-MI Heterozygous or homozygous
FV-Leiden (n = 250) FII g20210a (n = 67) FV-Leiden and FII g20210a (n = 23) Patients negative for FV-Leiden and FII g20210a DVT + PE (n = 574) Rec. VTE (n = 158)
228 (91.2%) 63 (94.0%) 21 (91.3%)
22 (8.8%) 4 (6%) 2 (8.7%)
530 (%) 140 (88.6%)
44 (8.3%) 18 (11.4%)
DVT: deep venous thrombosis; PE: pulmonary embolism; VTE venous thromboembolic events; rec.: recurrent.
fibrinolysis have been identified. Molecular biology–based methods for the detection and risk assessment of thrombophilia have gained widespread clinical use [27–31]. In this study, we have focused on the influence of the FSAP MarburgI sequence variation as a genetic thrombophilic risk factor on DVT, PE and recurrent thromboembolic disorders. According to the present results, FSAP Marburg I is a candidate marker for DVT and recurrent thromboembolic events, similar to the Factor V Leiden- and Prothrombin G20210A-variants. The FSAP Marburg I SNP was previously reported as an independent risk factor for VTE among Germans [19], but other studies have not confirmed this observation [18,20,21]. While in the initial investigation heterozygosity for FSAP MI was detected in 8/213 (3.8%) patients with VTE, the frequency was 2.3% within the control group (n = 213) [19]. Studies in Dutch cohort (LETS study) and in another German population also did not confirm the role of FSAP MI as a significant risk factor for DVT [20,21]. Specifically, Weisbach et al. [20] and van Minkelen et al. [21] reported higher frequencies of heterozygous FSAP MI carriers (6.3% in n = 471 and 6.4% in n = 241, respectively) within their control groups leading to non-significant associations between the Marburg-I polymorphism and VTE. Finally, Hoppe et al. determined the frequency of heterozygosity of FSAP MI to 3.2% in a healthy German population (n= 281) [32]. These incongruent observations have prompted us to enlarge our control group to n = 1283 for a more precise determination of the prevalence of FSAP MI in the average population. We found that the frequency of heterozygosity for the FSAP Marburg-I polymorphism is 5.14% in the healthy control group consistent with the data by Weisbach et al. that reported a frequency between 5.2-5.7% in healthy controls [21]. Analyzing our both cohorts statistically a significant association of FSAP MI with DVT (n = 780), DVT/PE (n = 891) and recurrent VTE (n = 272) can be demonstrated. The discrepancies to previously published manuscripts might be caused by specifics of the cohorts analysed on the one hand or by differently defined phenotypes and study designs applied. The study obtains an evaluation of the FSAPMI sequence variation in patients suffering from DVT, PE and recurrent thromboembolic disorders. It cannot be totally excluded that our findings were influenced by biases due to different selection criteria for cases and controls and local demographic factors of the patients included compared to other studies. To further validate the role of FSAP-MI in recurrent venous thromboembolism we compared patients displaying one with patients suffering from more than one thrombembolic event. Again a significant association of FSAP-MI with recurrent event could be demonstrated. Risk estimates for an additive model providing an OR for heterozygotes compared with wildtype carriers (OR 1.52 (1.08-2.17)) and homozygotes compared with wildtype carriers (2.22 (0.37- 13.33)) should only be regarded tentatively due to the small numbers available for calculation. In contrast to our findings, Gulesserian et al. could not demonstrate that FSAP-MI serves as a risk factor for recurrent venous thromboembolism [22]. This discrepancy might be explained by differences
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in the study design applied. The present study retrospectively analyses a patient cohort suffering from different thromboembolic disease entities and does not include a defined period of observation for recurrent VTE. Gulesserian et al. investigated 854 patients with a first unprovoked VTE for an average of 41 months after discontinuation of anticoagulation for the development of a secondary VTE [22]. Nevertheless they found that carriers of FSAP MI had a 1.5-fold higher risk of recurrent VTE than those without the mutation. The 95% confidence interval, however, was ranging widely from 0.6 to 2.8 in their study due to the relatively small number of carriers of the variation investigated [22]. In summary, the FSAP-MI polymorphism predisposes for VTE and recurrent VTE. In our study, 64.4% (574/891) of the DVT-patients and 58.1% (=158/272) of the patients suffering from recurrent VTE displayed no FV-Leiden or prothrombin mutation. In these cases, the determination of FSAP would make an additional contribution. In 7% and 10% of the cases, a genetic predisposition namely the FSAP-MI could be determined. Combination of FSAP-MI with FV-Leiden or prothrombin mutation might impose an additional risk. We suggest including FSAP MI determination next to FV-Leiden and Prothrombin g20210a-genotyping in patients with suspected thrombophilia or known DVT to determine their risk for thromboembolic events. Conflict of Interest Statement None. Acknowledgements We appreciate the excellent technical assistance of Romy Eichner. Appendix A. Supplementary Data Supplementary data to this article can be found online at doi:10. 1016/j.thromres.2012.02.009. References [1] http://www.gbe-bund.de. The Federal Health Monitoring of the Federal Ministry of Health- Federal Republic of Germany. http://www.gbe-bund.de. [2] Naess IA, Christiansen SC, Romundstad P, Cannegieter SC, Rosendaal FR, Hammerstrom J. Incidence and mortality of venous thrombosis: a population-based study. J Thromb Haemost 2007;5:692–9. [3] Heit JA, Silverstein MD, Mohr DN, Petterson TM, Lohse CM, O'Fallon WM, et al. The epidemiology of venous thromboembolism in the community. Thromb Haemost 2001;86:452–63. [4] Stein PD, Patel KC, Kalra NK, Petrina M, Savarapu P, Furlong Jr JW, et al. Estimated incidence of acute pulmonary embolism in a community/teaching general hospital. Chest 2002;121:802–5. [5] Emmerich J, Rosendaal FR, Cattaneo M, Margaglione M, De Stefano V, Cumming T, et al. Combined effect of factor V Leiden and prothrombin 20210A on the risk of venous thromboembolism–pooled analysis of 8 case-control studies including 2310 cases and 3204 controls. Study Group for Pooled-Analysis in Venous Thromboembolism. Thromb Haemost 2001;86:809–16. [6] Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;88:3698–703. [7] Rosendaal FR. Venous thrombosis: a multicausal disease. Lancet 1999;353:1167–73. [8] Rosendaal FR, Doggen CJ, Zivelin A, Arruda VR, Aiach M, Siscovick DS, et al. Geographic distribution of the 20210 G to A prothrombin variant. Thromb Haemost 1998;79:706–8.
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