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Keywords:

  • polymorphism;
  • recurrence;
  • SNP;
  • thrombophilia;
  • venous thrombosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interest
  9. References

Summary.  Background: Individual single nucleotide polymorphisms (SNPs) associated with an increased risk of a first venous thrombosis do not predict risk of recurrent thrombosis. Objective: To assess the risk of recurrent venous thrombosis associated with multiple SNPs. Patients/methods: Fifteen nucleotide polymorphisms (SNPs), either established or putative risk factors for venous thrombosis, were measured in 817 unselected patients presenting with a first episode of venous thrombosis. Data from patients enrolled in the Leiden Thrombophilia Study (LETS) (= 443) and the first Cambridge Prospective Cohort Study (= 374) were combined. Hazard ratios for recurrence of thrombosis were calculated for individual SNPs. Results: Of the total study population, 117 patients had a recurrent event after a mean follow-up of 4.6 years. The overall incidence rate was 30.8/1000 person years, corresponding with an annual risk of 3.1%. None of the individual SNPs was more than weakly associated with the risk of recurrent venous thrombosis. With addition of sequential SNPs, added in rank order of risk, the hazard ratios for recurrence increased by 1.7-fold for carriers (3.8% of all patients) of the first two SNPs, 2.7-fold for carriers of three (2.3%) and 5.1-fold for carriers of four (0.4%). With addition of each SNP the number of carriers rapidly reduced. Conclusions: Although there is a substantially increased risk of recurrent thrombosis for carriers of several genetic variants, the clinical utility of multiple SNP analysis at present would be limited to a small proportion of patients.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interest
  9. References

Each year venous thrombosis occurs in one to three per 1000 of Caucasian individuals [1]. Following a first episode of venous thrombosis patients are treated with a finite period of oral anticoagulation. When treatment is stopped the cumulative incidence of recurrence is between 5% and 20% after 2 years [2–5]. Continued treatment with oral anticoagulant therapy will prevent most episodes of recurrence but there is a significant risk of major hemorrhage associated with prolonged treatment [6,7]. In theory, anticoagulant therapy should be continued until the risk of hemorrhage outweighs the benefit of thrombosis prevention. However, the optimal duration of treatment for a patient is still uncertain, because the risk of bleeding associated with anticoagulation and the risk of recurrent venous thrombosis after stopping treatment are not easily predicted on an individual basis, although some patients with elevated D-dimer levels might benefit from prolonged oral anticoagulation therapy after an initial unprovoked thrombotic event [8]. Individual single nucleotide polymorphisms (SNPs) known to be associated with an increased risk of a first episode of venous thrombosis do not or only weakly predict risk of recurrent thrombosis [2,3,9,10]. This is probably because most patients with idiopathic venous thrombosis or thrombosis triggered by exposure to a minimal environmental risk factor have an underlying genetic predisposition that is determined by multiple genetic variations, including unknown polymorphisms. Consequently, the risk of recurrence due to a single known SNP in a symptomatic population is likely to be masked by a varying distribution of other causative SNPs in the study population.

Advances in genomic analysis now make measurement of the risk associated with multiple SNPs in an individual patient feasible. In order to examine the hypothesis that such an analysis predicts recurrence risk we conducted a proof-of-principle feasibility study. We analyzed recurrence in relation to multiple SNPs in a cohort of unselected patients following a first episode of venous thrombosis.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interest
  9. References

Data from patients enrolled in the Leiden Thrombophilia Study (LETS) and the first Cambridge Prospective Cohort Study were combined. Both studies have been described in detail previously [2,3]. In brief, the LETS comprises 474 patients (median age, 45; range, 15–69) with a first episode of deep venous thrombosis of the leg (95.6%) or deep venous thrombosis of the arm (4.4%). Follow-up of the patients started 90 days after the initial event, and extended up to 11.2 years (mean follow-up 7.3 years). The Cambridge study comprises 570 patients (median age, 67; range, 19–100) with deep venous thrombosis of the leg (69%), deep venous thrombosis of the arm (3%), or pulmonary embolism (29%). Follow-up of the patients started after cessation of anticoagulation therapy and extended up to 2 years (mean follow-up 1.4 years). In both studies all initial and recurrent thrombotic episodes were objectively confirmed; that is, deep venous thrombosis by Doppler ultrasound, venography or impedance plethysmography, and pulmonary embolism by ventilation perfusion lung scan, computerized tomographic scan or pulmonary angiogram. Patients with malignant disease at baseline and those continuing with anticoagulant therapy for other indications were excluded.

SNP analysis was performed using a multiplex platform that included 15 SNPs that were either established or putative risk factors for venous thrombosis (Table 1). Genotypes were determined using a multiplexing technique that combines PCR, allele-specific oligonucleotide ligation assays (OLA), and hybridization to Luminex®100TM xMAP microspheres (Luminex, Austin, TX, USA) [11]. Analysis was performed by Celera Inc. (Alameda, California, USA). The accuracy of the multiplex genotyping was determined to be at least 99.9%.

Table 1.  Hazard ratios associated with individual SNPs adjusted for study centre (= 817); in rank order for SNPs with the highest risk estimate
  PolymorphismGene symbolrs numberIndividual HR (95% CI) Sequential addition of SNPS HR (95% CI)
  1. FVL, factor V Leiden; MTHFR, methylenetetrahydrofolate reductase; FGB, fibrinogen β; HR2, factor V HR2 haplotype; TFPI, tissue factor pathway inhibitor; CYP2C9, cytochrome P450 2C9; PT, prothrombin; IL1β, interleukin 1β; ACE, angiotensin-converting enzyme; CBS, cystathionine beta-synthase.

  2. *The individual hazard ratio could not be calculated for these SNPs because none (CYP2C9 and CBS) or only two patients (TFPI) carried the rare allele.

1FVII10976G/AF7rs60461.3 (0.8–2.0)1.7 (0.8–3.6)2.7 (1.2–6.1)5.1 (0.7–36.9)
2FVL1691G/AF5rs60251.2 (0.8–1.9)
3MTHFR677C/TMTHFRrs18011331.1 (0.8–1.6) 
4FGB−455G/AFGBrs18007901.1 (0.7–1.6)  
5HR24070A/GF5rs18005951.1 (0.7–2.0)   
6TFPI874G/ATFPIrs59401.1 (0.5–2.4)   
7FXIII100G/TF13A1rs59851.0 (0.7–1.4)   
8CYP2C9430C/TCYP2C9rs17998531.0 (0.6–1.5)   
9PT2021020210G/AF2rs17999630.9 (0.4–2.1)   
10IL1B3954C/TIL1Brs11436340.8 (0.6–1.2)   
11ACEIntron 16 Ins/Del 287bpACErs46469940.8 (0.5–1.1)   
12CYP2C91075A/CCYP2C9rs10579100.8 (0.4–1.4)   
13CYP2C91080C/GCYP2C9rs28371686   
14CBS919G/ACBSNo rs number   
15TFPI*536C/TTFPINo rs number   

DNA samples were available for 374 patients of the Cambridge prospective follow-up study (66%) and 443 (93.5%) of the LETS. For the current study, analysis was restricted to subjects in whom all investigated SNPs were measured (= 817). The 817 patients were followed-up for a mean period of 4.6 years (0.02–11.7). Hazard ratios were calculated for heterozygous and homozygous carriers combined using Cox regression analysis. All hazard ratios were adjusted for study (i.e. the LETS or Cambridge study). SNPs were ranked according to individual hazard ratio for recurrence of deep venous thrombosis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interest
  9. References

The overall rate of recurrence was 30.8 per 1000 patient-years (95% CI, 25.5–37.0 per 1000 patient-years). This corresponds to an annual risk of recurrence of 3.1%.

Single SNP analysis

None of the individual SNPs was more than weakly associated with the risk of recurrent venous thrombosis (Table 1). The highest relative risk of recurrence was associated with factor (F) VII 10976G/A and the FV Leiden mutation with hazard ratios of 1.3 and 1.2, respectively.

Multiple SNP analysis

With addition of sequential SNPs, added in rank order of hazard ratios from the single SNP analysis, the hazard ratios for recurrence increased (Table 1). Carriers of both FVII 10976G/A and the FV Leiden (FVL) mutation had a 1.7-fold increased risk of recurrent venous thrombosis compared with individuals carrying only one or neither of these variants. Carriers of the first three (FVII, FVL and MTHFR 677T polymorphism) or carriers of the first four (FVII, FVL, MTHFR and FGB –455G polymorphism) had an even higher hazard ratio of 2.7 and 5.1, respectively. Exclusion of patients with a surgical trigger at the time of the first event only marginally changed the risk estimates. Similar risks were found when individuals carrying the first two, three or four variants were compared with individuals carrying none of the variants (FVII and FVL, 1.7; FVII, FVL and MTHFR, 2.8; FVII, FVL, MTHFR and FGB, 5.4). However, with addition of each SNP the number of carriers rapidly reduced, producing wide 95% confidence intervals for risk, with the combination of the top four ranked SNPs giving a confidence interval of 0.7 to 36.9 for the hazard ratio. The number of patients with combinations of SNPs was: FVII + FVL, 31 carriers of whom 7 had a recurrence; FVII + FVL + MTHFR, 19 carriers of whom 6 had a recurrence; FVII + FVL + MTHFR + FGB, 3 carriers of whom 1 had a recurrence.

Seven patients carried both the FV Leiden and the prothrombin 20210A mutation (i.e. were double heterozygotes). None of these patients had a recurrent thrombotic event.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interest
  9. References

Venous thrombosis is a multi-gene disorder often triggered by exposure to environmental risk factors [12].

Since the identification of the FV Leiden mutation many more candidate SNPs have been investigated, and some have been shown to be risk factors for a first venous thrombosis (e.g. the prothrombin G20210A mutation [13]), though none of them have been shown to confer a high risk of recurrent thrombotic events. The effect of inheritance of multiple potentially causative SNPs on risk of recurrent thrombosis has not been systematically examined. By combining two large follow-up studies we have been able to examine the hypothesis that multiple SNP analysis has clinical utility with regard to predicting risk of recurrent venous thrombosis and hence inform decisions regarding duration of anticoagulation.

We found that the relative risk of recurrent thrombosis was positively associated with the number of SNPs. However, the number of patients carrying more than one SNP was limited. Only 3.8% of patients (31/817) carried both FVII 10976G/A as well as the FV Leiden mutation, of whom seven patients developed recurrent venous thrombosis during follow-up. Nineteen patients (2.3%) carried the first three polymorphisms (FVII, FVL and MTHFR), of whom six had a recurrent thrombosis, and only three patients (0.4%) carried the first four polymorphisms (FVII, FVL, MTHFR and FGB), of whom one had a recurrent thrombotic event. Whilst a much larger study would be required to calculate hazard ratios with great certainty we have shown that testing for several SNPs in a large group of patients results in the identification of a small group of individuals that might benefit from prolonged anticoagulant therapy. Nearly 4% of patients carried two or more SNPs and their recurrence rate was 50.3 per 1000 patient-years (95% CI, 20.2–103.7); that is, substantially higher than in the remaining group of patients (30.1 per 1000 patient-years; 95% CI, 24.7–36.3), and at a level that might justify consideration of long-term anticoagulation.

With the rapid development of genome wide sequencing and association studies it is possible that many synonymous SNPs will be associated with venous thrombosis. As the ability to examine patient SNP profiles becomes faster and cheaper the possibility of estimating individual patient risk in relation to multiple SNPs becomes feasible. Studies of very large numbers of cases will be necessary to determine likelihood of risk associated with multiple combinations of SNPs that are identified from large population association studies that include many thousands of patients with a history of venous thrombosis. Studies will also be required to determine the interaction of clinical risk factors and SNP analysis for prediction of likelihood of recurrent thrombosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interest
  9. References

We are grateful to B. Young and A. Arellano of Celera Inc. for technical expertise with the SNP analysis. The LETS study was funded by grant 89.063 from the Netherlands Heart Foundation and the follow-up study was funded by grant 2827170 from the Prevention Fund/ZonMW. A. van Hylckama Vlieg was supported by a personal grant (VENI) from the Netherlands Organisation for Scientific Research.

Disclosure of Conflict of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interest
  9. References

L. Bare is an employee of Celera and was not involved in the analysis of recurrent thrombosis in relation to SNP profiling. None of the other authors have declared a conflict of interest.

References

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interest
  9. References
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