Targeted sequencing to identify novel genetic risk factors for deep vein thrombosis: a study of 734 genes.

Essentials Deep vein thrombosis (DVT) has a large unknown genetic component. We sequenced coding areas of 734 hemostasis-related genes in 899 DVT patients and 599 controls. Variants in F5, FGA-FGG, CYP4V2-KLKB1-F11, and ABO were associated with DVT risk. Associations in KLKB1 and F5 suggest a more complex genetic architecture than previously thought. SUMMARY: Background Although several genetic risk factors for deep vein thrombosis (DVT) are known, almost all related to hemostasis, a large genetic component remains unexplained. Objectives To identify novel genetic determinants by using targeted DNA sequencing. Patients/Methods We included 899 DVT patients and 599 controls from three case-control studies (DVT-Milan, Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis [MEGA], and the Thrombophilia, Hypercoagulability and Environmental Risks in Venous Thromboembolism [THE-VTE] study) for sequencing of the coding regions of 734 genes involved in hemostasis or related pathways. We performed single-variant association tests for common variants (minor allele frequency [MAF] ≥ 1%) and gene-based tests for rare variants (MAF ≤ 1%), accounting for multiple testing by use of the false discovery rate (FDR). Results Sixty-two of 3617 common variants were associated with DVT risk (FDR < 0.10). Most of these mapped to F5,ABO,FGA-FGG, and CYP4V2-KLKB1-F11. The lead variant at F5 was rs6672595 (odds ratio [OR] 1.58, 95% confidence interval [CI] 1.29-1.92), in moderate linkage with the known variant rs4524. Reciprocal conditional analyses suggested that intronic variation might drive this association. We also observed a secondary association at the F11 region: missense KLKB1 variant rs3733402 remained associated conditional on known variants rs2039614 and rs2289252 (OR 1.36, 95% CI 1.10-1.69). Two novel variant associations were observed, in CBS and MASP1, but these were not replicated in the meta-analysis data from the International Network against Thrombosis (INVENT) consortium. There was no support for a burden of rare variants contributing to DVT risk (FDR > 0.2). Conclusions We confirmed associations between DVT and common variants in F5,ABO,FGA-FGG, and CYP4V2-KLKB1-F11, and observed secondary signals in F5 and CYP4V2-KLKB1-F11 that warrant replication and fine-mapping in larger studies.

Discovery and characterization of an antibody directed against exosite I of thrombin.

UNLABELLED: ESSENTIALS: An IgA paraprotein with anti-thrombin activity was not associated with a severe bleeding phenotype. This observation challenges the paradigm that anticoagulant therapy necessarily increases bleeding risk. Characterization of the antibody showed that it specifically binds to thrombin exosite I. A therapeutic drug with the properties of this antibody might be an antithrombotic that doesn't cause bleeding. BACKGROUND: We report the case of a 54-year-old female who presented with a traumatic subdural hemorrhage. Coagulation tests were markedly prolonged due to the presence of an anti-thrombin IgA paraprotein at 3 g L(-1) . The patient made a complete recovery and has had no abnormal bleeding during a 7-year follow-up, despite the persistence of the paraprotein. OBJECTIVES: To determine how the paraprotein prolonged clotting tests by defining its target and its epitope. METHODS: The paraprotein was purified and added to normal pooled plasma for in vitro clotting assays. Binding studies were conducted to determine the affinity of the IgA for thrombin. The Fab was isolated and crystallized with thrombin. RESULTS: The purified IgA was sufficient to confer the patient's in vitro coagulation profile in normal pooled plasma, and was found to bind specifically and with high affinity to thrombin. A crystal structure of the Fab fragment in complex with thrombin revealed an exosite I interaction involving CDRH3 of the antibody. CONCLUSIONS: Although the patient originally presented with a subdural bleed, the hematoma resolved without intervention, and no other bleeding event occurred during the subsequent 7 years. During this period, the patient's IgA paraprotein levels have remained constant at 3 g L(-1) , suggesting that the presence of a high-affinity, exosite I-directed antibody is consistent with normal hemostasis. A therapeutic derivative of this antibody might therefore permit antithrombotic dose escalation without an associated increase in the risk of bleeding.

The risk of a first and a recurrent venous thrombosis associated with an elevated D-dimer level and an elevated thrombin potential: results of the THE-VTE study.

BACKGROUND: D-dimer and thrombin generation have been associated with the risk of recurrent venous thrombosis. However, for both measurements, different assays are available, and in vitro thrombin generation may be affected by the problem of contact activation during blood sampling. OBJECTIVES: To determine the association between hypercoagulability and first and recurrent thrombosis by the use of different D-dimer and thrombin generation assays, to assess whether the addition of corn trypsin inhibitor (CTI) prior to blood sampling to inhibit contact activation improved the association between thrombin generation and thrombosis risk, and to calculate the DASH score with two different D-dimer assays. METHODS: A case-control study (626 patients and 361 controls) with subsequent follow-up of the cases was performed (2987 patient-years after stopping of anticoagulant therapy). Blood was drawn 2-3 months after discontinuation of anticoagulation for the first event in citrate tubes with and without CTI. RESULTS/CONCLUSIONS: An elevated D-dimer level and elevated thrombin generation were associated with an increased risk of a first event regardless of the assay used (odds ratios: 1.8-3.4). An elevated D-dimer level but not elevated thrombin generation was associated with the risk of recurrence. Patients with elevated D-dimer levels had a more than two-fold increased recurrence rate (Vidas - hazard ratio [HR] 2.3, 95% confidence interval [CI] 1.4-3.8; HemosIL - HR 2.4, 95% CI 1.5-3.9; Thrombinoscope and Technoclone assay - HR 1.3). Elimination of contact factor activation did not improve the predictive value of thrombin generation. In patients with unprovoked first events, the DASH score had a similar predictive value for deep vein thrombosis and pulmonary embolism, both when calculated with Vidas D-dimer and when calculated with HemosIL D-dimer.

Venous thromboembolism in oesophago-gastric carcinoma: incidence of symptomatic and asymptomatic events following chemotherapy and surgery.

Venous thromboembolism (VTE) is a frequent cause of morbidity and mortality in patients with cancer and those having chemotherapy. However, the incidence of VTE during radical treatment for patients with oesophago-gastric cancer is poorly documented. The incidence of VTE was assessed in 200 consecutive patients with oesophago-gastric cancer having surgery with curative intent; 132 (66%) had neo-adjuvant chemotherapy, 37 (18.5%) had adjuvant chemotherapy and 64 (32%) had no chemotherapy. Patients received 40 mg of Enoxaparin subcutaneously daily during the peri-operative hospital stay. Asymptomatic VTE were detected by routine chest computed tomography (CT) pre and post surgery. Symptomatic patients with suspected VTE were investigated and treated as clinically appropriate. Twenty six patients (13%) developed VTE of which 14 (54%) were symptomatic; 12/26 (46%) VTE were detected pre-operatively, all during or after neo-adjuvant chemotherapy, and 14/26 (54%) post-operatively. There were two post-operative deaths caused by pulmonary emboli occurring at days 24 and 56 respectively despite peri-operative VTE prophylaxis. Multivariate analysis demonstrated that neo-adjuvant chemotherapy was the only factor that predicted pre-operative VTE (p = 0.073) and any VTE (p = 0.045). This study found a 13% incidence of VTE in patients undergoing therapy with curative intent for oesophago-gastric cancer and a statistically significant association between neo-adjuvant chemotherapy and VTE. Half of the patients with VTE were asymptomatic but two had fatal PE's. Current VTE prophylaxis regimens for this patient group may be inadequate.

Non-malignant haematology research in the UK: looking forward to new opportunities.

Over the last few years there has been rapid and radical change in the way clinical research in the UK is funded and supported within the NHS. This has resulted from restructuring and major new investment in research infrastructure, co-ordinated through Clinical Local Research Networks (CLRNs) and equivalent organisations in the devolved nations. CLRNs have resources to support local researchers undertake studies that have been adopted on to the national research portfolio. For example, CLRNs can help with gaining local approvals or provide research nurses to recruit patients, undertake study procedures and perform data entry. CLRNs can establish Local Speciality Groups in a number of areas of medicine, including nonmalignant haematology. These new networks offer non-malignant haematology access to significant new resources and a major opportunity to support clinical research for the benefit of our patients.

Proof of principle of potential clinical utility of multiple SNP analysis for prediction of recurrent venous thrombosis.

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) (n = 443) and the first Cambridge Prospective Cohort Study (n = 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.

Elevated endogenous thrombin potential is associated with an increased risk of a first deep venous thrombosis but not with the risk of recurrence.

Measurement of the thrombin generating potential could provide a method for quantifying the composite effect of multiple risk factors. This study assessed the risk of a first as well as a recurrent venous thrombotic event associated with an increased endogenous thrombin potential (ETP). Analyses were performed in 360 patients and 404 control subjects of the Leiden Thrombophilia Study. The ETP was measured directly using a fluorogenic assay (Thrombinoscope). Individuals with an increased ETP, i.e. above 90th percentile measured in control subjects (>2109.0 nM x min) had a 1.5-fold [95% confidence interval (CI): 0.9-2.3] increased risk of a first deep venous thrombosis. The risk was more pronounced after the analysis was restricted to idiopathic thromboses, i.e. 1.7-fold (95% CI: 1.0-2.8). Overall, the hazard ratio of a recurrent thrombotic event associated with a high ETP, adjusted for age, sex and oral anticoagulant use was 1.1 (95% CI: 0.5-2.2). Thus, a high ETP was not associated with an increased relative risk of recurrent venous thrombosis. At present, the clinical relevance of the thrombin generation assay in predicting recurrent venous thrombosis remains uncertain.

Predicting response to recombinant factor VIIa in non-haemophiliac patients with severe haemorrhage.

BACKGROUND: Despite increasing use of recombinant factor VIIa (rFVIIa) in non-haemophiliac patients, it is unclear when rFVIIa might be effective. METHODS: A single centre review of consecutive non-haemophiliac patients receiving rFVIIa for the management of severe haemorrhage. Treatments with rFVIIa were at a dose of 90 mug kg(-1) repeated at three hourly intervals at the clinicians' discretion. RESULTS: Eighteen patients received rFVIIa. Six patients survived to discharge and 12 patients died in hospital. The median (range) Sequential Organ Failure Assessment (SOFA) score at the time of administration of rFVIIa for the group that survived was 8.0 (5-12) compared with the group that died 12.0 (7.0-14.0) (P=0.03). One of the patients who survived (17%) had organ failure at the time of rFVIIa administration compared with 11 of those who died (92%) (P=0.004). Fifteen patients survived long enough to consider a second dose of rFVIIa, one patient who survived to discharge needed more than one dose (1/6, 17%), compared with seven of those who later died in hospital (7/9, 78%) (P=0.04). The survivors had a significant reduction in blood product requirements after rFVIIa, while patients who died did not. Neither the prothrombin time nor the activated partial thromboplastin time before or after rFVIIa predicted survival. CONCLUSIONS: High SOFA score and failure to respond to one adequate dose of rFVIIa appear to identify patients with poor prognosis. These observations may help in determining when rFVIIa treatment is likely to be futile.

Platelet size has diagnostic predictive value in patients with thrombocytopenia.

We studied 473 unselected patients with thrombocytopenia. The mean platelet volume (MPV) was 8.1 fl in patients with marrow disease and 9.8 fl in patients without marrow disease (P < 0.001). A total of 5% of patients with an MPV >or=10.5 fl have marrow disease (odds ratio 0.05, 95% CI 0.02-0.13). Conversely over three quarters of patients with an MPV of <8.0 fl have marrow disease (odds ratio 8.1, 95% CI 5.0-13.0). Therefore the MPV can strongly guide the clinician as to the likely presence or absence of bone marrow disease in thrombocytopenic patients.

Identification and removal of a promiscuous CD4+ T cell epitope from the C1 domain of factor VIII.

BACKGROUND: The development of inhibitors in hemophiliacs is a severe complication of factor VIII (FVIII) replacement therapy and is a process driven by FVIII specific T helper cells. OBJECTIVES: To finely map T cell epitopes within the whole FVIII protein in order to investigate the possibility of engineering FVIII variants with reduced propensity for inhibitor development. PATIENTS AND METHODS: T cell lines were generated from five patients with severe hemophilia who had developed inhibitors, and were screened for T cell proliferation against pools of overlapping peptides spanning the entire B domain deleted (BDD) FVIII sequence. Positive peptide pools were decoded by screening individual peptides against the T cell lines. Positive peptides, and mutants thereof, were tested for their ability to bind major histocompatibility complex (MHC) Class II and stimulate T cell proliferation in a panel of healthy donors. The activities of the corresponding mutant proteins were assessed via chromogenic assay. RESULTS: One peptide, spanning FVIII amino acids 2098-2112, elicited a vigorous response from one hemophiliac donor, induced strong T cell responses in the panel of healthy donors and bound to a number of HLA-DR alleles. Mutations were made in this peptide that removed its ability to stimulate T cells of healthy donors and to bind to MHC Class II while retaining full activity when incorporated into a mutant BDD-FVIII protein. CONCLUSIONS: Fine T cell epitope mapping of the entire FVIII protein is feasible, although challenging, and this knowledge may be used to create FVIII variants which potentially have reduced immunogenicity.

Travellers' thrombosis and economy class syndrome: incidence, aetiology and prevention.

In this article we review economy class syndrome or travellers' thrombosis as it should be more appropriately described. We review the evidence for and against the existence of this condition. Much of the evidence included in this article has been justifiably criticised but it is the best evidence available to date. We conclude that there is a probable link between thrombosis and long distance travel. However this association is confined to individuals with additional risk factors and fatal pulmonary embolus is very rare. The probable aetiology of this association is prolonged immobilisation in cramped conditions. Other factors are likely to play a minor role. When considering preventative measures, for the majority of individuals the risk is very small. Simple advise can be given with no requirement for additional intervention. Compression stockings should be considered for high-risk patients. In individuals planning prolonged travel with multiple risk factors for thrombosis low dose low molecular weight heparin should be considered. The criteria for defining risk groups remains unclear and we outline our recommendations. There is conflicting evidence with regard to the use of aspirin but considering the latest evidence we do not recommend it.