Jordi Fontcuberta

Genetic Determinants of Hemostasis Phenotypes in Spanish Families

BACKGROUND Recent studies have described genetic mutations that affect the risk of thrombosis as a result of abnormal levels of such hemostatic parameters as protein C, protein S, and the activated protein C resistance ratio. Although these mutations suggest that genes play a part in determining variability in some hemostasis-related phenotypes, the relative importance of genetic influences on these traits has not been evaluated. METHODS AND RESULTS The relative contributions of genetic and environmental influences to a panel of hemostasis-related phenotypes were assessed in a sample of 397 individuals in 21 extended pedigrees. The effects of measured covariates (sex, age, smoking, and exogenous sex hormones), genes, and environmental variables shared by members of a household were quantified for 27 hemostasis-related measures. All of these phenotypes showed significant genetic contributions, with the majority of heritabilities ranging between 22% and 55% of the residual phenotypic variance after correction for covariate effects. Activated protein C resistance ratio, activated partial thromboplastin time, and Factor XII showed the strongest heritabilities, with 71.3%, 83.0%, and 67.3%, respectively, of the residual phenotypic variation attributable to genetic effects. CONCLUSIONS These results clearly demonstrate the importance of genetic factors in determining variation in hemostasis-related phenotypes that are components of the coagulation and fibrinolysis pathways and that have been implicated in risk for thrombosis. The presence of such strong genetic effects suggests that it will be possible to localize previously unknown genes that influence quantitative variation in these hemostasis-related phenotypes that may contribute to risk for thrombosis.

Functional Effects of the ABO Locus Polymorphism on Plasma Levels of von Willebrand Factor, Factor VIII, and Activated Partial Thromboplastin Time

Lower levels of factor VIII and von Willebrand factor (vWF) have been reported in individuals with blood type O compared with individuals with other ABO blood types. However, this relationship has been demonstrated only by association studies and not by linkage studies. Also, it is not clear whether the ABO locus exerts a functional effect directly on these plasma factors or whether the ABO locus is in linkage disequilibrium with another locus that controls these factors. To distinguish between these 2 possibilities, we applied new statistical methods combining linkage and association tests in a pedigree-based sample. In contrast to most previous studies that used the ABO phenotypes, our study used the ABO genotypes, permitting us to distinguish AO from AA and BO from BB. Our results clearly showed significant linkage between the ABO locus and vWF antigen (P=0.00075). In addition, factor VIII coagulant activity and activated partial thromboplastin time showed suggestive linkage with the ABO locus (P=0.10 and P=0.13). All 3 plasma phenotypes showed significant differences between OO and non-OO genotypes. In addition, vWF antigen exhibited significant differences between O heterozygotes and non-OO homozygotes. This study is unique because it used a combined linkage and association test, which indicated that the ABO locus itself has a functional effect on these plasma phenotypes.

Risk of a first venous thrombotic event in carriers of a familial thrombophilic defect. The European Prospective Cohort on Thrombophilia (EPCOT)

Summary.  Background: Reliable risk estimates for venous thrombosis in families with inherited thrombophilia are scarce but necessary for determining optimal screening and treatment policies. Objectives: In the present analysis, we determined the risk of a first venous thrombotic event in carriers of a thrombophilic defect (i.e. antithrombin‐, protein C‐ or protein S deficiency, or factor V Leiden). Patients and methods: The asymptomatic carriers had been tested prior to this study in nine European thrombosis centers because of a symptomatic carrier in the family, and were followed prospectively for 5.7 years on average between March 1994 and January 2001. Annually, data were recorded on the occurrence of risk situations for venous thrombosis and events (e.g. venous thrombosis, death). Results: Twenty‐six of the 575 asymptomatic carriers (4.5%) and seven of the 1118 controls (0.6%) experienced a first deep venous thrombosis or pulmonary embolism during follow‐up. Of these events, 58% occurred spontaneously in the carriers compared with 43% in the controls. The incidence of first events was 0.8% per year (95% CI 0.5–1.2) in the carriers compared with 0.1% per year (95% CI 0.0–0.2) in the controls. The highest incidence was associated with antithrombin deficiency or combined defects, and the lowest incidence with factor V Leiden. Conclusions: The incidence of venous events in asymptomatic individuals from thrombophilic families does not exceed the risk of bleeding associated with long‐term anticoagulant treatment in the literature (1–3%).

Familial thrombophilia and lifetime risk of venous thrombosis

Summary  Background : We started a large multicenter prospective follow‐up study to provide reliable risk estimates of venous thrombosis in families with various thrombophilic defects.

Hereditary thrombophilia and fetal loss: a prospective follow-up study

Summary.  Background: As the placental vessels are dependent on the normal balance of procoagulant and anticoagulant mechanisms, inherited thrombophilia may be associated with fetal loss. Objectives: We performed a prospective study to investigate the relation between inherited thrombophilia and fetal loss, and the influence of thromboprophylaxis on pregnancy outcome. Patients and methods: Women were enrolled in the European Prospective Cohort on Thrombophilia (EPCOT). These included women with factor (F)V Leiden or a deficiency of antithrombin, protein C or protein S. Controls were partners or acquaintances of thrombophilic individuals. A total of 191 women (131 with thrombophilia, 60 controls) had a pregnancy outcome during prospective follow‐up. Risk of fetal loss and effect of thromboprophylaxis were estimated by frequency calculation and Cox regression modelling. Results: The risk of fetal loss appeared slightly increased in women with thrombophilia without a previous history of fetal loss who did not use any anticoagulants during pregnancy (7/39 vs. 7/51; relative risk 1.4; 95% confidence interval 0.4, 4.7). Per type of defect the relative risk varied only minimally from 1.4 for FV Leiden to 1.6 for antithrombin deficiency compared with control women. Prophylactic anticoagulant treatment during pregnancy in 83 women with thrombophilia differed greatly in type, dose and duration, precluding solid conclusions on the effect of thromboprophylaxis on fetal loss. No clear benefit of anticoagulant prophylaxis was apparent. Conclusions: Women with thrombophilia appear to have an increased risk of fetal loss, although the likelihood of a positive outcome is high in both women with thrombophilia and in controls.

Risk of Ischemic Stroke Associated With Functional Thrombin-Activatable Fibrinolysis Inhibitor Plasma Levels

Background and Purpose— Recently, a novel procarboxypeptidase B–like proenzyme, called thrombin-activatable fibrinolysis inhibitor (TAFI), has been described. It plays an important role in the delicate balance between coagulation and fibrinolysis. TAFI leads to potent inhibition of tissue plasminogen activator–induced fibrinolysis. The relevance of TAFI in thromboembolic disease is unclear. We have investigated the risk of ischemic stroke (IS) in relation to plasma levels of functional TAFI. Methods— In a case-control study, we enrolled 264 individuals; 114 had IS, and 150 were recruited as controls who were age and sex matched and had no history of arterial disease. The individuals supplied information on their personal and family histories of cardiovascular diseases and conventional cardiovascular risk factors. Functional TAFI assays were performed by use of a method based on the activation of TAFI with thrombin-thrombomodulin and the measure of the TAFI activity generated. Other hemostatic parameters assayed were factor VIIIc, anti-phospholipid antibodies,fibrinogen, factor V Leiden, and the prothrombin gene G20210A mutations (PT20210A). Results— Functional TAFI levels were significantly higher in patients with IS (113.7±25%; range, 57% to 209%) than in controls (102.6±19%). The odds ratio for IS in patients with functional TAFI levels >120% was 5.7 (95% confidence interval, 2.3 to 14.1). Conclusions— We found that functional TAFI levels in plasma (>120%) increased the risk of IS ≈6-fold. Further studies should elucidate the physiological role of TAFI in arterial disease and possibly provide clues to therapeutic approaches.

Homozygosity of the T Allele of the 46 C→T Polymorphism in the F12 Gene Is a Risk Factor for Ischemic Stroke in the Spanish Population

Background and Purpose— Ischemic stroke (IS) is a complex disease that involves genetic and environmental factors. In a family-based study (the Genetic Analysis of Idiopathic Thrombophilia [GAIT] Project) that included a genome-wide scan, we demonstrated that a common polymorphism (46 C→T) in the exon 1 of the F12 gene jointly influences variability of plasma Factor XII levels and susceptibility to thrombotic disease. We have investigated the risk of IS related to this polymorphism in a case–control study. Methods— We studied 436 individuals: 205 diagnosed with IS and 231 age–gender–ethnic control subjects. We measured Factor VIIIc, fibrinogen, and Factor XIIc levels, and we genotyped the 46 C→T polymorphism in the F12 gene. Results— There were 91 women and 114 men in the IS group and 109 women and 122 men in the control group. We confirmed our previous observation that individuals with different genotypes for the 46 C→T polymorphism showed significant differences in Factor XIIc levels. Most importantly, the mutated T allele in the homozygous state (genotype T/T) was associated with an increased risk of IS with an adjusted odds ratio of 4.1 (95% CI, 1.1 to 15.9). Conclusions— This study suggests that the 46 C→T polymorphism is a genetic risk factor for IS in the Spanish population. In addition, our results confirm that the use of genetic linkage studies along with a case–control association study is an extremely valuable approach for identifying DNA variants that affect complex diseases.

Low-molecular-weight heparin as bridging therapy during interruption of oral anticoagulation in patients undergoing colonoscopy or gastroscopy.

Nowadays, most patients under oral anticoagulant therapy (OAT) require invasive procedures such as colonoscopy (CC) or gastroscopy (GC). The goals of the management of OAT are to minimise the risk of thromboembolism and bleeding. We have performed the first prospective, observational study to evaluate these parameters using fixed‐dose high‐risk thromboprophylactic therapy with sodic bemiparin (Hibor®) as bridging therapy. From January 2004 to January 2005, patients under OAT were included. Periprocedure prophylaxis consisted of: Acenocumarol patients: Day –3: withdrawal acenocumarol. Days −2,−1,0: Hibor ®3500 UI/d sc and days +1,+2,+3: Hibor® 3500 U/I + acenocumarol. And day +5: acenocumarol only. Warfarin patients: Days −5,−4: withdrawal warfarin, −3,−2,−1, 0; Hibor® 3500 UI/day sc, days +1,+2,+3,+4: Hibor® 3500 UI/day sc and warfarin and day +5; warfarin only. Thromboembolic complications and bleeding were recorded in a 3 month follow‐up. We included 100 consecutive patients in the intention‐to‐treat group. The remaining 98 patients were 50 women and 48 men. Mean age of women was 71.1 (range: 46–87) years and 70.7 (range: 39–86) years in men. Eighty‐three took acenocumarol, and 15 warfarin. Thirty‐two gastroscopies and 61 colonoscopies were performed and in five patients both were performed. No thromboembolic and bleeding complications related to bemiparin were observed in the 103 endoscopies. Two patients developed pruritus at the punction site. Fixed‐dose high‐risk thromboprophilactic therapy with bemiparin (Hibor®) is safe and effective as a bridging therapy in patients under OAT who require GC or CC.

The F7 Gene and Clotting Factor VII Levels: Dissection of a Human Quantitative Trait Locus

Abstract Localization of human quantitative trait loci (QTLs) is now routine. However, identifying their functional DNA variants is still a formidable challenge. We present a complete dissection of a human QTL using novel statistical techniques to infer the most likely functional polymorphisms of a QTL that influence plasma levels of clotting factor VII (FVII), a risk factor for cardiovascular disease. Resequencing of 15 kb in and around the F7 gene identified 49 polymorphisms, which were then genotyped in 398 people. Using a Bayesian quantitative trait nucleotide (BQTN) method, we identified four to seven functional variants that completely account for this QTL. These variants include both rare coding variants and more common, potentially regulatory polymorphisms in intronic and promoter regions.

Protein C Levels Are Regulated by a Quantitative Trait Locus on Chromosome 16: Results from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project

Objective—Protein C (PC) is a component of the protein C anticoagulant pathway. PC deficiency is a risk factor associated with venous thromboembolism. As part of the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project, we conducted a genome-wide linkage scan to localize genes that influence variation in PC plasma levels. Methods and Results—PC levels were measured in 398 individuals belonging to 21 Spanish families. A total of 485 DNA microsatellite markers were genotyped to provide a 7.1-cM genetic map. Variance component linkage methods were used to evaluate linkage and to detect quantitative trait loci (QTL). A region on chromosome 16 (16q23), flanked by markers D16S3106 and D16S516, showed strong evidence of linkage with PC levels (LOD=3.69). This region contains 1 positional candidate gene, the NAD(P)H:dehydrogenase quinone 1 (NQO1), involved in vitamin K metabolism. The association of 1 SNP of this gene with PC levels (P =0.005) strongly supports the implication of NQO1 gene in the variability of PC levels. Conclusions—These results illustrate the application of genomic scans to identify the genetic determinants of quantitative variation in a component of the hemostatic pathways. They provide strong evidence for a locus (QTL) on chromosome 16 that influences PC levels.