Montserrat Borrell

Genetic Determinants of Hemostasis Phenotypes in Spanish Families

BACKGROUNDnRecent 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.nnnMETHODS AND RESULTSnThe 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.nnnCONCLUSIONSnThese 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.

A Quantitative Trait Locus Influencing Free Plasma Protein S Levels on Human Chromosome 1q Results From the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project

Objective—Protein S (PS) is a component of the protein C anticoagulant system. PS deficiency is associated with myocardial infarction and venous thromboembolism, two highly prevalent causes of death in industrialized nations. As part of the Genetic Analysis of Idiopathic Thrombophilia (GAIT) project, we conducted a genome-wide linkage screen to localize genes influencing variation in free PS (fPS) plasma levels. Methods and Results—fPS levels were measured in 397 individuals in 21 Spanish families. A total of 363 highly informative microsatellite markers were genotyped to provide a 10-cM genetic map, and variance component linkage methods were used. A region on chromosome 1q32, flanked by markers D1S425 and D1S213, showed strong evidence of linkage with fPS levels (LOD score, 4.07; nominal P =7.5×10−6; genome-wide P =0.0024). This region contains two positional candidate genes, the complement component 4-binding protein &agr; and &bgr; chains, which encode the principal binding protein for PS. Suggestive evidence for linkage was also observed on chromosomes 11p and 19p. Conclusions—These results represent one of the first genomic screens for quantitative variation in a component of the hemostatic pathway and provide strong evidence for a locus on chromosome 1q influencing fPS levels.

A locus on chromosome 2 influences levels of tissue factor pathway inhibitor: results from the GAIT study.

Objective—Levels of tissue factor pathway inhibitor (TFPI) have been associated with arteriosclerosis and thrombotic disease. Although a genetic component to variation in TFPI levels is well-documented, no systematic genome-wide screens have been conducted to localize genes influencing levels of TFPI. Methods and Results—We studied TFPI levels in 397 individuals in 21 Spanish families participating in the Genetic Analysis of Idiopathic Thrombosis (GAIT) study. Twelve families were selected through a proband with idiopathic thrombosis and 9 were ascertained without regard to phenotype. A genome scan was performed using microsatellite markers spaced at approximately 10 cM intervals. Standard multipoint variance component linkage methods were used. The heritability of TFPI levels was 0.52 (P<0.0001), with no evidence for shared household effects. In the genome screen, only 1 LOD score >2 was observed. On chromosome 2q, the maximum multipoint LOD score was 3.52 near marker D2S1384. This is near the structural gene for TFPI, which is located at 2q32. In follow-up association analyses, marginal evidence of association (P=0.04) was observed with the TFPI promoter variant C-399T. Conclusion—These results suggest that polymorphisms in and around the TFPI structural gene may be the major genetic determinants of variation in TFPI levels.

A Genome-Wide Association Study Identifies KNG1 as a Genetic Determinant of Plasma Factor XI Level and Activated Partial Thromboplastin Time

Objective—Elevated plasma levels of coagulation factor XI (FXI) are implicated in the pathogenesis of venous thromboembolism and ischemic stroke, and polymorphisms in the F11 gene are associated both with risk of venous thromboembolism and an elevated plasma FXI level. Methods and Results—Here, we report the first hypothesis-free genome-wide genetic analysis of plasma FXI levels. Two genome-wide significant loci were detected in the family-based Genetic Analysis of Idiopathic Thrombophilia 1 cohort: one located in the kininogen 1 gene (KNG1) (rs710446; P=7.98×10−10) and one located in the structural F11 gene (rs4241824; P=1.16×10−8). Both associations were replicated in a second population-based Swedish cohort. A significant effect on KNG1 mRNA expression was also seen for the 2 most robustly FXI-associated single nucleotide polymorphisms located in KNG1. Furthermore, both KNG1 single nucleotide polymorphisms were associated with activated partial thromboplastin time, suggesting that FXI may be the main mechanistic pathway by which KNG1 and F11 influence activated partial thromboplastin time and risk of thrombosis. Conclusion—These findings contribute to the emerging molecular basis of venous thromboembolism and, more importantly, help in understanding the biological regulation of a phenotype that has proved to have promising therapeutic properties in relation to thrombosis.

A genome-wide association study of the Protein C anticoagulant pathway.

The Protein C anticoagulant pathway regulates blood coagulation by preventing the inadequate formation of thrombi. It has two main plasma components: protein C and protein S. Individuals with protein C or protein S deficiency present a dramatically increased incidence of thromboembolic disorders. Here, we present the results of a genome-wide association study (GWAS) for protein C and protein S plasma levels in a set of extended pedigrees from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. A total number of 397 individuals from 21 families were typed for 307,984 SNPs using the Infinium® 317 k Beadchip (Illumina). Protein C and protein S (free, functional and total) plasma levels were determined with biochemical assays for all participants. Association with phenotypes was investigated through variance component analysis. After correcting for multiple testing, two SNPs for protein C plasma levels (rs867186 and rs8119351) and another two for free protein S plasma levels (rs1413885 and rs1570868) remained significant on a genome-wide level, located in and around the PROCR and the DNAJC6 genomic regions respectively. No SNPs were significantly associated with functional or total protein S plasma levels, although rs1413885 from DNAJC6 showed suggestive association with the functional protein S phenotype, possibly indicating that this locus plays an important role in protein S metabolism. Our results provide evidence that PROCR and DNAJC6 might play a role in protein C and free protein S plasma levels in the population studied, warranting further investigation on the role of these loci in the etiology of venous thromboembolism and other thrombotic diseases.

What is the clinical impact of low plasminogen activator inhibitor‐1 (PAI‐1) activity? A case report and study of the incidence of low PAI‐1 antigen in a healthy population

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin superfamily of protease inhibitors, and is the primary inhibitor of tissue-type plasminogen activator in plasma. Platelets, vascular endothelial cells and smooth muscle cells contain PAI-1, suggesting that it functions as an important regulator of fibrinolysis at sites of vascular injury and thrombus formation [1–4]. Epidemiological studies have identified an association between elevated plasma PAI-1 and thrombotic vascular disease in humans [5]. However, some case reports have indicated that low levels of PAI-1 activity may be a rare cause of bleeding [1–7]. Surprisingly, a recent study [8] reported a high incidence of low levels of PAI-1 activity in the general population, without any bleeding disorders. Because low levels of PAI-1 activity may induce a broad spectrum of clinical symptoms, it is difficult to know whether it is actually diagnostic of a bleeding disorder, or just a risk factor. In addition, the actual role of PAI-1 in patients with bleeding diatheses remains unclear. We report a case of a woman with low levels of PAI-1 activity, and with an atypical bleeding diathesis. We also report the incidence of low levels of PAI-1 in a healthy population.We studied a 49-year-old woman who had a life-long history of bleeding tendency that included mild epistaxis, gingival bleeding and microscopic hematuria. Three years prior to this study, she experienced a spontaneous and autolimited mild bleeding from her navel and 1 year later she experienced two episodes of bleeding from her right nipple. The patient had no family history of bleeding, except for menorrhagia in one of her daughters. Serial mammographies were normal. As her symptoms suggested that she had a bleeding disorder, blood analyses were performed. These showed hemoglobin levels of 141 g L, a platelet count of 152 · 10 L and a leukocyte count of 5.34 · 10 L. Her renal and hepatic functions were normal. The hepatitis serology was negative. Coagulation tests were also performed as described elsewhere [9]. These tests included activated partial thromboplastin time (ratio 0.94; range: 0.75– 1.30), International Normalized Ratio (1.07; 0.75–1.20), thrombin time (22.4 s; 17–24 s), fibrinogen levels (3.12 g L; 1.5–4 g L), factor VIII coagulant activity (129%; 45–175%), von Willebrand factor levels (130%; 55–150%), and ristocetin cofactor activity (98%). Platelet function studied using the PFA-100 instrument (Dade Behring, Marburg, Germany) was normal; aggregometry was also normal. We measured some of the more unusual causes of bleeding such as a2-antiplasmin and PAI-1 plasma levels. The a2-antiplasmin was normal (107%; range: 80–105%). PAI-1 activity was measured by an immunoactivity assay (Chromolize PAI-1, Biopool, Umeå, Sweden). The sensitivity of the kit was between 2.0 and 50 IU mL. As levels <2 IU mL were indistinguishable from 0 UI mL PAI-1, we set the lower limit below 2.0 IU mL. The patient showed low levels of PAI-1 activity (0 IU mL; range: 0–30 IU mL). We also performed two tests to assess the global procoagulant and fibrinolytic capacity of her plasma. All parameters measured by the thromboelastogram (TEG) test of whole blood, which evaluates the viscoelastic properties during blood clot formation (ROTEM , Pentapharm GmbH, Munich, Germany), were normal. The results of the clot lysis time [10], which measures the plasma fibrinolytic potential, were normal (51 min; range: 27–72 min). To manage the patient clinically, we measured her biological response to treatment with tranexamic acid in case of bleeding, particularly during surgery. We added tranexamic acid (0.3 mg mL blood) in vitro and the TEG test was repeated. No differences were observed between the TEG tests either with or without tranexamic acid. Thus, no clinical response to antifibrinolytic treatment was observed. One daughter had no history of bleeding and yet her PAI-1 levels were abnormally low (0.6 IU mL). The other daughter who had menorrhagias also had abnormally low PAI-1 levels (0.7 IU mL). We studied the levels of PAI-1 activity in 66 blood donors (41 women and 25 men) from a healthy population. The mean age was 45 years (range 18–64 years). These donors were anonymous and no additional data were available, although blood donors who had a history of bleeding diathesis were not included. Fasting blood samples were obtained in the morning. Nine volumes of blood were collected in tubes with one volume Correspondence: Amparo Santamarı́a Ortiz, Department of Haematology, Hospital de la Santa Creu i Sant Pau, C/ Sant Antoni Ma Claret, 167, 08025-Barcelona, Spain. Tel: +34 93 291 91 93; fax: +34 93 291 91 92; e-mail: msantamaria@ santpau.es

Genetic determinants of plasma β2‐glycoprotein I levels: a genome‐wide association study in extended pedigrees from Spain

β2‐Glycoprotein I (β2‐GPI), also designated apolipoprotein H, is a 50‐kDa protein that circulates in blood at high concentrations, playing important roles in autoimmune diseases, hemostasis, atherogenesis, and angiogenesis, as well as in host defense against bacteria and in protein/cellular waste removal. Plasma β2‐GPI levels have a significant genetic component (heritability of ~ 80%).

Higher risk of ischaemic stroke associated with factor XI levels in dyslipidaemic patients

Background:u2002 Ischaemic stroke (IS) is a complex disease that involves genetic and environmental factors. The role of factor XI (FXI) in arterial thrombosis is unclear. We have investigated the risk of IS related to FXI levels in a case–control study.