M. C. H. De Visser

Haplotypes of the EPCR gene, plasma sEPCR levels and the risk of deep venous thrombosis.

Background: Binding of protein C (PC) to the endothelial cell PC receptor (EPCR) stimulates PC activation by increasing the affinity of PC for the thrombin‐thrombomodulin complex. A soluble form of this receptor (sEPCR) circulates in plasma and inhibits both PC activation and APC anticoagulant activity. Objectives: The aim of this study was to investigate whether variations in the EPCR gene or plasma sEPCR levels are risk factors for deep venous thrombosis (DVT). Patients/methods: In a large case‐control study, the Leiden Thrombophilia Study (LETS), sEPCR levels were measured by ELISA. All subjects were genotyped for three haplotype‐tagging SNPs, enabling us to detect all four common haplotypes of the EPCR gene. Results: The distribution of sEPCR levels in the control population was trimodal and was genetically controlled by haplotype 3 (H3). This haplotype explained 86.5% of the variation in sEPCR levels. Carriers of two H3 alleles had higher sEPCR levels (439 ng mL−1) than carriers of one H3 allele (258 ng mL−1), which had higher levels than non‐H3 carriers (94 ng mL−1). Haplotype 4 was associated with a slightly increased risk (OR = 1.4, 95%CI:1.0–2.2). The risk of subjects with sEPCR levels in the top quartile (≥ 137 ng mL−1) was increased compared to that of subjects in the first quartile (< 81 ng mL−1), but since there was no dose–response effect, it is most likely that low sEPCR levels reduce the risk of DVT. Conclusions: Our data do not suggest a strong association between EPCR haplotypes and thrombosis risk, but low sEPCR levels appear to reduce the risk of DVT.

ABO blood group genotypes and the risk of venous thrombosis: effect of factor V Leiden

ABO blood group and more recently high von Willebrand factor (VWF) and factor (F)VIII levels have been associated with thrombotic disease. An excess of non-O blood group has long been recognized in patients with ischemic heart disease [1] and venous thrombosis [2]. In 1995, we demonstrated that nonO blood group, high VWF levels and high FVIII levels all increased the risk of deep vein thrombosis [3]. In multivariate analysis only FVIII remained a risk factor, whereas the thrombosis risk associated with VWF and ABO blood group largely disappeared. Since then, several other studies have identified high FVIII levels as a risk factor for venous thrombosis [4–7]. Usually blood group phenotypes are used to study the associationbetweenbloodgroupandvenous thrombosis.Blood group genotypes may be more informative since genotypes can distinguish between heterozygous and homozygous carriers of A, B and O alleles and between A and A alleles. Therefore we studied the effect ofABOgenotype on thrombosis risk in a large population-based case–control study of venous thrombosis (Leiden Thrombophilia Study, LETS). This study, which included 474 patients and 474 control subjects, has been previously described [3]. For the present study DNA was available for 471 patients and 471 control subjects. Blood was collected into 0.1 volume 0.106 mol L trisodium citrate. Plasma was prepared by centrifugation for 10 min at 2000 · g at room temperature and stored at )70 C. FVIII coagulant activity (FVIII:C), FVIII:Ag, VWF:Ag and blood group phenotype were measured as previously reported [3–5]. High-molecular-weight DNA was isolated from leukocytes and stored at 4 C. Polymerase chain reaction (PCR) was designed to amplify exons 6 and 7 of the ABO blood group gene in two separate reactions. The sequences of the primers have been described previously [8]. The amplified DNA fragments corresponding to exons 6 and 7 were digested with Acc65I (MBI Fermentas) or MspI (MBI Fermentas), respectively, in two separate reactions and separated by electrophoresis on 3.5% agarose gels. With this method we discriminated A, A, B, O and O alleles. There was 99% agreement between ABO blood group phenotype and genotypes in all patients and controls. Table 1 (upper part) shows the frequency of the ABO blood group genotypes in patients and controls. Odds ratios (OR) were calculated as estimates of the relative risk by anunmatched method. Ninety-five percent confidence intervals were assessed according to Woolf [9]. All non-OO genotypes except A homozygotes or A–O combinations, i.e. AO/AO/AA, were associated with an increased thrombosis risk when compared with OO genotypes. This reinforces the concept that blood group exerts its thrombotic risk largely via FVIII levels, since AO/AA genotypes correspond to the lowest FVIII levels among non-OO genotypes (data not shown). Adjustment for age and sex did not alter the risk estimates. Because blood group is known to affect plasma levels of VWF and FVIII and because VWF and FVIII levels influence thrombosis risk, we adjusted the thrombosis risk associated

Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen γ′/γA mRNA ratio

Summary.  Background: Fibrinogen gamma haplotype 2 (FGG‐H2) is associated with reduced fibrinogen γ′ levels and fibrinogen γ′/total fibrinogen ratios and with an increased deep‐venous thrombosis (DVT) risk. Two FGG‐H2 tagging single nucleotide polymorphisms (SNPs), 9615C>T and 10034C>T, are located in the region of alternative FGG pre‐mRNA processing. 10034C>T is located in a GT‐rich downstream sequence element (DSE) that comprises a putative cleavage stimulation factor (CstF) binding site.Objectives: To investigate the functionality of SNPs 9615C>T and 10034C>T, and the importance of the DSE containing 10034C>T.Methods: Different minigene constructs containing FGG exon 9, intron 9, exon 10 and the 3′ region were transiently transfected into HepG2 cells and quantitative real‐time polymerase chain reaction was used to measure relative polyadenylation (pA) signal usage (pA1/pA2 ratio).Results: Compared with the reference construct CC (9615C–10034C; FGG‐H1; pA1/pA2 ratio set at 100%), the pA1/pA2 ratio of construct TT (9615T–10034T; FGG‐H2) was 1.4‐fold decreased (71.5%, P = 0.015). The pA1/pA2 ratio of construct CT (9615C–10034T) was almost 1.2‐fold decreased (85.3%, P = 0.001), whereas the pA1/pA2 ratio of construct TC (9615T–10034C) did not differ significantly from the reference construct (101.6%, P = 0.890). Functionality of the putative CstF binding site was confirmed using constructs in which this site was deleted or its sequence altered by point mutations.Conclusions: SNP 10034C>T is located in a GT‐rich DSE involved in regulating the usage of the pA2 signal of FGG, which may represent a CstF binding site. We propose that the 10034C>T change is the functional variation in FGG‐H2 that is responsible for the reduction in the fibrinogen γ′/total fibrinogen ratio and the increased DVT risk.

Gross deletions/duplications in PROS1 are relatively common in point mutation-negative hereditary protein S deficiency

Hereditary protein S (PS) deficiency is an autosomal disorder caused by mutations in the PS gene (PROS1). Conventional PCR-based mutation detection identifies PROS1 point mutations in approximately 50% of the cases. To verify if gross copy number variations (CNVs) are often present in point mutation-negative hereditary PS deficiency we used multiplex ligation-dependent probe amplification (MLPA) as a detection tool in samples from individuals with a high probability of having true PS deficiency. To this end, DNA samples from nine PS deficient probands with family members (seven type I and two type III) and nine isolated probands (three type I and six type III), in whom PROS1 mutations were not found by DNA sequencing, were evaluated. An independent quantitative PCR (qPCR) was performed to confirm the findings of the MLPA assay. Family members were also tested when DNA was available. Gross abnormalities of PROS1 were found in six out of eighteen probands. In three probands complete deletion of the gene was detected. Two probands had a partial deletion involving different parts of the gene (one from exon 4 through 9 and another from exon 9 through 11). One family showed a duplication of part of PROS1. qPCR analysis was in accordance with these results. In conclusion, this study substantiates that gross gene abnormalities in PROS1 are relatively common in hereditary PS deficient patients and that MLPA is a useful tool for direct screening of CNVs in PROS1 point mutation-negative individuals.

Linkage analysis of factor VIII and von Willebrand factor loci as quantitative trait loci

Summary.  Elevated factor (F)VIII levels contribute to venous thrombotic risk. FVIII levels are determined to a large extent by levels of von Willebrand factor (VWF), its carrier protein which protects FVIII against proteolysis. VWF levels are largely dependent on ABO blood group. Subjects with blood group non‐O have higher VWF and FVIII levels than individuals with blood group O. Apart from ABO blood group no genetic determinants of high FVIII levels have been identified, whereas clustering of FVIII levels has been reported within families even after adjustment for ABO blood group and VWF levels. We investigated the FVIII and VWF loci as possible quantitative trait loci (QTL) influencing FVIII and VWF levels. Two sequence repeats in the FVIII gene and three repeats in the VWF gene were typed in 52 FV Leiden families. Multipoint sib‐pair linkage analysis was performed with the MAPMAKER/SIBS program. FVIII levels adjusted for VWF levels and age, and VWF levels adjusted for ABO blood group and age, were used for this linkage analysis. No linkage of FVIII levels to the FVIII locus was found, whereas we found evidence that the VWF locus contains a QTL for VWF levels [maximum likelihood no dominance variance lod score = 0.70 (P = 0.04) and non‐parametric Z‐score = 1.92 (P = 0.03)]. About 20% of the total variation in VWF levels may be attributed to this VWF locus.

Haplotypes of the fibrinogen gamma gene do not affect the risk of myocardial infarction

As the precursor of fibrin, fibrinogen plays an important role in hemostasis [1]. Of all the components of the coagulation system, elevated plasma fibrinogen levels have most consistently been shown to be associated with occlusive vascular disorders [2–5]. Several polymorphisms in the fibrinogen genes (FGA, FGB, FGG) have been reported to be associated with fibrinogen levels. Most studies focused on FGB polymorphisms. However, results are not consistent and none of these polymorphisms has been found to be associated with an increased risk of venous [6,7] or arterial thrombosis [7]. Recently, we reported that a specific haplotype (H2) of the fibrinogen gamma gene (FGG) was associated with a 2.4-fold increased risk of deep venous thrombosis [95% confidence intervals (CI): 1.5–3.9] [6]. In the same study, we found that another haplotype (FGG-H3) was associated with a slight reduction in risk [odds ratio (OR) 1⁄4 0.8, 95% CI: 0.6–1.0]. None of these haplotypes was associated with total fibrinogen levels. However, the FGG-H2 haplotype was associated with reduced levels of fibrinogen c¢, a product of alternative splicing of the FGG gene. In a recent report, Mannila et al. [8] studied the effect of haplotypes across the fibrinogen gene cluster on the risk of myocardial infarction (MI). They determined the *216C > T polymorphism, which is identical to FGG-H2 tagging SNP 10034 C/T [rs2066865]. In their study, the FGGH2 haplotype was not associated with the risk ofMI. However, they found a significant difference in the frequency distribution of the minor allele of the 1299 + 79T > C polymorphism between patients and controls (0.294 vs. 0.342, P 1⁄4 0.04), which suggests that this haplotype might be protective against the development of MI. This polymorphism is identical to FGG-H3 tagging SNP 9340 T/C [rs1049636]. The aim of the present study was to investigate the effect of the four most common haplotypes of the FGG gene on the risk of MI. For this study, a large population-based case–control study, Study of Myocardial Infarctions Leiden (SMILE) was used. Full details of the SMILE study have been described Table 1 Variant allele and haplotype group frequencies for VKORC1 and CYP2C9 in selected populations (actual SNP locations in parentheses). Haplotype groups A and B are based on classifications fromReider et al. [1] where haplotype A represents individuals at risk for excessive anticoagulation with standard warfarin dosing, and haplotype B represents individuals at risk for subtherapeutic anticoagulation from standard warfarin dosing. Overall low dose group defines individuals with at least one haplotype A and/or at least one CYP2C9 variant allele. Combined haplotype A and CYP2C9 group defines individuals with at least one haplotype A in combination with at least one CYP2C9 variant allele. The functional significance of haplotypes not in group A or group B (other) is currently unknown. IVS is standard nomenclature for intronic sequence

Genome-wide linkage scan in affected sibling pairs identifies novel susceptibility region for venous thromboembolism: Genetics In Familial Thrombosis study

Venous thromboembolism (VTE) is a multicausal disorder involving environmental and genetic risk factors. In many thrombophilic families the clustering of thrombotic events cannot be explained by known genetic risk factors, indicating that some remain to be discovered.

High levels of protein C are determined by PROCR haplotype 3

Summary.  Background: Genetic determinants of plasma levels of protein C (PC) are poorly understood. Recently, we identified a locus on chromosome 20 determining high PC levels in a large Dutch pedigree with unexplained thrombophilia. Candidate genes in the LOD‐1 support interval included FOXA2, THBD and PROCR. Objectives: To examine these candidate genes and their influence on plasma levels of PC. Patients/Methods: Exons, promoter and 3′UTR of the candidate genes were sequenced in 12 family members with normal to high PC levels. Four haplotypes of PROCR, two SNPs in the neighboring gene EDEM2 and critical SNPs encountered during resequencing were genotyped in the family and in a large group of healthy individuals (the Leiden Thrombophilia Study (LETS) controls). Soluble endothelial protein C receptor (sEPCR) and soluble thrombomodulin (sTM) plasma levels were measured in the family. Results:PROCR haplotype 3 (H3) and FOXA2 rs1055080 were associated with PC levels in the family but only PROCR H3 was also associated with plasma levels in the healthy individuals. Carriers of both variants had higher PC levels than carriers of only PROCR H3 in the family but not in healthy individuals, suggesting that a second determinant is present. EDEM2 SNPs were associated with PC levels, but their effect was small. PC and sEPCR levels were associated in both studies. sTM was not associated with variations of THBD or PC levels. Conclusions: Chromosome 20 harbors genetic determinants of PC and sEPCR levels and the analysis of candidate genes suggests that the PROCR locus is responsible.

Selectin haplotypes and the risk of venous thrombosis: influence of linkage disequilibrium with the factor V Leiden mutation

Summary.  Background: Selectins (E‐, L‐ and P‐selectin) and their most important counter‐receptor P‐selectin glycoprotein ligand (SELPLG) facilitate the interaction of platelets, leukocytes and endothelial cells at inflammatory sites. Selectin polymorphisms/haplotypes have been associated with cardiovascular disease. Objectives: We investigated the association between haplotypes (H) of these four genes and deep venous thrombosis (DVT) risk. We additionally explored the effect of linkage disequilibrium (LD) with the nearby Factor V Leiden mutation (FVL). Furthermore, interactions between SELPLG polymorphisms and selectin polymorphisms were investigated. Patients/methods: Leiden Thrombophilia Study (LETS) subjects were genotyped for 24 polymorphisms by TaqMan or PCR–RFLP, detecting all common haplotypes in four blocks. P‐selectin was analyzed in two blocks, upstream (SELPup) and downstream (SELPdown) of the recombination hotspot. Results: In E‐ and L‐selectin, none of the haplotypes was associated with DVT risk. In SELPup, H2‐carriers had a 1.3‐fold increased risk (95% CI, 1.0–1.7), whereas H4‐carriers had a 1.4‐fold decreased risk (95% CI, 0.5–1.0). In SELPdown, H2‐carriers had a 1.3‐fold increased risk (95% CI, 1.0–1.7). Because of LD with FVL, we subsequently excluded all FVL‐carriers and all risks disappeared. Mutual adjustment within a logistic regression model resulted in disappearance of the risks for the SELP haplotypes, whereas FVL risk remained. Conclusions: After adjustment for LD with FVL, none of the selectin haplotypes was associated with DVT risk, showing that the increased risks of the selectin haplotypes were a reflection of the effect of FVL on thrombosis risk.

Sequence variants and haplotypes of the factor IX gene and the risk of venous thrombosis.

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