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

BACKGROUND: 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. OBJECTIVES: We aimed to identify novel thrombosis susceptibility alleles in a large panel of small thrombophilic families: the Genetics In Familial Thrombosis (GIFT) study. PATIENTS/METHODS: In the GIFT study, 201 families were recruited consisting of 438 siblings with an objectively confirmed VTE at a young age. Multipoint linkage analysis (402 SSR markers) and fine mapping were performed, followed by genotyping of tagging SNPs in positional candidate genes. RESULTS: Established genetic risk factors such as factor V Leiden, ABO blood group non-O, prothrombin 20210A, fibrinogen gamma 10034T and deficiencies of antithrombin, protein C and protein S were more frequent in GIFT patients than in unselected VTE patients. Linkage supported the presence of novel thrombosis susceptibility loci on 7p21.3-22.2 (LOD score = 3.23) and Xq24-27.3 (LOD score = 1.95). Simulation analysis showed that the chr7 signal was genome-wide statistically significant (P = 0.022). Tagging SNPs (n = 157) in eight positional candidate genes (LOD drop 1.5 regions) were genotyped in GIFT patients and 332 healthy controls. Five chr7 SNPs associated with VTE. SNP THSD7A rs2074597 was responsible for part of the chr7 signal. CONCLUSIONS: The GIFT panel is rich in established genetic risk factors for VTE, but genetic factors remain unidentified in many families. Genome-wide linkage failed to identify the previously established genetic risk factors for VTE, but identified a novel VTE susceptibility locus on chr7.

High levels of protein C are determined by PROCR haplotype 3.

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.

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.

The -589C>T polymorphism in the interleukin-4 gene (IL-4) is associated with a reduced risk of myocardial infarction in young individuals.

BACKGROUND: Inflammatory reactions contribute to the development of arterial disease. We investigated the role of interleukin-4 (IL-4) in the development of myocardial infarction (MI) by genotyping patients with MI and control subjects for the -589C>T (rs2243250) single nucleotide polymorphism (SNP), which tags a functional haplotype of IL-4. METHODS AND RESULTS: Study of Myocardial Infarctions Leiden (SMILE) included 560 men with a first MI and 646 control subjects. The Valencia study included 305 patients with MI at T genotype was found [odds ratio (OR) 0.84; 95% CI 0.37-1.95 for -589TT and 0.82; 95% CI 0.62-1.07 for -589CT compared with -589CC]. In patients younger than 50 years, carriership of one or two -589T alleles was associated with a reduced risk of MI (OR 0.57: 95% CI 0.34-0.95). This result was replicated in the Valencia study, where carriers of one or two -589T alleles had a reduced risk of MI (OR 0.67: 95% CI 0.47-0.95), with a strong protective effect of the -598T allele in homozygous -589T (OR 0.33: 95% CI 0.10-1.05). In the control subjects of the Valencia study, the -589T allele was associated with reduced levels of F1+2. CONCLUSION: Our data indicate that the IL-4 haplotype tagged by the -589T allele reduces the risk of MI in young individuals.

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

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.

Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen gamma'/gammaA mRNA ratio.

BACKGROUND: Fibrinogen gamma haplotype 2 (FGG-H2) is associated with reduced fibrinogen gamma' levels and fibrinogen gamma'/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 gamma'/total fibrinogen ratio and the increased DVT risk.

Determinants of the APTT- and ETP-based APC sensitivity tests.

BACKGROUND: A reduced sensitivity for activated protein C (APC) is associated with an increased risk of venous thrombosis even in the absence of the factor (F)V Leiden mutation. This risk has been demonstrated with two APC sensitivity tests, which quantify the effects of APC on the activated partial thromboplastin time (APTT) and the endogenous thrombin potential (ETP), respectively. OBJECTIVES: We examined determinants of both APC sensitivity tests in the control group of the Leiden Thrombophilia Study (LETS). METHODS: Multiple linear regression analysis was performed with normalized APC-SR(APTT) or APC-SR(ETP) as dependent variable and putative determinants [levels of FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII A subunit, FXIII B subunit, protein S total, protein S free, protein C, tissue factor pathway inhibitor (TFPI) total, TFPI free, antithrombin and fibrinogen] as independent variables. RESULTS AND CONCLUSIONS: The major determinant of the APTT-based test was FVIII level, followed by FII level. The ETP-based test was influenced most by free protein S and free TFPI levels. In both tests FXa formation plays a major role, as the effect of FVIII and TFPI on the tests seems to be executed via FXa. The ETP-based test was also strongly influenced by oral contraceptive use, even when we adjusted for all the clotting factors listed above. This means that the effect of oral contraceptives on the ETP-based test is not fully explained by the changes of coagulation factor levels investigated in this study, and that the molecular basis of acquired APC resistance during use of oral contraceptives remains to be established.

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.

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

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.