Genetic variation associated with plasma von Willebrand factor levels and the risk of incident venous thrombosis

In a recent genome-wide association study, variants in 8 genes were associated with VWF level, a risk factor for venous thrombosis (VT). In an independent, population-based, case-control study of incident VT, we tested hypotheses that variants in these genes would be associated with risk. Cases were 656 women who experienced an incident VT, and controls comprised 710 women without a history of VT. DNA was obtained from whole blood. Logistic regression was used to test associations between incident VT and single nucleotide polymorphisms (SNPs) in 7 genes not previously shown to be associated with VT. Associations with P < .05 were candidates for replication in an independent case-control study of VT in both sexes. Two of the 7 SNPs tested yielded P < .05: rs1039084 (P = .005) in STXBP5, a novel candidate gene for VT, and rs1063856 (P = .04) in VWF, a gene whose protein level is associated with VT risk. Association results for the remaining 5 variants in SCARA5, STAB2, STX2, TC2N, and CLEC4M were not significant. Both STXBP5 and VWF findings were replicated successfully. Variation in genes associated with VWF levels in the genome-wide association study was found to be independently associated with incident VT.     

Factor V leiden increases plasma F1+2 levels both in normal and deep venous thrombosis subjects

BACKGROUND AND OBJECTIVE: A simple approach to understanding molecular mechanisms leading to thrombosis is the definition of how genetic factors influence biochemical parameters of coagulation. Conflicting data have been reported regarding the role that the genotype of factor V plays in the control of plasma F1+2 levels. The aim of this study was to test whether the factor V Leiden mutation affects F1+2 levels. DESIGN AND METHODS: We studied the effect of factor V Leiden mutation (detected by the polymerase chain reaction technique) on plasma F1+2 levels in 418 normal subjects and 39 subjects affected by deep venous thrombosis. RESULTS: In both normal subjects and those with venous thrombosis, heterozygotes for the Leiden mutation showed significantly higher plasma levels of F1+2 (p<0.0001 and p<0.005, respectively). Subjects with venous thrombosis had a higher allelic frequency of the Leiden mutation than normal subjects (11.5% and 3.1%, respectively). INTERPRETATION AND CONCLUSIONS: The results indicate that the genotype of factor V is a determinant of plasma F1+2 concentration. The allelic frequency of Leiden mutation in our normal subjects is higher than that found in other Italian populations but similar to that reported for populations of north- and middle-Europe. This finding is consistent with the peculiar ancestry and history of Friuli (the area in which subjects for this study were recruited), with respect to other Italian regions.     

Genetic variation in the first-pass metabolism of ethinylestradiol,sex hormone binding globulin levels and venous thrombosis risk

BackgroundUse of ethinylestradiol, one of the active ingredients in combined oral contraceptives, affects the incidence of venous thrombosis. To explain why some women develop thrombosis when using oral contraceptives and others do not, we hypothesized a role for the first-pass metabolism of ethinylestradiol in the liver. We set out to determine the association between genetic variation in the first-pass metabolism of ethinylestradiol, venous thrombosis risk and the effect on Sex-hormone-binding-globulin (SHBG) levels.MethodsPremenopausal women were included from two case-control studies: LETS (103 cases; 159 controls) and MEGA (397 cases; 796 controls). Haplotype-tagging SNPs were selected in 11 candidate genes; COMT, CYP1A2, CYP2C9, CYP3A4, CYP3A5, SULT1A1, SULT1E1, UGT1A1, UGT1A3, UGT1A9, UGT2B7. Venous thrombosis risk was expressed as odds ratios (OR) with 95% confidence intervals (CI). For SHBG levels, mean differences with 95%CI were estimated in combined oral contraceptive-using control subjects from the MEGA study.ResultsTwo copies of haplotype D in the UGT2B7 gene increased venous thrombosis risk (ORLETS: 3.78; OR_MEGA: 2.61) as well as SHBG levels (mean difference 27.6 nmol/L, 95%CI: − 61.7 to 116.9 compared with no copies) in oral contraceptive users and not in non-users. In oral contraceptive users, haplotype A and B in the CYP3A4 gene were associated with venous thrombosis risk, but not in non-users; however, the effect on SHBG levels was not directional with the risk. None of the other haplotypes were associated with venous thrombosis.ConclusionGenetic variation in the UGT2B7 gene may, in part, explain venous thrombosis risk in combined oral contraceptive users.     

Candidate gene polymorphisms and the risk for pregnancy-related venous thrombosis

Venous thrombosis (VT) is one of the leading causes of maternal death in the western world, but the genetic causes of pregnancy-related VT are insufficiently understood. The aim of this study was to investigate the association between common genetic variations in candidate genes and pregnancy-related VT. We undertook a hospital based case-control study of women with VT during pregnancy or puerperium; controls were women giving birth without having VT. Single nucleotide polymorphisms (SNPs) were selected in 49 pre-specified candidate genes involved in coagulation, inflammation, and hormonal metabolism in 313 cases and 353 controls. We found new associations between SNPs and total pregnancy-related VT in the genes encoding coagulation factors V and VIII, and p-selectin. Additional new associations between SNPs and antenatal VT were found in the genes encoding the epidermal growth factor receptor, the pregnane X receptor, and protein S. Of 21 SNPs previously associated with thrombotic disease, rs2289252 in F11 and rs3917643 in F3 were associated with pregnancy-related VT, while rs4524 in F5 was associated with antenatal VT.     

Activated protein C resistance, Leiden mutation, anticoagulant proteins and fibrinogen levels in patients with deep venous thrombosis

Resistance to activated protein C (APC-R) is the leading cause of deep vein thrombosis (DVT). Deficiencies of protein C (PC) or its cofactor protein S (PS) are less frequent. Resistance usually results from nucleotide substitution (1691 GA) in the factor V gene (Leiden mutation). We searched for APC-R and the Leiden mutation (FVL) and measured the activities of PC, PS, antithrombin III (AT III) and Fb levels in patients with DVT. The results were analyzed against a history of thrombosis (idiopathic, risk factor related). We enrolled 29 patients aged 50 years or younger, with first symptoms of thrombosis detected before the age of 40 years. The control group consisted of 25 healthy volunteers of similar age. APC-R was established using Accelerimat (bioMerieux) assays. APC-R was diagnosed when the normalized sensitivity coefficient "r" was < 0.9. FVL was detected with the SSP-PCR (sequence specific primers-polymerase chain reaction) method. Abnormal APC-R "r" values were found in seven DVT patients (24%). Heterozygous (G/A genotype) form of the Leiden mutation was confirmed in six of them (all had a history of recurrent thrombosis). FVL carriers demonstrated lower PC levels in comparison with controls and DVT without FVL. Eight patients (27%) had PS activities below the cut-off point (60%). The deficiency in six of them was not associated with other abnormalities. Patients with recurrent thrombosis had markedly higher concentrations of Fb (usually without FVL) and reduced AT III activities. The study has shown that the APC-R test is useful for FVL screening. The Leiden mutation, elevated levels of Fb and reduced activities of PC are the main factors predisposing to DVT and its recurrence. Reduced activity of PS is usually an isolated abnormality tending to predispose to a single DVT episode.     

A nonsense polymorphism in the protein Z-dependent protease inhibitor increases the risk for venous thrombosis

The protein Z-dependent protease inhibitor (ZPI) is a hemostatic serpin with anticoagulant activity. As for antithrombin, deficiency of ZPI could have relevant thrombotic consequences. We have studied 6 genetic modifications affecting the ZPI gene, identifying 5 haplotypes. Haplotype H5 is featured by a stop codon at position 67. The relevance of these genetic modifications and haplotypes in venous thrombosis was evaluated in a case-control study including 1018 patients and 1018 age- and sex-matched controls. Surprisingly, the H5 haplotype was found in 0.9% of controls, supporting that the Arg67Stop change is a low frequency nonsense polymorphism. The prevalence of this haplotype increased significantly in patients (3.0%), one of whom was in a homozygous state. Multivariate analysis confirms that carriers have a 3.3-fold risk of developing venous thrombosis (P = .002; 95% CI: 1.5-7.1). Moreover, we observed a significant association of this polymorphism with familial history of thrombosis (P < .001). Our study supports that the ZPI Arg67Stop nonsense polymorphism might be an independent genetic risk factor for venous thrombosis. This polymorphism has slightly lower prevalence but similar thrombotic risk than the FV Leiden or prothrombin 20210A. Although further studies are required, all available data support that the ZPI is a candidate to play a significant role in thrombosis and should be evaluated in thrombophilic studies.     

Incidence and risk factors for bilateral deep venous thrombosis of the lower limbs

The incidence of bilateral deep venous thrombosis in patients with single limb or bilateral symptoms was determined using duplex scan examination. In a prospective study, 157 inpatients with clinical suspicion of deep venous thrombosis underwent duplex scan evaluation of the lower extremities. Demographic characteristics, physical examination data, and risk factor information were collected. In all, 57 (36.3%) patients evaluated presented echographic evidence of acute deep venous thrombosis. Forty-six individuals presented unilateral thrombosis, and 11 patients presented bilateral disease (19.3% of all thrombosis, 7.0% of all patients). Sensitivity and specificity of clinical examination in identifying bilateral thrombosis was 27.2% and 93.3%, respectively. For the risk factors evaluated, active human immunodeficiency virus disease and iliofemoral thrombosis presented an increased risk for bilateral thrombosis (P = .045 and P = .049, respectively). The high incidence of bilateral deep venous thrombosis justifies bilateral duplex scan examination. Active human immunodeficiency virus disease and proximal thrombosis were risk factors for bilateral disease.     

Reduced plasma fibrinolytic potential is a risk factor for venous thrombosis

The role of the fibrinolytic system in the development of deep vein thrombosis (DVT) is unclear. We determined the plasma fibrinolytic potential of patients enrolled in the Leiden Thrombophilia Study (LETS), a population-based case-control study on risk factors for DVT. Plasma fibrinolytic potential was determined in 421 patients and 469 control subjects by means of a tissue factor-induced and tissue-type plasminogen activator (tPA)-induced clot lysis assay. Using clot lysis times above the 70th, 80th, 90th, 95th, and 99th percentiles of the values found in control subjects as cut-off levels, we found a dose-dependent increase in risk for DVT in patients with hypofibrinolysis (odds ratios of 1.4, 1.6, 1.9, 2.1, and 2.2, respectively). This indicates a 2-fold increased risk of DVT in subjects with clot lysis times above the 90th percentile. The risk increase was not affected by age or sex (adjusted odds ratio for 90th percentile, 2.0), and after correction for all possible confounders (age, sex, and levels of procoagulant proteins shown to associate with clot lysis times in the control population), the risk estimate was marginally reduced (odds ratio, 1.6 for 90th percentile). Taken together, these results indicate that plasma hypofibrinolysis constitutes a risk factor for venous thrombosis, with a doubling of the risk at clot lysis times that are present in 10% of the population.     

Inferior vena cava malformation as a risk factor for deep venous thrombosis in the young

Conditions which result in hypercoagulable blood or venous stasis may predispose to the development of deep vein thrombosis (DVT). Most of the recently described risk factors for DVT induce a hypercoagulable state. Over a 3-year period we have observed anomaly of the inferior vena cava (IVC) in four young patients presenting with spontaneous unprovoked DVT. This is a greater than expected rate (5% observed versus 0.5% expected). Further, bilateral DVT, which constitutes less than 10% of cases in most series, was present in three of the four cases. Anomaly of the IVC is a rare example of a prevalent congenital condition that predisposes to DVT, presumably by favouring venous stasis. This diagnosis should be considered in young patients with spontaneous and bilateral DVT.     

Identification of polymorphisms in the 5'-untranslated region of the TAFI gene: relationship with plasma TAFI levels and risk of venous thrombosis.

BACKGROUND AND OBJECTIVES. Thrombin activatable fibrinolysis inhibitor (TAFI) plays an important role in hemostasis, functioning as a potent fibrinolysis inhibitor. TAFI gene variations may contribute to plasma TAFI levels and thrombotic risk. DESIGN AND METHODS. We sequenced a 2083-bp region of the 5'-regulatory region of the TAFI gene in 127 healthy subjects searching for variations, and correlated identified polymorphisms with plasma TAFI levels. TAFI polymorphisms were examined as risk factors for venous thrombosis by determining their prevalence in 388 patients with deep venous thrombosis (DVT) and in 388 controls. RESULTS. Seven novel polymorphisms were identified: -152 A/G, -438 A/G, -530 C/T, -1053 T/C, -1102 T/G, -1690 G/A, and -1925 T/C. -152 A/G, -530 C/T and -1925 T/C were found to be in strong linkage disequilibrium, as were the -438 A/G, -1053 T/C, -1102 T/G and -1690 G/A. Plasma TAFI levels were higher in -438GG/-1053CC/-1102GG/-1690AA homozygotes than in -438AG/-1053TC/-1102TG/-1690GA heterozygotes, and -438AA/-1053TT/-1102TT/-1690GG homozygotes had the lowest TAFI levels (p=0.0003). TAFI concentrations in -152AA/-530CC/-1925TT homozygotes were somewhat higher but not significantly different from levels observed for -152AG/-530CT/-1925TC heterozygotes. Taken in combination, -438AG/-1053TC/-1102TG/-1690GA and -438AA/-1053TT/-1102TT/-1690GG yielded an OR for DVT of 0.8 (95%CI: 0.6-1). In subjects aged <35 years the OR was 0.7 (95%CI: 0.5-1.1). The OR for -152AG/-530CT/-1925TC was 1 (95%CI: 0.5-2.2) in the whole group of patients and controls, whereas in subjects aged <35 years the OR was 0.1 (95%CI: 0.02-0.9). INTERPRETATION AND CONCLUSIONS. Polymorphisms in the TAFI promoter determine plasma antigen levels and may influence the risk of venous thrombophilia.