Factor V Leiden and Factor V R2 Allele: High-throughput Analysis and Association with Venous Thromboembolism

SummaryThrombophilia is a multigenic disease in which the combination of genetic polymorphisms increases the risk of deep vein thrombosis (DVT). The rapid identification of these genetic combinations requires high-throughput analysis of single nucleotide polymorphisms (SNPs). The TaqMan® fluorogenic 5’→3’ nuclease assay (PE/Applied Bio-systems, Foster City, CA) with custom-designed primers, probes and controls has provided a highly efficient platform for high throughput. This assay was used to rapidly detect two SNPs, FV Leiden (G1691A) and FV A4070G (R2 allele), in a study of 6295 subjects. With one thermal cycler, we completed sample set-up, PCR and analysis on 84 samples in 3 h with an additional 12 wells containing 4 “no template controls” (NTC), 4 “allele-1 controls”, and 4 “allele-2 controls” in a 96-well plate. When additional thermal cyclers were used and more assays were set up while the initial sets of reactions were in the PCR machines, the output could correspondingly be increased. The TaqMan® assay was extremely accurate, avoided contamination by using uracil-N-glycolase (UNG) in a single, closed tube, and offered the possibility for additional automation with robotic equipment to implement the PCR. This TaqMan® assay facilitates high throughput to screen large populations quickly and economically while utilizing a simple protocol that requires minimal expenditure of personnel time. Our results demonstrated a prevalence of the R2 allele of 11.9% in U.S. Caucasians, 5.6% in African-Americans, 13.4% in Asian or Pacific Islanders and 11.3% in Hispanics. No association between venous thromboembolism and the R2 allele was noted, and furthermore no interaction with FV Leiden was observed.

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Influence of single nucleotide polymorphisms on thrombin generation in factor V Leiden heterozygotes

Thrombosis and Haemostasis ◽

10.1160/th13-05-0360 ◽

2014 ◽

Vol 111 (03) ◽

pp. 438-446 ◽

Cited By ~ 8

Author(s):

Olivier Segers ◽

Paolo Simioni ◽

Daniela Tormene ◽

Elisabetta Castoldi

Keyword(s):

Thrombin Generation ◽

Factor V Leiden ◽

Individual Variability ◽

Intermediate Phenotype ◽

Individual Risk ◽

Factor V ◽

Nucleotide Polymorphisms ◽

Factor X ◽

Large Variability ◽

Fv Leiden

SummaryCarriership of the factor V (FV) Leiden mutation increases the risk of venous thromboembolism (VTE) ~4-fold, but the individual risk of each FV Leiden carrier depends on several co-inherited risk and protective factors. Under the hypothesis that thrombin generation might serve as an intermediate phenotype to identify genetic modulators of VTE risk, we enrolled 188 FV Leiden heterozygotes (11 with VTE) and determined the following parameters: thrombin generation in the absence and presence of activated protein C (APC); plasma levels of prothrombin, factor X, antithrombin, protein S and tissue factor pathway inhibitor; and the genotypes of 24 SNPs located in the genes encoding these coagulation factors and inhibitors. Multiple regression analysis was subsequently applied to identify the (genetic) determinants of thrombin generation. The endogenous thrombin potential (ETP) showed a striking inter-individual variability among different FV Leiden carriers and, especially when measured in the presence of APC, correlated with VTE risk. Several SNPs in the F2 (rs1799963, rs3136516), F10 (rs693335), SERPINC1 (rs2227589), PROS1 (Heerlen polymorphism) and TFPI (rs5940) genes significantly affected the ETPAPC and/or the ETP+APC in FV Leiden carriers. Most of these SNPs have shown an association with VTE risk in conventional epidemiological studies, suggesting that the genetic dissection of thrombin generation leads to the detection of clinically relevant SNPs. In conclusion, we have identified several SNPs that modulate thrombin generation in FV Leiden heterozygotes. These SNPs may help explain the large variability in VTE risk observed among different FV Leiden carriers.

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Association of common genetic variations and idiopathic venous thromboembolism

Thrombosis and Haemostasis ◽

10.1160/th09-07-0430 ◽

2010 ◽

Vol 103 (06) ◽

pp. 1161-1169 ◽

Cited By ~ 11

Author(s):

Aurélien Delluc ◽

Lénaïck Gourhant ◽

Karine Lacut ◽

Bernard Mercier ◽

Marie-Pierre Audrezet ◽

...

Keyword(s):

Risk Factors ◽

Venous Thromboembolism ◽

Factor V Leiden ◽

Coagulation Cascade ◽

Factor V ◽

Cascade Process ◽

Nucleotide Polymorphisms ◽

Factor Xi ◽

Bonferroni Adjustment ◽

Adrenergic Systems

SummaryVenous thromboembolism (VTE) is a multifactorial disease, caused by interacting environmental and genetic risk factors. Gene-centric geno-typing strategy is one of the approaches to explore unexplained associations between risk factors and VTE. It was the objective of this study to evaluate, using a gene-centric genotyping strategy, polymorphisms in genes involved in the following pathways: coagulation cascade process, renin-angiotensin or adrenergic systems, lipid metabolism, platelet aggregation. Allele frequency was compared between 677 cases with idiopathic VTE and their matched controls. After Bonferroni adjustment, four single nucleotide polymorphisms (SNPs) were significantly associated with VTE: Factor XI rs925451 polymorphism, factor XI rs2289252 polymorphism, factor II rs1799963 (G20210A) polymorphism and factor V Leiden rs6025. An additive mode of inheritance fitted best both factor XI polymorphisms. In this hospital-based case-control study, two polymorphisms located on the factor XI gene were significantly associated with VTE. Other newly investigated polymorphisms with potentially false negatives may warrant further analyses.

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Coexistence of Factor V Leiden and Factor II A20210 Mutations and Recurrent Venous Thromboembolism

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614882 ◽

1999 ◽

Vol 82 (12) ◽

pp. 1583-1587 ◽

Cited By ~ 71

Author(s):

Giovanna D’Andrea ◽

Donatella Colaizzo ◽

Giuseppe Cappucci ◽

Annamaria del Popolo ◽

Vincenzo Brancaccio ◽

...

Keyword(s):

Venous Thromboembolism ◽

Antiphospholipid Antibodies ◽

Gene Mutations ◽

Factor V Leiden ◽

Factor V ◽

Factor Ii ◽

Recurrent Venous Thromboembolism ◽

Fv Leiden ◽

Gene Defects ◽

Work Up

SummaryPatients carrying the FV Leiden or the FII A20210 mutation have a high risk of venous thromboembolism. Among 542 patients with a documented diagnosis of deep venous thrombosis in one leg consecutively referred for a thrombophilic work-up, we have retrospectively assessed the rate of objectively documented previous recurrence in carriers of both FV Leiden and FII A20210 mutations. Eighty-two patients had experienced 115 episodes of recurrent venous thromboembolism. The rate of recurrent venous thromboembolism was 29.2% among subjects with and 14.5% in those without deficiencies of natural anticoagulant proteins (p = 0.055), and 24.6% among patients with and 14.0% in those without antiphospholipid antibodies (p = 0.036). The frequency of having a recurrent thromboembolism was 16.2%, 20.0%, and 36.4% among carriers of FV Leiden, FII A20210 mutation, or both gene defects, respectively, and 12.8% in subjects carrying neither mutation (p for trend = 0.004). When adjusted for age, sex, and thrombophilic risk factors, the rate was higher among patients with than in those without deficiencies of natural anticoagulant proteins (OR: 3.0; 95% CI: 1.2-7.5), aPL 2.5 (95% CI: 1.3-4.9), or both FV Leiden and FII A20210 gene mutations (OR 4.8; 95% CI: 1.9-12.2).The rate of previous recurrent venous thromboembolism was significantly higher in subjects carrying both FV Leiden and FII 20210 mutations and was comparable to that observed in subjects with deficiencies of natural anticoagulant proteins or antiphospholipid antibodies.

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Probability of Recurrence of Thrombosis in Patients with and without Factor V Leiden

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650249 ◽

1996 ◽

Vol 75 (02) ◽

pp. 229-232 ◽

Cited By ~ 56

Author(s):

C Rintelen ◽

I Pabinger ◽

P Knöbl ◽

K Lechner ◽

Ch Mannhatter

Keyword(s):

Venous Thromboembolism ◽

Recurrence Rate ◽

Factor V Leiden ◽

Observation Time ◽

Control Group ◽

Factor V ◽

Apc Resistance ◽

History Of ◽

Fv Leiden

SummaryActivated protein C (APC) resistance is a common risk factor for venous thromboembolism and is associated with the replacement of Arg 506 by Gin in the factor V gene (factor V Leiden). We investigated the risk of recurrence of venous thromboembolism in APC resistant patients heterozygous and homozygous for FV Leiden and compared these patient groups with a group of patients, who had a history of venous thromboembolism, but had neither APC resistance nor the FV Leiden mutation. APC resistance was determined in frozen blood samples from patients with a history of venous thromboembolism, who were not receiving oral anticoagulant (OAC) treatment. The plasma samples were collected between 1984 and 1991. Twenty-one patients in whom APC resistance was found in the stored plasma samples were reinvestigated in 1994 (5 males, 16 females, median [m] age 49 years, range 21-71 years). Twenty-one sex and age matched patients with venous thromboembolism (5 males, 16 females, age m = 50 years, range 25-73 years) investigated during the same time period who had a normal APC resistance test served as a control group. Patients with APC resistance as well as controls were reinvestigated for the presence of FV Leiden by genetic analysis in 1994. Of the 21 APC resistant patients, 5 were homozygous and 16 heterozygous for FV Leiden. Before the study entry homozygous patients had a significantly higher recurrence rate (5/5 patients) compared to the control group, in heterozygous patients (9/16) and controls (9/21) the recurrence rate was not significantly different.The total observation time was 21 years in patients with homozygous FV Leiden, 83 years in patients with heterozygous FV Leiden and 108 years in controls, excluding the time when patients were on OAC treatment. During the observation time the recurrence rate was highest in patients with homozygous FV Leiden (9.5 % per patient per year), but was similar in patients with heterozygous FV Leiden (4.8% per patient per year) and controls (5% per patient per year). Two of five (40%) homozygous patients, 4/16 (25%) heterozygous and 5/21 (24%) controls had at least one recurrent event during the observation period. The probability for development of thrombosis in the Kaplan-Meyer-Plot analysis was not different between the three groups.Bearing limitations of our study in mind (retrospective design, relatively small patient number) we conclude that the risk of recurrence after a thromboembolic event is not higher in patients with heterozygous FV Leiden than in patients without this mutation. Thus, it appears that the identification of heterozygous FV Leiden mutation is not an indication for long-term OAC treatment. Also, long-term OAC treatment cannot generally be recommended for homozygous patients with a single thromboembolic event. More definitive conclusions will require larger prospective studies.

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Factor V Leiden Is Associated with Repeated and Recurrent Unexplained Fetal Losses

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656060 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0822-0824 ◽

Cited By ~ 145

Author(s):

Elvira Grandone ◽

Maurizio Margaglione ◽

Donatella Colaizzo ◽

Marina d'Addedda ◽

Giuseppe Cappucci ◽

...

Keyword(s):

Protein C ◽

Strong Association ◽

Activated Protein C ◽

Fetal Loss ◽

Factor V Leiden ◽

Factor V ◽

Significant Difference ◽

History Of ◽

Fv Leiden ◽

Caucasian Women

SummaryActivated protein C resistance (APCR) is responsible for most cases of familial thrombosis. The factor V missense mutation Arg506>Gln (FV Leiden) has been recognized as the commonest cause of this condition. Recently, it has been suggested that APCR is associated with second trimester fetal loss. We investigated the distribution of FV Leiden in a sample (n = 43) of Caucasian women with a history of two or more unexplained fetal losses. A group (n = 118) of parous women with uneventful pregnancies from the same ethnical background served as control. We found the mutation in 7 cases (16.28%) and 5 controls (4.24%; p = 0.011). A statistically significant difference between women with only early fetal loss vs those with late events (p = 0.04) was observed. Our data demonstrate a strong association between FV Leiden and fetal loss. Furthermore, they indicate that late events are more common in these patients.

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Prevalence of Factor V Leiden Mutation in Non-European Populations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655963 ◽

1997 ◽

Vol 77 (02) ◽

pp. 329-331 ◽

Cited By ~ 55

Author(s):

Guglielmina Pepe ◽

Olga Rickards ◽

Olga Camacho Vanegas ◽

Tamara Brunelli ◽

Anna Maria Gori ◽

...

Keyword(s):

Factor V Leiden ◽

Indirect Evidence ◽

Factor V ◽

Factor V Leiden Mutation ◽

The World ◽

Fv Leiden ◽

Sub Saharan ◽

Low Prevalence ◽

European Populations ◽

Apparently Healthy

SummaryA difference in the prevalence of venous thromboembolism (TE) in major human groups has been described and an uneven distribution of FV Leiden mutation over the world has recently been reported.We investigated FV Leiden mutation in 584 apparently healthy sub#jects mostly from populations different from those previously investi#gated: 170 Europeans (Spanish, Italians), 101 sub-saharan Africans (Fon, Bariba, Berba, Dendi), 115 Asians (Indonesians, Chinese, Tharus), 57 Amerindians (Cayapa), 84 Afroamericans (Rio Cayapa, Viche), and 57 Ethiopians (Amhara, Oromo).The mutation was detected in only 1/115 Asian (Tharu) and in 5/170 Europeans (4 Italians, 1 Spanish).These data confirm that in non-Europeans the prevalence of FV mutation is at least 7 times lower than in Europeans and provide indirect evidence of a low prevalence not only of the FV Leiden gene but also of other genes leading to more severe thrombophilia. Finally, findings from the literature together with those pertaining to this study clearly show a marked heterogeneity among Europeans.

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