scholarly journals Detection of the Factor V Leiden Mutation by a Modified Photo-Cross-Linking Oligonucleotide Hybridization Assay

2004 ◽  
Vol 50 (2) ◽  
pp. 296-305 ◽  
Author(s):  
Cynthia French ◽  
Conan Li ◽  
Charles Strom ◽  
Weimin Sun ◽  
Reuel Van Atta ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Factor V Leiden ◽  
Fold Increase ◽  
Clinical Samples ◽  
Cross Linking ◽  
Target Sequence ◽  
Factor V ◽  
Capture Probe ◽  
Factor V Leiden Mutation ◽  
Oligonucleotide Hybridization

Abstract Background: Our previously developed assay for detection of the factor V Leiden mutation (G1691A) based on a nucleic acid photo-cross-linking technology used two allele-specific capture probes and six fluorescein-modified signal-generating reporter probes. We wished to improve the sensitivity and performance of the method. Methods: We developed new reporter probes with ∼10-fold more fluorescein molecules than the original probes. The single, cross-linker-modified capture probe was replaced by a three-probe system, separating the probe–target cross-linking function and the allelic differentiation function. The capture probe cross-linked to either or both of two flanking probes through stem structures at the capture-probe/flanking-probe junctions. The flanking probes cross-linked to target DNA through two cross-linking sites each. Genomic DNA was extracted from 0.2 mL of whole blood and restriction-enzyme digested to create a defined 677 bp target sequence. Preliminary genotype ranges were determined for the assay by testing of pretyped samples. We then tested 1054 clinical samples, using an automated sample processor. Results: The new assay had a 10-fold increase in signal-to-background ratio. Genotype results for 1039 of 1054 clinical samples (98.6%) agreed with those of a PCR-based method. Of the 15 remaining samples, 10 produced an indeterminate result outside the defined genotype ranges, 2 yielded insufficient signal to be genotyped, and 3 gave a discordant result. All 15 samples were genotyped correctly after reextraction of genomic DNA and retesting. Conclusion: The modified photo-cross-linking assay for factor V Leiden detection is a sensitive non-PCR-based assay with potential for use in high-throughput clinical laboratories.

scholarly journals Cross-linking hybridization assay for direct detection of factor V Leiden mutation

1997 ◽  
Vol 43 (9) ◽  
pp. 1703-1708 ◽  
Author(s):  
James Zehnder ◽  
Reuel Van Atta ◽  
Carol Jones ◽  
Howard Sussman ◽  
Michael Wood
Keyword(s):  
Direct Detection ◽  
Factor V Leiden ◽  
Hybridization Assay ◽  
Cross Linking ◽  
Factor V ◽  
Wild Type ◽  
Mutation Site ◽  
Factor V Leiden Mutation ◽  
Allele Specific ◽  
Factor V Gene

Abstract A nucleic acid photocross-linking technology was used in the development of a direct assay for factor V Leiden, a point mutation in the factor V gene (G1691A) that is the most common inherited risk factor for thrombosis. This cross-linking hybridization assay included two allele-specific capture probes and six signal-generating reporter probes; all were modified with a photoactivated cross-linking compound. By using two different capture probes complementary to a 16-base sequence at the factor V Leiden mutation site, but differing in the nucleotide opposite the mutation site (C vs T), wild-type and factor V Leiden alleles were differentiated in purified DNA specimens. The assay was also successfully applied to genomic DNA in leukocytes isolated from whole blood; the factor V status of 122 patients as determined by this method was in complete concordance with a standard PCR-based assay and clearly discriminated between healthy individuals and factor V Leiden heterozygotes.

Thrombophilia and risk of VTE recurrence according to the age at the time of first VTE manifestation

VASA ◽  
2015 ◽  
Vol 44 (4) ◽  
pp. 313-323 ◽  
Author(s):  
Lea Weingarz ◽  
Marc Schindewolf ◽  
Jan Schwonberg ◽  
Carola Hecking ◽  
Zsuzsanna Wolf ◽  
...  
Keyword(s):  
Factor V Leiden ◽  
Cox Proportional Hazards ◽  
First Episode ◽  
Factor V ◽  
Factor V Leiden Mutation ◽  
Younger Patients ◽  
Risk Of Recurrence ◽  
G20210a Mutation ◽  
Increased Risk ◽  
The Impact

Abstract. Background: Whether screening for thrombophilia is useful for patients after a first episode of venous thromboembolism (VTE) is a controversial issue. However, the impact of thrombophilia on the risk of recurrence may vary depending on the patient’s age at the time of the first VTE. Patients and methods: Of 1221 VTE patients (42 % males) registered in the MAISTHRO (MAin-ISar-THROmbosis) registry, 261 experienced VTE recurrence during a 5-year follow-up after the discontinuation of anticoagulant therapy. Results: Thrombophilia was more common among patients with VTE recurrence than those without (58.6 % vs. 50.3 %; p = 0.017). Stratifying patients by the age at the time of their initial VTE, Cox proportional hazards analyses adjusted for age, sex and the presence or absence of established risk factors revealed a heterozygous prothrombin (PT) G20210A mutation (hazard ratio (HR) 2.65; 95 %-confidence interval (CI) 1.71 - 4.12; p < 0.001), homozygosity/double heterozygosity for the factor V Leiden and/or PT mutation (HR 2.35; 95 %-CI 1.09 - 5.07, p = 0.030), and an antithrombin deficiency (HR 2.12; 95 %-CI 1.12 - 4.10; p = 0.021) to predict recurrent VTE in patients aged 40 years or older, whereas lupus anticoagulants (HR 3.05; 95%-CI 1.40 - 6.66; p = 0.005) increased the risk of recurrence in younger patients. Subgroup analyses revealed an increased risk of recurrence for a heterozygous factor V Leiden mutation only in young females without hormonal treatment whereas the predictive value of a heterozygous PT mutation was restricted to males over the age of 40 years. Conclusions: Our data do not support a preference of younger patients for thrombophilia testing after a first venous thromboembolic event.

Coagulation Factor V Leiden Mutation Was Detected in the Patients with Activated Protein C Resistance in Thailand

1998 ◽  
Vol 80 (08) ◽  
pp. 344-345 ◽  
Author(s):  
Pasra Arnutti ◽  
Motofumi Hiyoshi ◽  
Wichai Prayoonwiwat ◽  
Oytip Nathalang ◽  
Chamaiporn Suwanasophon ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Factor V Leiden Mutation ◽  
Coagulation Factor V

Low Prevalence of the Factor V Leiden Mutation Among “Severe” Hemophiliacs with a “Milder” Bleeding Diathesis

1995 ◽  
Vol 74 (05) ◽  
pp. 1255-1258 ◽  
Author(s):  
Arnaldo A Arbini ◽  
Pier Mannuccio Mannucci ◽  
Kenneth A Bauer
Keyword(s):  
Hemophilia A ◽  
Factor V Leiden ◽  
Protein S ◽  
Factor Ix ◽  
Hemophilia B ◽  
Factor V ◽  
Bleeding Diathesis ◽  
Mutation Site ◽  
Spontaneous Bleeding ◽  
Factor V Leiden Mutation

SummaryPatients with hemophilia A and B and factor levels less than 1 percent of normal bleed frequently with an average number of spontaneous bleeding episodes of 20–30 or more. However there are patients with equally low levels of factor VIII or factor IX who bleed once or twice per year or not at all. To examine whether the presence of a hereditary defect predisposing to hypercoagulability might play a role in amelio rating the hemorrhagic tendency in these so-called “mild severe” hemophiliacs, we determined the prevalence of prothrombotic defects in 17 patients with hemophilia A and four patients with hemophilia B selected from 295 and 76 individuals with these disorders, respectively, followed at a large Italian hemophilia center. We tested for the presence of the Factor V Leiden mutation by PCR-amplifying a fragment of the factor V gene which contains the mutation site and then digesting the product with the restriction enzyme Mnll. None of the patients with hemophilia A and only one patient with hemophilia B was heterozygous for Factor V Leiden. None of the 21 patients had hereditary deficiencies of antithrombin III, protein C, or protein S. Our results indicate that the milder bleeding diathesis that is occasionally seen among Italian hemophiliacs with factor levels that are less than 1 percent cannot be explained by the concomitant expression of a known prothrombotic defect.

Detection of the Factor V Leiden Mutation Using Whole Blood PCR

1996 ◽  
Vol 75 (03) ◽  
pp. 520-521 ◽  
Author(s):  
D C Rees ◽  
M Cox ◽  
J B Clegg
Keyword(s):  
Whole Blood ◽  
Factor V Leiden ◽  
Factor V ◽  
Factor V Leiden Mutation

“Pseudo Homozygous” Activated Protein C Resistance due to Double Heterozygous Factor V Defects (Factor V Leiden Mutation and Type I Quantitative Factor V Defect) Associated with Thrombosis: Report of Two Cases Belonging to Two Unrelated Kindreds

1996 ◽  
Vol 75 (03) ◽  
pp. 422-426 ◽  
Author(s):  
Paolo Simioni ◽  
Alberta Scudeller ◽  
Paolo Radossi ◽  
Sabrina Gavasso ◽  
Bruno Girolami ◽  
...  
Keyword(s):  
Protein C ◽  
Dna Analysis ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Type I ◽  
Factor V ◽  
Factor V Leiden Mutation ◽  
Thrombotic Risk ◽  
Apc Resistance ◽  
Quantitative Factor

SummaryTwo unrelated patients belonging to two Italian kindreds with a history of thrombotic manifestations were found to have a double heterozygous defect of factor V (F. V), namely type I quantitative F. V defect and F. V Leiden mutation. Although DNA analysis confirmed the presence of a heterozygous F. V Leiden mutation, the measurement of the responsiveness of patients plasma to addition of activated protein C (APC) gave results similar to those found in homozygous defects. It has been recently reported in a preliminary form that the coinheritance of heterozygous F. V Leiden mutation and type I quantitative F. V deficiency in three individuals belonging to the same family resulted in the so-called pseudo homozygous APC resistance with APC sensitivity ratio (APC-SR) typical of homozygous F. V Leiden mutation. In this study we report two new cases of pseudo homozygous APC resistance. Both patients experienced thrombotic manifestations. It is likely that the absence of normal F. V, instead of protecting from thrombotic risk due to heterozygous F. V Leiden mutation, increased the predisposition to thrombosis since the patients became, in fact, pseudo-homozygotes for APC resistance. DNA-analysis is the only way to genotype a patient and is strongly recommended to confirm a diagnosis of homozygous F. V Leiden mutation also in patients with the lowest values of APC-SR. It is to be hoped that no patient gets a diagnosis of homozygous F. V Leiden mutation based on the APC-resi-stance test, especially when the basal clotting tests, i.e., PT and aPTT; are borderline or slightly prolonged.

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