A Hemi-nested, Allele Specific, Whole Blood PCR Assay for the Detection of the Factor V Leiden Mutation

1997 ◽  
Vol 77 (06) ◽  
pp. 1154-1155 ◽  
Author(s):  
Gary D Sinclair ◽  
Sandra Low ◽  
Man-Chiu Poon
Keyword(s):  
Whole Blood ◽  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Restriction Enzyme Digestion ◽  
Factor V ◽  
Pcr Assay ◽  
Specific Primers ◽  
Factor V Leiden Mutation ◽  
Allele Specific

SummaryWe describe a novel hemi-nested, allele specific whole blood PCR assay for detection of the factor V Leiden mutation associated with the plasma defect, activated protein C resistance. This assay utilizes 5 μl of whole blood without prior DNA extraction. The hemi-nested design, employing an outer primer pair in combination with nested, allele specific primers obviates the need for restriction enzyme digestion. PCR reactions are analysed directly on agarose or polyacrylamide minigels. The assay confirmed the genotypes of 50 individuals previously categorized by PCR and Mnll digestion, and has been subsequently utilized in the genotyping of 445 individuals referred for thrombosis studies.

The Factor V Leiden Mutation which Predisposes to Thrombosis Is Not Common in Patients with Antiphospholipid Syndrome

1995 ◽  
Vol 74 (04) ◽  
pp. 1029-1031 ◽  
Author(s):  
Donna Dizon-Townson ◽  
Christopher Hutchison ◽  
Robert Silver ◽  
D Ware Branch ◽  
Kenneth Ward
Keyword(s):  
Antiphospholipid Syndrome ◽  
Protein C ◽  
Restriction Analysis ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Factor V ◽  
Factor V Leiden Mutation ◽  
Specific Hybridization ◽  
Allele Specific ◽  
Protein Resistant

SummaryAntiphospholipid syndrome is associated with venous, arterial, and placental thrombosis, possibly through autoantibody impairment of phospholipid-dependent protein C activation. Recently, a missense mutation in the factor V gene (1691 G → A) has been identified that results in an abnormal factor V product (1). This mutation, known as the Leiden mutation, causes an amino acid substitution of glutamine for arginine at position 506 in the factor V molecule and renders the protein resistant to proteolytic inactivation by activated protein C and thus predisposes to thrombosis (2, 3). We hypothesized that some individuals with antiphospholipid syndrome may also carry the Leiden mutation, and thus have a “second hit” predisposition to thrombosis. To test this hypothesis, allele-specific hybridization and allele-specific restriction analysis were used to test for the Leiden mutation in thirty women with the antiphospholipid syndrome, 10 of whom had a history of thrombosis. None of the women were heterozygous or homozygous for the factor V mutation. We conclude that the presence of the factor V Leiden mutation is not a prerequisite for the thrombotic events in patients with antiphospholipid syndrome, due to the occurrence of thrombosis seen in patients lacking the factor V mutation.

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

“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.

scholarly journals Activated Protein C Resistance and Factor V Leiden in Mexico

2007 ◽  
Vol 14 (4) ◽  
pp. 428-437 ◽  
Author(s):  
Abraham Majluf-Cruz ◽  
Manuel Moreno-Hernández ◽  
Adriana Ruiz-de-Chávez-Ochoa ◽  
Rosario Monroy-García ◽  
Karim Majluf-Cruz ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Factor V Leiden Mutation ◽  
Common Cause ◽  
Factor V Gene ◽  
V Gene ◽  
Low Prevalence

A common cause of hereditary thrombophilia is activated protein C resistance (APCR), and most cases result from factor V Leiden mutation. An APCR phenotype without association with factor V Leiden has been described. This transversal, observational, nonrandomized study evaluated these 2 phenomena in healthy indigenous and mestizo Mexican subjects (n = 4345), including 600 Mexican natives. No indigenous subjects had APCR, but 82 mestizo subjects did. After retesting, 50 subjects had a negative test. The remaining 32 subjects had factor V Leiden, giving a 0.85% prevalence of factor V Leiden in the mestizo Mexican population. Only 31% of APCR carriers had factor V Leiden. These results show a very low prevalence of APCR and factor V Leiden in Mexico. Except for factor V Leiden, there are no other mutations in the factor V gene responsible for the APCR phenotype. Acquired APCR is nearly twice as prevalent as the inherited variant.

The Polymerase Chain Reaction with Sequence Specific Primers for the Detection of the Factor V Mutation Associated with Activated Protein C Resistance

1995 ◽  
Vol 74 (03) ◽  
pp. 874-878 ◽  
Author(s):  
Nancy E Kirschbaum ◽  
Paul A Foster
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Chain Reaction ◽  
Specific Primers ◽  
Allele Specific ◽  
Fv Leiden ◽  
Polymerase Chain

SummaryThe prevalence of the Factor V (FV) mutation associated with activated protein C resistance (FV Leiden) and its significance as a genetic risk factor for venous thrombosis have necessitated the development of a simple, rapid, and accurate assay for its detection. The polymerase chain reaction with sequence specific primers (PCR-SSP) provides a powerful technique for the discrimination of alleles resulting from single base substitutions. PCR amplification was performed using a sense primer complementary to both FV alleles coupled with either of two antisense allele specific primers, one complementary to the normal FV allele and one complementary to the FV Leiden allele. PCR conditions were developed that favored amplification only in the case of perfect complementation between template DNA and allele specific primer. The FV genotype was assigned based on whether or not each allele specific primer set produced an amplified product. Assignment of genotypes correlated 100% with those determined by the method of PCR amplification followed by MnII digestion. PCR-SSP allows the rapid and accurate identification of carriers of the Factor V Leiden mutation by a simple PCR reaction without the need for the usual post-amplification specificity step.

A Reduced Sensitivity for Activated Protein C in the Absence of Factor V Leiden Increases the Risk of Venous Thrombosis

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1271-1276 ◽  
Author(s):  
Marieke C.H. de Visser ◽  
Frits R. Rosendaal ◽  
Rogier M. Bertina
Keyword(s):  
Risk Factor ◽  
Confidence Interval ◽  
Venous Thrombosis ◽  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Factor V ◽  
Factor V Leiden Mutation ◽  
Apc Resistance ◽  
Increased Risk

Abstract Activated protein C (APC) resistance caused by the factor V Leiden mutation is associated with an increased risk of venous thrombosis. We investigated whether a reduced response to APC, not due to the factor V point mutation, is also a risk factor for venous thrombosis. For this analysis, we used the Leiden Thrombophilia Study (LETS), a case-control study for venous thrombosis including 474 patients with a first deep-vein thrombosis and 474 age- and sex-matched controls. All carriers of the factor V Leiden mutation were excluded. A dose-response relationship was observed between the sensitivity for APC and the risk of thrombosis: the lower the normalized APC sensitivity ratio, the higher the associated risk. The risk for the lowest quartile of normalized APC-SR (<0.92), which included 16.5% of the healthy controls, compared with the highest quartile (normalized APC-SR > 1.05) was greater than fourfold increased (OR = 4.4; 95% confidence interval, 2.9 to 6.6). We adjusted for VIII:C levels, which appeared to affect our APC resistance test. The adjusted (age, sex, FVIII:C) odds ratio for the lowest quartile was 2.5 (95% confidence interval, 1.5 to 4.2). So, after adjustment for factor VIII levels, a reduced response to APC remained a risk factor. Our results show that a reduced sensitivity for APC, not caused by the factor V Leiden mutation, is a risk factor for venous thrombosis.

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