FV Leiden

Overview of Factor V Leiden

• Definition: Factor V Leiden is a specific genetic mutation in the F5 gene (encoding Factor V) that results in resistance to inactivation by Activated Protein C (APC)
• Significance: The most common inherited thrombophilia, associated with an increased risk of venous thromboembolism (VTE), such as deep vein thrombosis (DVT) and pulmonary embolism (PE)
• Key Points:
  • Molecular Test: Diagnosis is made by detecting the presence of the Factor V Leiden mutation using DNA-based molecular assays
  • No Cure: Having the mutation isn’t always symptomatic, but it’s essential to know that if the mutation is present, a higher level of care is required
  • Increased Risk: Those with it are more prone to clotting

The F5 Gene and Factor V Leiden Mutation

• The F5 Gene:
  • Location: Chromosome 1 (1q24.2)
  • Encodes: Factor V, a key coagulation factor that acts as a cofactor to Factor Xa in the prothrombinase complex, which converts prothrombin to thrombin
• The Factor V Leiden Mutation:
  • Description: A point mutation in the F5 gene (G1691A)
  • Amino Acid Substitution: Results in a glutamine (Q) to arginine (R) substitution at amino acid position 506 (R506Q)
  • Effect on APC Cleavage Site: This substitution alters one of the cleavage sites on Factor V that is normally recognized by activated protein C (APC)
• Mechanism of Resistance to APC:
  • Normal Inactivation of Factor V: APC, along with its cofactor protein S, normally inactivates Factor Va by cleaving it at specific sites
  • Factor V Leiden Resistance: The R506Q mutation makes Factor V resistant to inactivation by APC, leading to prolonged activity of Factor Va and increased thrombin generation

Testing Principles

• Molecular Testing is Required: The Factor V Leiden mutation is detected using DNA-based molecular assays. This can be done with a simple blood draw as this mutation is present from birth
• These tests analyze the patient’s DNA to determine if the mutation is present, or not

Molecular Testing Procedures

The general procedure for detecting the Factor V Leiden mutation involves the following steps:

1. DNA Extraction:
   • Extract DNA from a blood sample (typically EDTA whole blood) using a commercially available DNA extraction kit
   • Ensure that the DNA is of high quality and free from inhibitors
2. PCR Amplification:
   • Use the polymerase chain reaction (PCR) to amplify a specific region of the F5 gene that contains the R506Q mutation
   • Use primers that flank the region of interest and are specific for the F5 gene
3. Mutation Detection: Use various techniques to detect the presence of the Factor V Leiden mutation:
   • Allele-Specific PCR (AS-PCR):
     • Uses primers that are specific for either the normal sequence or the mutated sequence
     • The presence of a PCR product indicates the presence of that specific allele
   • Restriction Fragment Length Polymorphism (RFLP) Analysis:
     • Uses a restriction enzyme that recognizes and cuts the normal DNA sequence but does not cut the mutated sequence
     • The PCR product is digested with the restriction enzyme, and the resulting fragments are analyzed by gel electrophoresis
     • The presence of different fragment sizes indicates the presence of the Factor V Leiden mutation
   • Real-Time PCR (qPCR):
     • Uses fluorescent probes to detect the amplified DNA product in real time
     • Can be used to quantify the amount of the normal and mutated alleles
   • Next-Generation Sequencing (NGS):
     • Can be used to sequence the entire F5 gene and identify both known and novel mutations
     • More commonly used when other genetic abnormalities are suspected

Interpretation of Results

• Normal (Homozygous Wild-Type): The patient has two copies of the normal F5 gene and does not carry the Factor V Leiden mutation
• Heterozygous: The patient has one copy of the normal F5 gene and one copy of the Factor V Leiden mutated gene
  • Increased risk of venous thrombosis
• Homozygous: The patient has two copies of the Factor V Leiden mutated gene
  • Higher risk of venous thrombosis compared to heterozygotes
• Important Note: The interpretation of results should always be made in the context of the patient’s clinical history, family history, and other laboratory findings

Key Considerations:

• Results are qualitative, which will not determine treatment

Factors Affecting Test Results

• Pre-Analytical Variables:
  • Sample Collection:
    • Collect blood in an EDTA (purple-top) tube
    • Avoid clots or hemolysis
  • Sample Storage:
    • Store samples properly to prevent DNA degradation
• Analytical Variables:
  • Reagent Quality:
    • Use high-quality reagents and ensure that they are not expired
  • Proper Controls:
    • Include positive and negative controls in each PCR run to monitor the performance of the assay
  • Contamination:
    • Take precautions to prevent contamination of the PCR reagents and samples
• Patient-Related Variables:
  • Recent Blood Transfusion:
    • May affect results if the transfused blood contains a different F5 genotype than the patient’s own cells
    • Wait several weeks after transfusion before performing the test
  • Hematopoietic Stem Cell Transplantation (HSCT):
    • The F5 genotype may reflect the donor’s genotype rather than the patient’s original genotype

Troubleshooting Erroneous Results

• If the molecular test results are unexpected or inconsistent with the patient’s clinical presentation:

  • Verify the sample identity and chain of custody
  • Repeat the test on a fresh sample
  • Check the quality control data for the assay
  • Investigate potential interfering substances or technical errors
  • Consider the possibility of a rare or novel F5 mutation

Key Terms

• F5 Gene: The gene that encodes Factor V
• Factor V Leiden: A specific mutation in the F5 gene (R506Q) that causes resistance to activated protein C
• Hypercoagulability (Thrombophilia): An increased tendency to develop blood clots
• Venous Thromboembolism (VTE): A condition that includes deep vein thrombosis (DVT) and pulmonary embolism (PE)
• Allele-Specific PCR (AS-PCR): A PCR technique that uses primers specific for either the normal or mutated sequence
• Restriction Fragment Length Polymorphism (RFLP): A technique that uses restriction enzymes to cut DNA at specific sites
• Real-Time PCR (qPCR): A PCR technique that allows for the quantification of the amplified DNA product