PT 20210

Overview of Prothrombin G20210A Mutation Testing

• Definition: Molecular testing for the prothrombin G20210A mutation involves detecting a specific genetic variation in the prothrombin (F2) gene
• Clinical Significance:
  • Assesses the Prothrombin Mutation in the Body
  • Evaluation of Thrombotic Risk
• Key Points:
  • Molecular Test: The most common way to detect and analyze
  • No Cure: Not everyone gets all of the issues with the disorder, it just implies that it is more easy for patients with the mutation to develop

The F2 Gene and Prothrombin G20210A Mutation

• The F2 Gene:
  • Location: Chromosome 11 (11p11.2)
  • Encodes: Prothrombin (Factor II), a vitamin K-dependent coagulation factor that is converted to thrombin by the prothrombinase complex
• The Prothrombin G20210A Mutation:
  • Description: A point mutation in the 3’-untranslated region (3’UTR) of the F2 gene
  • Nucleotide Change: A guanine (G) to adenine (A) substitution at position 20210 (G20210A)
  • Effect on Prothrombin Levels: The mutation leads to increased prothrombin mRNA stability, resulting in elevated plasma prothrombin levels

Molecular Testing Procedures

The general procedure for detecting the prothrombin G20210A 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 F2 gene that contains the G20210A mutation
   • Use primers that flank the region of interest and are specific for the F2 gene
3. Mutation Detection:
   • 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 prothrombin G20210A 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 F2 gene and identify both known and novel mutations
     • Less commonly used for routine prothrombin G20210A testing, but may be useful in cases with atypical presentations

Interpreting Results

• Normal (Homozygous Wild-Type): The patient has two copies of the normal F2 gene and does not carry the prothrombin G20210A mutation
• Heterozygous: The patient has one copy of the normal F2 gene and one copy of the prothrombin G20210A mutated gene
  • Increased risk of venous thrombosis
• Homozygous: The patient has two copies of the prothrombin G20210A mutated gene
  • Higher risk of venous thrombosis compared to heterozygotes
• Important Notes:
  • The interpretation of results should always be made in the context of the patient’s clinical history, family history, and other laboratory findings
  • Patients with prothrombin G20210A mutation are at risk for high prothrombin levels
  • Increased risk of venous thromboembolism
  • Pregnancy loss
  • Arterial thrombotic events (less common)

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

• Patient-Related Variables:

  • Recent Blood Transfusion:
    • May affect results if the transfused blood contains a different F2 genotype than the patient’s own cells
    • Wait several weeks after transfusion before performing the test

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 F2 mutation

Reflex Testing

There generally isn’t a reflex test, as the F2/Prothrombin mutation is generally either present or not. However, after finding the mutation, you might: * Run protein levels to evaluate how much Prothrombin the patient is making * Other tests may be used for other thrombophilias

Key Terms

• F2 Gene: The gene that encodes prothrombin (Factor II)
• Prothrombin G20210A Mutation: A genetic mutation in the 3’-untranslated region of the F2 gene
• Thrombophilia (Hypercoagulable State): 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