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Hematology

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 & 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