Molecular

Overview of Molecular Testing in Hypercoagulability Assessment

• Purpose: Molecular testing is performed to identify genetic mutations that are associated with an increased risk of thrombosis (hypercoagulable state)
• When to Consider Molecular Testing:
  • Unexplained or recurrent venous thromboembolism (VTE)
  • VTE at a young age (<50 years)
  • Family history of thrombophilia
  • Thrombosis in unusual sites (e.g., mesenteric, cerebral)
  • Recurrent pregnancy loss
• Important Considerations:
  • Molecular testing is generally performed after an acute thrombotic event has resolved and the patient is not on anticoagulant therapy
  • The decision to perform molecular testing should be based on clinical judgment and consideration of the patient’s risk factors and the likelihood of finding a clinically significant mutation
  • A positive result on molecular testing does not guarantee that the patient will develop thrombosis, but it can provide useful information for risk stratification and management decisions

Key Molecular Tests

Here’s a detailed breakdown of the common molecular tests used to assess inherited thrombophilias:

• Factor V Leiden Mutation Analysis (F5 Gene):

  • Background: Factor V Leiden is the most common inherited thrombophilia

• Prothrombin G20210A Mutation Analysis (F2 Gene):

  • Background: The second most common inherited thrombophilia

General Molecular Testing Procedures

Regardless of the specific mutation being tested, the general procedure for molecular testing involves:

1. DNA Extraction: Extract DNA from a blood sample
2. PCR Amplification: Use PCR to amplify the region of the gene that contains the mutation
3. Mutation Detection: Use various techniques to detect the presence of the mutation:

• Allele-Specific PCR (AS-PCR): Uses primers that are specific for the mutated sequence
• Restriction Fragment Length Polymorphism (RFLP) Analysis: Uses restriction enzymes to cut the DNA at specific sites, creating fragments of different lengths depending on the presence or absence of the mutation
• Real-Time PCR: Uses fluorescent probes to detect the amplified DNA product and quantify the amount of the mutated sequence
• Next Generation Sequencing: If negative for Factor V Leiden and Prothrombin, then this is a good option. This can test many other mutations associated with thrombophilia

Key Terms

• Thrombophilia (Hypercoagulable State): A condition with an increased risk of thrombosis
• Factor V Leiden: A common genetic mutation that causes resistance to activated protein C
• Prothrombin G20210A Mutation: A genetic mutation that leads to increased prothrombin levels
• Mutation: A change in the DNA sequence of a gene