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Resistance to Activated Protein C: Comparison of Three Different PCR Methods for Detection FV R506Q
1Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA
Molecular Diagnosis : a Journal Devoted to the Understanding of Human Disease Through the Clinical Application of Molecular Biology
|December 1, 1996
Summary
Detecting the Factor V Leiden mutation (FV R506Q) is crucial for identifying inherited thrombophilia risk. Three PCR-based methods showed high concordance in identifying this mutation, with Mnl I RFLP proving highly reliable.
Area of Science:
- Genetics
- Molecular Biology
- Hematology
Background:
- Activated protein C (APC) resistance is a primary risk factor for inherited thrombophilia.
- Over 90% of APC resistance stems from the FV R506Q mutation in the Factor V gene.
- This mutation results in a hypercoagulable state due to impaired Factor Va inactivation.
Purpose of the Study:
- To compare the efficacy of three distinct polymerase chain reaction (PCR)-based methods for detecting the FV R506Q mutation.
- To evaluate diagnostic concordance and reliability across different PCR techniques.
Main Methods:
- Genotyping of 67 patient blood samples using three PCR-based approaches.
- Method 1: Restriction-fragment-length polymorphism (RFLP) analysis detecting loss of a Mnl I recognition site.
- Method 2: Primer-engineered RFLP creating a novel Nla III site; Method 3: Allele-specific PCR.
Main Results:
- Sixty-five out of 67 samples (97%) yielded concordant genotype results across all three methods.
- Minor non-diagnostic results were observed in two samples for allele-specific PCR or primer-engineered RFLP.
- The Mnl I RFLP method showed no false positive or negative results.
Conclusions:
- Primer-engineered RFLP and allele-specific PCR are viable alternatives or confirmatory tests for Mnl I RFLP in FV R506Q detection.
- All three PCR-based methods demonstrated high diagnostic concordance for identifying the FV R506Q mutation.
- Mnl I RFLP is a highly accurate and reliable method for detecting the Factor V Leiden mutation.