Factor V Leiden
Resistance to Activated Protein C [ARCr] was first assessed used a modified APTT. Subsequent studies have shown that in the majority of cases of APCr arise from a G→A missense mutation at nucleotide 1691 in the F5 gene leading to an Arg506Gln mutation in which the arginine residue at position 506 in Factor Va is replaced by an glutamine and this abolishes an inactivation cleavage site for Activated Protein C [APC.] In the diagram below the cleavage sites for APC, FXa and Thrombin [T] in FVa are shown .
In purified reaction mixtures, Gln506-FVa in comparison to normal FVa shows reduced susceptibility to APC, because it is inactivated approximately 10-fold slower than normal Arg506-FVa. Cleavage of Arg506 alone does not inactivate FV but decreases the rate at which Arg306 are cleaved.
Principles & Method
A number of methods exist for the detection of the FV Leiden mutation. The majority of methods depend on PCR to amplify genomic DNA followed by a method to identify the presence or absence of the missense mutation e.g. restriction enzyme digestion and mapping.
A wide vareity of methods are in use:
- Multiplexed techniques e.g. for the FVL and Prothrombin G20210A mutations
- Direct sequencing, either single pass or bidirectional
- Spectrophotometric evaluation of binding to allele-specific oligonucleotides in microtitre plates
- Enzyme immunoassay for the PCR product
- Luciferase-linked DNA polymerase-mediated depolymerisation
- PCR-independent oligonucleotide-generated fluorescence
- Direct fluorogenic probe-based PCR assay
- Real-Time PCR
- Nanochip micro electronic arrays
Most techniques can rapidly distinguish heterozygous individuals from homozygous mutant or wild type [i.e. normal] It is important to remember that the the majority of tests only screen for the presence or absence of the Arg506Gln mutation and although an abnormal APC ratio may be obtained in a screening test for APC resistance, in the rare cases in which this is due to a mutation in an APC inactivation cleavage sites other than at Arg506 this/these will not be detected unless specific approaches to identify these are undertaken.
Similarly the Factor V HR2 haplotype has been demonstrated to cause mild APC resistance and to interact synergistically with the Arg506Gln mutation leading to severe APC resistance [ie. pseudo homozygosity] in individuals who are heterozygous for the Arg506Gln mutation.
There are no reference ranges for a genetic test - the mutation is either present in a heterozygous or homozygous form or it is not.
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