Inherited Thrombophilia: Factor V Leiden and Prothrombin

Thrombophilia testing

Venous thromboembolism (deep vein thrombosis, pulmonary embolism) has many causes; inherited variants are one. Two common variants are routinely genotyped: factor V Leiden and prothrombin G20210A. Both are single-nucleotide changes, so they make good teaching examples of SNV genotyping.

Factor V Leiden

Activated factor V (Va) speeds thrombin production. Activated protein C (APC) turns it off by cutting at arginine 506. Factor V Leiden replaces that arginine with glutamine (Arg506Gln, R506Q), so APC inactivates factor V more slowly, and clotting proteins persist longer ¹. One copy raises clot risk; two copies raise it further ¹.

Notation: traditionally G1691A; in current HGVS numbering F5 c.1601G>A, p.(Arg534Gln), because the protein number now includes the 28-amino-acid signal peptide.

It’s common in people of European ancestry (a few percent are heterozygous) and rare elsewhere. A functional APC resistance assay (a clotting test) screens; DNA testing confirms and distinguishes heterozygotes from homozygotes.

Prothrombin G20210A

Prothrombin G20210A lies in the 3’ untranslated region of the prothrombin gene (F2). It doesn’t change the protein; it causes cells to make too much prothrombin, which means more thrombin and more clotting ². (HGVS: F2 c.*97G>A.)

Unlike most thrombophilias, there’s no functional test for it: prothrombin levels overlap too much with normal. It’s detected only by DNA testing.

Testing methods

Both variants are typically tested together from whole blood in EDTA:

• Real-time PCR with allele-specific probes (hydrolysis probes, one for each allele labeled with a different dye): the ratio of signals calls the genotype.
• Melt-curve analysis with hybridization probes (see below).
• Invader, microarray, or sequencing methods.
• Historically, PCR-RFLP: the Leiden change abolishes an MnlI site, and the prothrombin assay uses a mutagenic primer to create a HindIII site.

Reading a melt curve

In a melt-curve assay, a fluorescent probe matching one allele (here wild type) hybridizes over the variant site. After PCR, the instrument heats slowly and records fluorescence as the probe melts off; the software plots the negative derivative (-dF/dT), which peaks at each melting temperature:

• Normal (homozygous wild type): one peak at the higher temperature (perfect match).
• Homozygous variant: one peak at a lower temperature (one-base mismatch destabilizes the probe).
• Heterozygote: two peaks.

Each run includes controls of all three genotypes, so peak positions can be read against known samples.

Interpretation

Reports state the genotype (no variant, heterozygous, homozygous) and an interpretation. These variants raise relative risk, but most carriers never have a clot, and other factors (surgery, pregnancy, estrogen, immobility, cancer) contribute. Testing is generally reserved for situations where the result changes management. MTHFR variant testing for thrombophilia is not recommended.

What to take from this

Factor V Leiden (R506Q; c.1601G>A in HGVS) alters the APC cleavage site so factor Va resists inactivation. Prothrombin G20210A sits in the F2 3’ UTR and raises prothrombin, with no functional test available. Both are genotyped by allele-specific real-time PCR or melt curves, where a heterozygote shows two melting peaks. They raise relative risk but don’t predict clots on their own.