Factor V Leiden

Clinical Usage

  • Identify a cause of thrombosis in affected patients.
  • To determine a genetic cause for recurrent second- or third-trimester pregnancy loss.
  • Evaluation of individuals with a family history of thrombosis or a family member identified to have Factor V Leiden

Background Information

Normal Factor V, coded by the Factor V gene, is a 330 kDa single chain glycoprotein comprised of 2,224 amino acid residues that is part of the coagulation system.  It is made primarily in the liver and circulates in the blood in inactive form until activated, usually by an injury to blood vessels.  Factor V functions as a cofactor to allow factor Xa to activate the enzyme thrombin. Thrombin in turn cleaves fibrinogen to form fibrin, which polymerizes to form the dense meshwork that makes up the majority of a clot.

Factor V also has a role in regulating the coagulation system; activated Protein C (aPC) cleaves Factor V and thus inactivates and degrades it.  This inactivation slows the clotting process and prevents clots from growing too large.  In addition, cleavage of Factor V at a position 506, allows it to work with aPC to inactivate factor VIIIa, which is another protein essential for normal clotting.

The Factor V Leiden mutation results in a variant protein that cannot be as easily degraded by aPC.  The gene mutation, a single nucleotide polymorphism (SNP), changes an arginine to glutamine at amino acid position 506 or 534. Since this amino acid is normally the cleavage site for aPC, the mutation prevents efficient inactivation of factor V. The resistance of Factor V Leiden to aPC cleavage also results in more active factor VIIIa.  As a result, both the activated form of coagulation factor V and coagulation factor VIIIa persist longer in circulation, which may facilitate overproduction of thrombin leading to generation of excess fibrin and excess clotting.  This tendency to form abnormal clots that can block blood vessels is known as thrombophilia.

This mutation is associated with inherited predisposition to venous thrombosis, but many people carrying the Factor V Leiden mutation do not suffer any consequences.  The excessive clotting that occurs in this disorder is almost always restricted to the veins, where the clotting may cause a deep vein thrombosis (DVT). If the venous clots break off, these clots can travel through the right side of the heart to the lung where they block a pulmonary blood vessel and cause a pulmonary embolism. It is extremely rare for this disorder to cause the formation of clots in arteries that can lead to stroke or heart attack, though a “mini-stroke”, known as a transient ischemic attack, is more common. Individuals who are homozygous for the mutated allele are at a heightened risk for the events detailed above versus those that are heterozygous for the mutation.

The presence of the A19911G mutation of Factor II gene, in addition to the Factor V Leiden mutation is additive and increases the risk of venous thrombosis even further.  Other genetic risk factors associated with Factor II and MTHFR may also be additive.

The presence of additional risk factors such as older age, surgery, obesity, prolonged travel, immobility, hospitalization, oral contraceptive use, hormonal replacement therapy, pregnancy, and malignancy may exacerbate the predisposition to the development of clot. Testing is recommended in patients with a symptomatic venous thrombotic event.

Gene Information

The Factor V gene is located on the long (q) arm of chromosome 1 at position 23.  More precisely, the Factor V gene is located from base pair (bp) 169,481,191 to base pair 169,555,768 on chromosome 1.  This is a gene of 72,313 bp with a 6,672 bp coding region consisting of 25 exons.

The Factor V Leiden mutation is a single point mutation, or single nucleotide polymorphism (SNP) with the replacement of a G for an A in exon 10 of the gene.  Depending on the chosen start, the position of the nucleotide variant is either at position 1691 or 1746.

This substitution changes the Factor V’s amino acid from arginine to glutamine.  The Factor V Leiden variant thus has glutamine at amino acid position 506 (or 534 with altered nucleotide 1746) rather than the arginine that occurs in normal Factor V.

Population Information

Factor V Leiden thrombophilia is the most common inherited form of thrombophilia. The prevalence varies by population.

In the US, the prevalence of the Factor V Leiden mutation reflects the world distribution.  Factor V Leiden allele is found in the following frequencies depending on ethnic origins.

  • 5.3% of Americans of European origin
  • 2.2% of Hispanic Americans
  • 1.2% of African Americans
  • 0.45% of Asian Americans
  • 1.25% of Native Americans

The frequency of homozygosity for factor V Leiden is approximately 0.02%
Factor V Leiden heterozygosity occurs in 3% – 8% of the general US population.Factor V Leiden is extremely rare in the indigenous Australian population.
Interestingly, the prevalence of the Factor V Leiden mutation varies from 10% – 15% in Sweden and Turkey to 2% – 3% in Spain and Portugal. Higher frequencies are found in Germany, the British islands, Italy and Greece.  Lower frequencies are found in a cluster of southern Slavic countries and also Russia, Finland and other countries in north eastern Europe, as well as France and The Netherlands.

Test Method

These assays were developed using CLSI guidelines.  Control DNA samples of known genotype are tested together with each patient sample to ensure correct results.  Genomic DNA is extracted from the submitted buccal swab  or blood sample and subjected to polymerase chain reaction (PCR).  The Factor V Leiden (A) allele was detected.  The normal or wild type Factor V allele is called the G allele.

Specimen

Rejection Criteria

• Buccal swab:
i. Physical damage
ii. Specimen appears to have microbial contamination or other visible  contamination
iii. The name on the tube does not match the name on the paperwork.
iv. It is older than 10 days.
• Blood specimen:
i. It is collected in a heparin-containing tube because heparin can  inhibit the PCR reaction.
ii. It leaked in the shipping container.
iii. The name on the tube does not match the name on the paperwork.
iv. It is older than 10 days.

Interpretation

-Homozygous wild type Factor V, G/G:
Normal Factor V with normal function and normal blood clotting.
-Heterozygous Factor V Leiden, G/A:
This causes a resistance to activated protein C.  These individuals have  a five- to ten-fold increased risk of venous thrombosis.  If the A19911G mutation of Factor II is present with Factor V Leiden mutation, then the risk of venous thrombosis is even greater.                                      -Homozygous Factor V Leiden, A/A:                                                                                                                                  This genotype causes great resistance to activate protein C. These individuals have a 50- to  100-fold increased risk of venous thrombosis. If the A19911G mutation of Factor II is present with the Factor V Leiden mutation, then the risk of venous thrombosis is even greater. 

This genotype result is but one factor affecting thrombosis risk, and other genetic and clinical factors should also be considered.  Multiple thrombosis risk factors are usually additive.   Additional risk factors for development of thrombosis include:  older age, surgery, obesity, prolonged travel, immobility, hospitalization, oral contraceptive use, hormonal replacement therapy, and pregnancy, and malignancy.

CPT code

81241

Test Limitations

Other genetic variants of the Factor V gene that are not detected in the assay may influence Factor V activity. Other genetic and nongenetic factors may also influence the balance of proper coagulation.  The detection of genetic variants does not replace the need for appropriate clinical monitoring by the health care provider.  These tests were developed and the performance characteristics were determined by MDL.  This test has not been cleared or approved by the US Food and Drug Administration.  The FDA has determined that such approval is not necessary.

This test is approved for use on New York state residents.

References

1. Blondon M, Hwang M, Smith NL.  Genotyping in Prothrombotic States:  Implications for the Clinician.  Curr Cardiovasc Risk Rep.  5: 525-532.  2011.
2. Gohil, Reya., Peck, George., Sharma, Panjak.   The Genetics of Venous Thromboembolism: A meta-analysis involving ~120,000 cases and ~180,000 controls. Journal of Thrombosis and Haemostasis, 102: 360-370. doi: 10.1160/TH09010013.   2009
3. Grody WW, et al.  American College of Medical Genetics Consensus Statement on Factor V Leiden Mutation Testing.  Genetics in Medicine. 3(2): 139-148.  2001
4. Reiner, Alex P., et al.  Common Hemostasis and Inflammation Gene Variants and Venous Thrombosis in Older Adults from the Cardiovascular Health Study. Journal of Thrombosis and Haemostasis, 7(9): 1499-1505. doi: 10.1111/j.1538-7836.03522.x.  2009
5. Spector, Elaine B., et al.  Technical Standards and Guidelines: Venous Thromboembolism (Factor V Leiden and Prothrombin 2010G>A Testing): A Disease-Specific Supplement to the Standards and Guidelines for Clinical Genetics Laboratories.  Retrieved from http://www.acmg.net/Pages/ACMG_Activities/stds-2002/fv-pt.htm     
2006
6. Zee YL, Glynn RJ, Cheng S, Steiner L, Rose L, Ridker PM.  An Evaluation of Candidate Genes of Inflammation and Thrombosis in Relation to the Risk of Venous Thromboembolism.  Circulation Cardiovascular Genetics.  2: 57-62.  2009