Verigene^® F5 Nucleic Acid Test (IVD)

Background

The F5 gene, located on chromosome 1q23, encodes a protein that plays a role in the formation of blood clots (i.e., coagulation). The activity of the F5 protein is controlled by activated protein C (APC), which regulates F5 protein levels by cleaving it at particular sites, simultaneously eliminating its activity and marking it for degradation by the cell¹.

Different ethnic groups can carry a mutation in one or both copies of their F5 gene that changes a single DNA base from a G to an A (1691G>A). The prevalence of the mutation varies between ethnic groups as demonstrated in Table 1.  This mutation (commonly referred to as Factor V Leiden) causes an amino acid substitution at one of the cleavage sites in the F5 protein that makes it resistant to cleavage by APC. The result is an increase in F5 activity leading to a state of hypercoagulation (i.e., thrombophilia).

The relative risk for deep vein thrombosis or its recurrence is directly associated with an individual’s F5 genotype. Individuals with one copy of the Factor V Leiden mutation (i.e., heterozygous) are at a 4-8 fold relative risk for venous thrombosis compared to individuals with no mutation. Individuals with 2 copies of the Factor V Leiden mutation (i.e., homozygous mutant) are at an 80-fold risk for venous thrombosis as compared to individuals with no mutation¹.

In 2001 and 2005, the American College of Medical Genetics (ACMG) issued guidelines for F5 and F2 testing (see below), indicating that testing in certain ethnic groups may have utility in the following circumstances^1,2:

• Age < 50, any venous thrombosis.
• Venous thrombosis in unusual sites (such as portal hepatic, mesenteric, and cerebral veins).
• Recurrent venous thrombosis.
• Venous thrombosis and a strong family history of thrombotic disease.
• Venous thrombosis in pregnant women or women taking oral contraceptives.
• Myocardial infarction in female smokers under age 50.
  

Testing may also be considered in the following situations:

• Venous thrombosis, age >50, except when active malignancy is present.
• Relatives of individuals known to have the F5 1691G>A mutation (Factor V Leiden).
• Women with recurrent pregnancy loss or unexplained severe preeclampsia, placental abruption, intrauterine fetal growth retardation, or stillbirth.
  
  

Intended Use

The Verigene^® F5 Nucleic Acid Test is an in vitro diagnostic for the detection and genotyping of a single point mutation (G to A at position 1691; also known as Factor V Leiden) of the human Factor V gene (F5; Coagulation Factor V gene) in patients with suspected thrombophilia, from isolated genomic DNA obtained from whole blood samples. The test is intended to be used on the Verigene^® System.

Clinical Study Highlights

Accuracy of the Verigene^® F5 / F2 / MTHFR Nucleic Acid Tests was assessed at three sites using two hundred eighty seven (n=287) samples, sixty-eight percent (68%) from patients undergoing “rule-out thrombophilia” testing, and comparing results to bi-directional sequencing analysis performed by an independent reference laboratory. The percent agreement between the methods was 100%.

Two studies were designed to assess reproducibility of the Verigene^® F5 / F2 / MTHFR Nucleic Acid Tests. In the first study, three DNA samples that had been whole genome amplified were tested in duplicate twice per day by two operators at each of three test sites using two different lots of cartridges at each site (i.e., six lots total). One site performed this testing for 10 non-consecutive days; the other two sites performed the testing for 5 non-consecutive days. The qualitative reproducibility between all sites, lots, and operators demonstrated 100% agreement between the calls made and expected results based on bi-directional DNA sequencing.

The second reproducibility study was comprised of 4 parts, each using a different DNA sample extracted from whole blood. The studies examined intra- and inter- operator and laboratory reproducibility as well as lot-to-lot reproducibility. Again the qualitative reproducibility between all sites, lots, and operators demonstrated 100% agreement between the calls made and expected results based on bi-directional DNA sequencing.

Literature Cited

1. Grody WW, Griffin JH, Taylor AK, et al. Genet Med 2001; 3(2):  139-148.
2. Spector EB, Grody WW, Matteson CJ, et al. Genet Med 2005; 7(6):  444-453.

 

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