Verigene^® F2 Nucleic Acid Test (IVD)

Background

The F2 gene, located on the short arm of human chromosome 11 (11p11-q12), encodes a protein that plays a role in the formation of blood clots (i.e., coagulation) in response to injury. The F2 protein is an inactive precursor of thrombin, which upon activation by coagulation factors Xa and V, phospholipid, and calcium, is converted to thrombin and helps initiate the clotting cascade.

The most common recurring mutation in the F2 gene occurs in the 3' untranslated region of the gene, where there is a G to A base change at position 20210. This mutation increases the efficiency of translation of the F2 mRNA transcript¹. The result is an increase in the amount of prothrombin transcript that is available for translation into functional prothrombin protein, which creates a hypercoaguable (i.e., thrombotic) state.

Like the F5 1691G>A, the F2 20210G>A mutation exhibits semi-dominant expression in that both heterozygotes and homozygotes are at increased risk of occurrence/recurrence of venous thrombosis. However, the effect of the F2 mutation is not as strong. Individuals with one copy of the F2 20210G>A mutation (i.e., heterozygous) are at a 2-4 fold relative risk for venous thrombosis compared to individuals with no mutation. Individuals with 2 copies of the F2 20210G>A mutation (i.e., homozygous mutant) are at a >4-fold risk for venous thrombosis. Individuals carrying both the Factor V Leiden and F2 20210G>A mutations (i.e., a compound heterozygote) have a 20-fold more likely chance of having a venous thrombosis than individuals without either mutation².

The F2 20210G>A mutation is present in about 2% of the general population. It has been observed in most ethnic groups but it is less common than the F5 1691G>A mutation (Table 1).

 

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^2,3.

• 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^® F2 Nucleic Acid Test is an in vitro diagnostic for the detection and genotyping of a single point mutation (G to A at position 20210) of the human Factor II gene (F2; prothrombin 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. Gehring NH, Frede U, Neu-Yilik G, et al. Nat Genet 2001; 28:  389-392.
2. Grody WW, Griffin JH, Taylor AK, et al. Genet Med 2001; 3(2):  139-148.
3. Spector EB, Grody WW, Matteson CJ, et al.  Genet Med 2005; 7(6):  444-453.

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