F5/F2/MTHFR *

Factor V Leiden

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.

Factor II

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 transcript³. 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 mutation1.

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^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.

MTHFR

The MTHFR gene, located on human chromosome 1p36.3, encodes an enzyme that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, the primary circulating form of folate. This substrate is vital for both DNA synthesis⁴ and the methionione synthase-catalyzed conversion of homocysteine to methionine.

Several polymorphisms in the MTHFR gene have been reported, two of which have been studied in detail: 677C>T and 1298A>C. The 677C>T is a missense mutation that creates a thermolabile form of the enzyme such that heterozygotes exhibit ~65% of normal enzyme activity levels in vitro and homozygous mutants exhibit ~30% of normal enzyme activity levels in vitro5. Both heterozygotes and homozygous mutants show elevated levels of plasma homocysteine⁶. The 1298A>C mutation has been studied according to similar parameters and measures as the 677C>T mutation, but its association with decreased enzyme activity in vitro is less strong, as is its effect on plasma homocysteine levels. Therefore, the Verigene® MTHFR Nucleic Acid Test assesses the 677C>T genotype. The MTHFR 677C>T mutation has been observed in all ethnic groups and varies widely in prevalence, from low levels (~6.6%) in Africa to high levels (~44.9%) in certain populations of South America⁵.

In Recommendation 4 of the ACMG statement1 on Factor V Leiden mutation testing, it is noted that hyperhomocysteinemia is considered a potential risk factor for thrombophilia. Patients with classic homocystinuria are at extremely elevated risk of thromboembolism. Since MTHFR mutations can lead to elevated levels of plasma homocysteine and subsequent higher risk of thrombosis, laboratorians and physicians have requested the addition of the MTHFR 677C>T polymorphism to F5 and F2 tests. Although homocysteine levels can be measured directly, if MTHFR is genotyped concurrently with F5 and F2, a more complete picture of the heritable component of the thrombophilic phenotype can be made. Homozygosity of this mutation accounts for about a third of cases of hyperhomocysteinemia¹. Hyperhomocysteinemia interacts synergistically with coexisting Factor V Leiden to increase the relative risk of venous thrombosis to 20-fold greater than individuals without either risk factor¹.

In a meta-analysis of 24 retrospective (n=3289) and 3 prospective studies (n=476), the association between homocysteine and thrombosis was evaluated⁷. Then, in a separate meta-analysis of 53 studies (n=8634), the potential causal association between MTHFR 677TT (homozygous mutant) genotype and venous thrombosis was studied. The authors found a 5 µmol/L higher homocysteine level was associated with a 27% (odds ratio [OR]=1.27 [95%Cl=1.01-1.59]) higher risk of venous thrombosis in the 3 prospective studies and a 60% (OR=1.60 [95%Cl=1.10-2.34]) higher risk in the 24 retrospective studies. The MTHFR 677TT genotype was associated with a 20% (OR=1.20 [95%Cl=1.08-1.32]) higher risk of venous thrombosis compared with MTHFR 677CC (homozygous wildtype) genotype. The conclusion states that the elevated risk associated with the MTHFR 677TT genotype provides some support for causality.

Elevated plasma homocysteine has also been associated with an increased risk of cardiovascular disease and neural tube defects in pregnant women^5,8,9.

References

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.
3. Gehring NH, Frede U, Neu-Yilik G, et al. Nat Genet 2001; 28:  389-392.
4. Sharp L & Little J. Am Jour Epidemiol 2004; 159(5): 423-443.
5. Rozen R. Thromb Haemost 1997; 78: 523-6.
6. Schneider JA, Rees DC, Liu Y-T et al. Am J Hum Genet 1998; 62: 1258-1260.
7. den Heijer M, Lewington S, Clarke R. Nat Genet 2001; 28: 389-392.
8. Frosst P, Blom HJ, Milos R et al. Nat Genet 1995; 111-113.
9. Wilcken DEL. Lancet 1997; 350: 603-604.