Reverse hybridization kit for the determination of the mutation FV:Q506 in the Factor V gene, the polymorphism 20210A in the Factor ll gene and for the detection of the MTHFR risk allele for Coronary Artery Disease

Clinic

Factor V

Thromboses are connected with disorders of the human coagulation system and can even cause symptoms as serious as pulmonary embolism. One of the factors regulating the coagulation system is the activated protein C (APC), a serine protease which, together with a co-factor, influences the clotting process by the proteolytic inactivation of two components of the coagulation cascade, factors Va and Vllla. Factor Va is developed by splitting from the factor V protein, a process brought about by the factor lla.

According to the present state of knowledge, the APC resistance is the most frequent genetic risk factor for venous clotting disorders. This is caused by a point mutation in the factor V gene, in which at position 1691 a guanine (G) is replaced by an adenine (A). As a result of this, the 506th amino acid (arginine) is replaced by glutamine in the translated protein (FV:Q506). The factor V genetic product, changed in this way, is then described as the Factor V-Leiden (FVL). Because the mutated amino acid is localized with the APC protein in the combining site, the FVL variant cannot be sufficiently splitted from the APC and inactivated. This leads to an accumulation of the factor Va, resulting in a raised tendency to coagulation, which in its turn causes a higher risk of thrombosis.

Approximately 5% of the population have a higher APC resistance, and the rate in patients with a personal or familial previous history of thrombosis is 20-60%. The allelic frequency of the mutation described is therefore ten times higher than in all other genetic risk factors of thrombosis, such as a deficiency of protein C or anti thrombin. Homozygous patients with these characteristics have a 50-100 times greater risk of thrombosis, this risk is in heterozygous patients 5-10 times greater. In combination with other risks, the probability of suffering from thrombosis rises further.

For example, oral contraceptives lead to a five times greater risk of thrombosis, this risk in combination with a heterozygous factor V mutation is then 35 times greater.

Measuring the APC resistance is an important laboratorial diagnostic parameter in identifying functional disorders of the coagulation system, and can point to the presence of a FVL genotype. In order to ascertain the genotype exactly, especially to differentiate between heterozygous and homozygous, an analysis at DNA level is necessary.

Factor II

At the end of 1996 a point mutation in the prothrombin gene could be identified which is also connected with a higher risk of thrombosis (by factor 2-3). This mutation is a G�A replacement at position 20210 in the 3' untranslated regulatory region of the gene, causing an elevated prothrombin plasma activity. Prothrombin (factor ll) is the preliminary stage of the active coagulative enzyme thrombin (factor lla), which has a key function in regulating coagulation (for example the development of factor V into factor Va). According to the first results, the heterozygous form of the mutation has a prevalence of 1 - 2%.

Individual cases of mutation cannot be proved by the determination of prothrombin activity in plasma; this risk factor can only be detected at DNA level.

This DNA probe test enables a simple and reliable identification of homozygous and heterozygous cases of significant mutations in the factor V and factor ll genes. The test is suitable as a prognostic parameter in cases of a tendency to thrombosis and can also be carried out during oral anticoagulative treatment. The risk in cases of both mutations (FVL and prothrombin) has yet to be assessed.

MTHFR

Hyperhomocysteinemia has emerged as a risk factor for coronary artery disease (1).

The metabolism of homocysteine occurs through two pathways. Genetic defects in genes of the involved enzymes are associated with hyperhomocysteinemia. One of the pathways contains the enzyme methylenetetrahydrofolate reductase (MTHFR), which catalyses the remethylation of homocysteine to methionine.

A common polymorphism in the MTHFR gene correlates with the characteristics of a thermolabile MTHFR protein. The heat-sensitive variant of the enzyme has only 50% of the activity of the normal enzyme (2). The mutation responsible for this form is a C (cytosine) to T (thymidine) substitution at nucleotide 677 of the coding sequence which converts an alanine to a valine residue (3).

The plasma homocysteine levels in individuals homozygous for this mutation were significantly higher than those of other individuals (3).

The MTHFR-C677T polymorphism is very common. About 40% of the population are homozygous for the wild type C677, 45% are heterozygous for both alleles C677 and T677 and about 15% are homozygous carrier for the T677 mutated allele (4).

Several investigations have shown that the homozygous mutated genotype is a independent risk factor for coronary artery disease (3, 5-7).