Verigene^® MTHFR Nucleic Acid Test (IVD)

Background

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 synthesis¹ 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 vitro². Both heterozygotes and homozygous mutants show elevated levels of plasma homocysteine³. 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 America².

In Recommendation 4 of the ACMG statement⁴ 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 hyperhomocysteinemia⁴. 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 factor⁴.

In a meta-analysis of 24 retrospective (n=3289) and 3 prospective studies (n=476), the association between homocysteine and thrombosis was evaluated⁵. 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^2,6,7.

Intended Use

The Verigene^® MTHFR Nucleic Acid Test is an in vitro diagnostic for the detection and genotyping of a single point mutation (C to T at position 677) of the human 5,10 methylenetetrahydrofolate reductase gene (MTHFR) 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. Sharp L & Little J. Am Jour Epidemiol 2004; 159(5): 423-443.
2. Rozen R. Thromb Haemost 1997; 78: 523-6.
3. Schneider JA, Rees DC, Liu Y-T et al. Am J Hum Genet 1998; 62: 1258-1260.
4. Grody WW, Griffin JH, Taylor AK, et al. Genet Med 2001; 3(2):139-148.
5. den Heijer M, Lewington S, Clarke R. Nat Genet 2001; 28: 389-392.
6. Frosst P, Blom HJ, Milos R et al. Nat Genet 1995; 111-113.
7. Wilcken DEL. Lancet 1997; 350: 603-604.

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