Methylenetetrahydrofolate Reductase (MTHFR)

Clinical Usage

Identify a cause for arteriosclerotic vascular disease or venous thrombosis, especially in individuals with hyperhomocysteinemia or a family history of that disease.

Background Information

Methylene tetra-hydrofolate reductase (MTHFR) is an enzyme involved in folate metabolism which catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.  MTHFR participates in a multistep process that converts  homocysteine to methionine. A decrease in the function or amount of MTHFR causes increased blood levels of homocysteine.  Increased homocysteine blood concentrations are associated with an increased risk for cardiovascular disease including venous thrombosis, atherosclerosis, stroke, and peripheral artery disease.  Genetic, environmental (diet) and physiologic factors affect homocysteine levels.  Folate, vitamin B6 and vitamin B12 are involved in regulating homocysteine levels; dietary supplementation can decrease blood homocysteine concentration.

Two mutations of the MTHFR gene, C677T and A1298C, cause decreased MTHFR enzyme activity and are fairly common.  The C677T mutation encodes an alanine to valine substitution at amino acid 223 of the MTHFR enzyme. This mutation results in an enzyme with only 45% – 50% of the normal activity, and the enzyme is inactivated by heat (thermolabile).   The A1298C MTHFR variant results in a glutamate to alanine substitution of the MTHFR protein, and the enzyme activity is decreased to 68% of the wild type enzyme.

Studies on the correlation of MTHFR mutations, blood homocysteine concentration, and cardiovascular disease have mixed results.  Some studies show a correlation of the MTHFR mutations with blood homocysteine levels and cardiovascular disease risk, while others show no correlation.  MTHFR genotyping has been used for some time as one of the risk factors for venous thrombosis and atherosclerosis.  However, based on the mixed results of research studies and that several factors influence homocysteine levels, the American college of Medical Genetics and Genomics’ 2013 practice guideline is that MTHFR genotyping has minimal clinical utility.
MTHFR mutations have also been associated with increased risk of bearing children with neural tube defects, recurrent pregnancy loss, and colon cancer.

Gene Information

The MTHFR gene is located on the short (p) arm of chromosome 1 at position 36.3.  More precisely, the MTHFR gene is located from base pair 11,845,786 to base pair 11,866,159 on chromosome 1.

The C677T mutation is at base pair position 677 of the MTHFR gene and is a replacement of a C for a T. 

The A1298C mutation is in exon 7 at base pair position 1298 in the MTHFR gene and is a replacement of an A for a C.

Population Information

The frequency of the C677T variant is higher in those of Mediterranean descent.  The frequency of this mutation in those of Asian descent is variable.  Japanese reportedly have a frequency of the homozygous mutation of about 30%.  Han Chinese from the north have a homozygous frequency as high as 60% and from the south a homozygous frequency of 6%.  The incidence of the C677T mutation is very low in Pacific islanders and native Americans.

The frequency of the MTHFR A1298C variant is higher in individuals of Indian descent.  Interestingly, Han Chinese have the homozygous mutant form of A1298C in 25% of the population living in southern China, and have only 2% of the population with the homozygous mutant form in northern China.  The incidence of the A1298C mutation is very low in native Americans.

Test Method

These assays were developed using CLSI guidelines.  Control DNA samples of known genotype are tested together with each patient sample to ensure correct results.  Genomic DNA is extracted from the submitted buccal swab or blood sample and subjected to polymerase chain reaction (PCR).  The C677T and A1298C alleles of MTHFR were detected.



• Buccal swab
• Whole blood,  2-5 mL, into:
i. EDTA-containing tube (purple or lavender top), or
ii. ACD-containing tube (yellow top), or
iii. Citrate-containing tube (blue top).
• Store at 2-8°C.  Ship by overnight carrier at ambient temperature.

Rejection Criteria

• Buccal swab:
i. Physical damage
ii. Specimen appears to have microbial contamination or other visible  contamination
iii. The name on the tube does not match the name on the paperwork.
iv. It is older than 10 days.
• Blood specimen:
i. It is collected in a heparin-containing tube because heparin can inhibit  the PCR reaction.
ii. It leaked in the shipping container.
iii. The name on the tube does not match the name on the paperwork.
iv. It is older than 10 days.


CPT code


Test Limitations

Other mutations for the MTHFR gene will not be detected in this test.  The detection of genetic variants does not replace the need for appropriate clinical monitoring by the health care provider.  These tests were developed and the performance characteristics were determined by MDL.  This test has not been cleared or approved by the US Food and Drug Administration. The FDA has determined that such approval is not necessary.

This test is approved for use on New York state residents


1. Alfirevic Z, et al.  Frequency of Factor II G20210A, Factor V Leiden, MTHFR C677T and PAI-1 5G/4G olymorphism in patients with veous thromboembolism:  Croatian case-control study.  Biochemia Medica  20(2): 229-235.  2010
2. Castro R, et al.  5,10-Methylenetetrahydrofolate reductase 677CT and 1298AC mutations are genetic determinants of elevated homocysteine.  Q J Med 96: 297-303.  2003
3. Den Heijer M, Lewington S, Clarke R.  Homocysteine, MTHFR and Risk of Venous Thrombosis:  A Meta-Analysis of Published Epidemiological Studies.  J Thrombosis and Haemostasis  3:  292-299.  2005
4. Hanson NQ, Aras O, Yang F, Tsai MY.  C677T and A1298C Polymorphisms of the Methylenetetrahydrofolate Reductase Gene:  Incidence and Effect of Combined Genotypes on Plasma Fasting and Post-Methionine Load Homocysteine in Vascular Disease.  Clinical Chemistry  47(4):  661-666.   2001
5. Hickey SE, Curry CJ, Toriello HV.  ACMG Practice Guideline:  Lack of evidence for MTHFR polymorphism testing.  Genetics in Medicine  15(2): 153-156.  2013
6. Leclerc D, Sibani S, Rozen R.  Molecular Biology of Mehylenetetrahydrofolate Reductase (MTHFR) and Overview of Mutations/Polymorphisms.  In MTHFR Polymorphisms and Disease.  Per Magne Ueland and Rima Rozen, eds.  Landes Bioscience.  ISBN:1-58707-217-8.  2005
7. Masz A, Melegh B.  Three periods of one and a half decade of ischemic stroke susceptibility gene research:  lessons we have learned.  Genome Medicine 2: 64-75.   2010
8. Morelli VM, et al.  Hyperhomocysteinemia increases the risk of venous thrombosis independent of the C677T mutation of the methylenetetrahydrofolate reductase gene in selected Brazilian patients. Blood Coagulation and Fibrinolysis 13: 271-275.  2002
9. Salomon O, et al.  Single and Combined Prothrombotic Factors in Patients with Idiopathic Venous Thromboembolism.  Arterioscler Thromb Vasc Biol.  19:  511-518.  1999
10. Schwahn BC, Rozen R.  Methylenetetrahydrofolate Reductase Polymorphisms:  Pharmacogenetic Effects.  In MTHFR Polymorphisms and Disease.  Per Magne Ueland and Rima Rozen, eds.  Landes Bioscience.  ISBN:1-58707-217-8.  2005
11. Spiroski I, et al.  Methylenetetrahydrofolate reductase (MTHFR-677 and MTHFR-1298) genotypes and haplotypes and plasma homocysteine levels in patients with occlusive artery disease and deep venous thrombosis.  Acta Biochimica Polonica  55 (3): 587-594.  2008    
12. Varga EA, Sturm AC, Misita CP, Moll S.  Homocysteine and MTHFR Mutations.  Relation to Thrombosis and Coronary Arery Disease.  Circulation  111: 289-293.   2005
doi:  10.1161/01.CIR.0000165142.37711.E7    
13. Wilcken B, et al.  Geographical and ethnic variation of the 677C>T allele of 5,10-methylenetetrahydrofolate reductase (MTHFR):  findings from over 7000 newborns from 6 areas world wide.  J Med Genet 40:619-625.  2003
14. Yang B, et al.  Geographical Distribution of MTHFR C677T, A1298C and MTRR A66G Gene Polymophisms in China:  Findings from 15357 Adults of Han Nationality. PLOS  8 (3): e57917.  9pp.  2013
15. Yang Q, Bailey L, Clarke R, Flanders WD, Liu T, Yesupriya A, Khoury MJ, Friedman JM.  Prospective study of methylenetetrahydrofolate reductase (MTHFR) variant C677T and risk of all-cause and cardiovascular disease mortality among 6000 US adults.  Am J Clin Nutr 95: 1245-1253.  2012