MTHFR Clinical Considerations: A Review
Genetic Testing
MTHFR Clinical Considerations: A Review
By Traci Pantuso ND, MS
Adjunct Faculty, Bastyr University, Seattle, WA
Dr. Pantuso reports no financial relationships relevant to this field of study.
Summary Points
- C677T and A1298C polymorphisms are common variants in the MTHFR gene.
- At this point, only C677T homozygous variant with elevated plasma homocysteine level is clinically significant.
- Patients with a C677T homozygous without elevated plasma homocysteine level do not have an increased risk of venous thromboembolism or recurrent pregnancy loss related to their MTHFR status.
- Patients with a C677T The methylenetetrahydrofolate reductase (MTHFR) enzyme is centrally involved with both folate and homocysteine metabolism (see Figure 1)homozygous variant with elevated plasma homocoysteine level do have a slightly increased risk of venous thromboembolism, and recurrent pregnancy loss
. The MTHFR enzyme irreversibly converts 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is the primary circulatory form of folate. 5-methyltetrahydrofolate is also a cosubstrate in the remethylation of homocysteine to produce methionine.1-3 Methionine is the precursor to S-Adenosyl methionine (SAMe) and is one of the essential amino acids in that it is required through dietary intake.1-3 SAMe is a major methyl donor and is involved in numerous biological reactions including the epigenetic mechanism of DNA methylation.2
Figure 1. Schematic Representation of Homocysteine and Folate Metabolism |
The MTHFR (methylenetetrahydrofolate reductase) enzyme converts 5,10 methylenetetrahydrofolate (5,10 methylene-THF) into 5-methyltetrahydrofolate (5-MTHF), which requires B2 (riboflavin) as a cofactor. 5-MTHF donates a methyl group in the reaction converting homocysteine to methionine, which requires B12 as a cofactor. B6 (pyridoxine) is required in the conversion of tetrahydrofolate (THF). |
Polymorphisms of the MTHFR gene are a major cause of hyperhomocystinemia of unknown clinical significance.3,4 At first, elevated homocysteine levels were thought to be a major risk factor for cardiovascular disease (CVD),3-5 but this has softened lately. Recent research has demonstrated that moderately elevated homocysteine levels are weakly correlated with coronary heart disease risk. A recently published meta-analysis that investigated unpublished data sets demonstrated that moderately elevated homocysteine levels do not affect coronary heart disease risk.6
MTHFR gene polymorphisms also have been associated with a long list of disease conditions including autism, cancer, hypertension, aneurysm, recurrent pregnancy loss, peripheral artery disease, migraine, and neuropsychiatric disease.1-3,5
Most of the association data with MTHFR are contradictory and there is no clear evidence to suggest a causal relationship.1,5,7 Nonetheless, MTHFR gene testing has become exponentially more popular in the last few years.
With the introduction of genetic testing that is increasingly affordable, more patients are approaching their health care providers about such testing or with results from their direct-to-consumer genetic tests. Research findings linking MTHFR gene polymorphisms and disease risk have been conflicting. This has led to challenges as far as interpreting such data.7
This review article will provide the following:
- Background on the MTHFR gene and its known functions
- MTHFR polymorphisms and mutations and their association to diseases
- Bottom line recommendations on ordering MTHFR genetic testing.
BACKGROUND
The MTHFR gene is located on chromosome 1 and expression of the MTHFR enzyme has been found in most body tissues.3 In 1972, Mudd et al found that a patient with homocystinuria had a rare and severe deficiency in the MTHFR enzyme.8 In 1988, the thermolabile variant of MTHFR enzyme was isolated from lymphocytes in patients with CVD who also had a mild-to-moderate increase in homocysteine, which at the time was an emerging risk factor for CVD.9 Frosst et al first demonstrated the association between homozygous C677T genotype and mild hyperhomocysteinenia in the NHLBI Heart Study.7 This correlation between the C677T homozygous variant and elevated homocysteine was found only to occur in the presence of low folate status.7
MTHFR POLYMORPHISMS
According to the National Center for Biotechnology Information of the National Institutes of Health, there are nine common polymorphic variants of MTHFR, and 34 rare and deleterious mutations are documented.3 The clinical presentation of MTHFR deficiency resulting from these mutations is highly variable and may include everything from neurological deterioration and death in infancy to mild thrombophilia in adults.10-12
The two most common polymorphic variants that affect enzyme activity are the C677T and the A1298C variants.3 The C677T and the A1298C variants are the polymorphisms that are analyzed through most laboratory MTHFR genetic tests in the United States.5,7 The 34 rare and deleterious mutations require gene sequencing, which is not widely available.
Because there are two copies of each gene, an individual can be homozygous for the C677T variant. This can cause hyperhomocysteinemia if the individual also has a low plasma folate.3-6 Individuals who are heterozygous for C677T have one copy of the C677T variant and one "wild type" or normal copy. These C677T heterozygous individuals do not demonstrate elevated homocysteine. C677T homozygous variant enzyme is thermolabile and demonstrates 70% reduced enzyme activity in vitro.3 The heterozygous C677T MTHFR enzyme has 35% reduced activity in vitro.3 It is estimated that 32% of Mexicans, 10-15% of North American Caucasians, and 6% of people of African descent are C677T homozygous.3
Another common variant is the A1298C polymorphism. Neither A1298C homozygosity nor heterozygosity causes elevated homocysteine levels.3,5 The A1298C variant has demonstrated decreased enzyme activity in vitro; however, it has not demonstrated thermolability. C677T is inherited in an autosomal recessive pattern.5 The C677T and A1298C variants are in linkage disequilibrium with each other, making compound heterozygosity an infrequent occurrence.13
DISEASE CONNECTIONS
There have been numerous, often contradictory, studies investigating the role of MTHFR variant genotype status, but different medical conditions with no clear positive relationship has been demonstrated.1,3,5
In a recent meta-analysis comparing unpublished data with published data investigating the role of MTHFR polymorphism and coronary heart disease, the authors found no relationship between MTHFR polymorphism status and risk of coronary heart disease.6 A recent study reported that elevated plasma homocysteine did increase the risk of coronary heart disease. However, this was independent of the MTHFR genotype.14
Research has demonstrated that there is increased risk (odds ratio, 1.6) of a woman who is homozygous for the C677T variant having offspring with a neural tube defect.5,15 The risk of neural tube defects are further increased if both the mother and the fetus are homozygous for the C677T variant.5,15
Another area of research that may demonstrate importance in the future is investigating the differential effects of certain pharmaceutical drugs on MTHFR genotype status.3 Of particular interest in this field of research is whether there is a differential effect of anti-folate medications on individuals with MTHFR polymorphisms and mutations.1,3,5
EFFECT OF DIET ON MTHFR GENE EXPRESSION
Plasma homocysteine levels are dependent on nutritional, genetic, and environmental factors, and this may be mediated through MTHFR. In addition, the vitamins folate, B12, and B6 are required for homocysteine metabolism (see Figure 1). An Italian study investigating forms of folate (dietary, 5-MTHF, and folic acid) and effect on total plasma homocysteine levels found that all three experimental groups had lowered plasma homocysteine levels compared to the controls. Italy has not mandated folic acid food fortification, so the average folic acid intake through diet is 220 µg/day.16 The study showed that a folic acid-enriched food diet (400 µg/day), supplemental folate in the form of 5-MTHF (200 µg/day), and folic acid (200 µg/day) are comparable in reducing total homocysteine levels irrespective of MTHFR genotype status.16 Although supplementation of folic acid, B12, and B6 may lower plasma homocysteine, it does not appear that they affect CVD risk.17
Folic acid, also known as vitamin B9, is a synthetic vitamin that is not found in large amounts in food. Folic acid is stable while the natural forms of folate are unstable.18-20 There are numerous forms of dietary folate. The folate that is most commonly found in foods is in the form of polyglutamates and must be converted into the monoglutamate form at the brush border of the intestine to be absorbed. Folic acid is already in the monoglutamate form so it is easily absorbed. Folic acid must be converted into tetrahydrofolate (THF) by an enzyme named dihydrofolate reductase. THF then must undergo a number of steps to be finally converted into the active form of 5-MTHF by the enzyme MTHFR. 5-MTHF is the most common form found in the bloodstream.18-20 Research is ongoing to determine whether supplementation of folic acid or 5-MTHF is preferred. Numerous enzymes and pathways are involved in folate absorption, metabolism, and trafficking throughout the body. The best form of supplemental folate is unresolved; folic acid or 5-MTHF appear to both be good choices.17 More research is needed to understand any differences in absorption, metabolism, and trafficking of folic acid vs 5-MTHF.
RIBOFLAVIN
The MTHFR enzyme requires the cofactor flavin adenine dinucleotide (FAD). FAD has been shown to stabilize the MTHFR enzyme in vitro.3,21 As riboflavin is the precursor to FAD, a diet rich in riboflavin may help to stabilize the MTHFR enzyme.
BETAINE/CHOLINE
Betaine and choline also act as methyl donors to convert homocysteine to methionine and have demonstrated total plasma homocysteine lowering effects in humans.19 Betaine supplementation has been shown to lower homocysteine levels in both healthy subjects and patients with severe decreased MTHFR activity.22-23
SUMMARY
The MTHFR gene is complex and how it influences health requires much more research to fully understand the functions of the gene and how it is regulated. There is an MTHFR mouse model that may also help to shed light on the role of the MTHFR genotypes and their functions in other diseases.3
TESTING
Most experts recommend to not test for MTHFR polymorphisms C677T and A1298C in the general population. The American College of Medical Genetics (ACMG) does not recommend that MTHFR polymorphism testing be performed for the evaluation of thrombophilia or recurrent pregnancy loss. ACMG also recommends that clinicians do not order testing for at-risk family members. The ACMG recommends that women of childbearing age take daily folic acid according to the general population guidelines to prevent neural tube defects regardless of MTHFR status.
None of the following medical associations recommend ordering C677T polymorphism testing with or without elevated homocysteine in persons with inherited thrombophilia or thrombosis:
American College of Medical Genetics American College of Obstetrics and Gynecology College of American Pathologists American College of Chest Surgeons British Hematology Standards CommitteeOnly the American Heart Association states that it is optional whether to order a C677T polymorphism test if elevated homocysteine is present.7
Despite the recommendations of numerous medical associations, MTHFR testing is one of the most commonly ordered molecular pathology tests in the United States.7 In one study, only 14.5% of MTHFR gene polymorphism testing was conducted after a total plasma homocysteine test was found elevated and for thrombophilia or thrombosis.7 Sixty-four laboratories in North America, along with a number of direct-to-consumer laboratories, offer the MTHFR testing.3,7
According to ACMG practice guidelines, "it is not uncommon that medical problems are incorrectly attributed to positive MTHFR status and that the geneticist should ensure that patients have received thorough and appropriate evaluations for their symptoms."5
Total plasma homocysteine can be ordered and if it is elevated, dietary interventions should be initiated. It is important to note that total homocysteine levels increase with age and are lower in pregnant populations. Total homocysteine levels are also influenced by ethnicity to an unknown degree, so caution is warranted when interpreting results.
Some clinicians only use such predictive genetic tests when the result will significantly influence the clinical management of a patient. An example of this is ordering a genetic test because a treatment is available for the medical condition or disease caused by the mutation. It can be difficult to predict the many downstream effects of some genetic tests, MTHFR included, making a strong case to involve genetic counselors or clinicians with specialized training when determining the appropriateness of gene analysis in a given patient circumstance.
RECOMMENDATION
According to research and the numerous medical organizations, there is insufficient evidence to support testing for the MTHFR polymorphisms C677T and A1298C in most cases. However, if a clinician suspects that one of these genetic tests is warranted, a referral to a genetic counselor could be considered. One of the reasons for such a referral is that the MTHFR polymorphism testing for C677T and A1298C does not rule out rare and severe mutations of the gene, and gene sequencing would then be required. Also, unnecessary testing can carry risks. For example, patients receiving a homozygous C677T or A1298C result may experience psychological stress, even though the overwhelming body of evidence is inconclusive.5
A healthy diet containing folate, choline, betaine, riboflavin, B6, and B12 should be recommended for everyone, especially individuals who have MTHFR polymorphisms (see Table 1). One of the reasons for this, as described above, is that the C677T homozygous polymorphism combined with low dietary folate leads to elevated homocysteine, a situation that can be reversed with a change in diet. Folate-rich foods include dark green leafy vegetables and legumes. Choline-rich foods include high-quality organic/grass fed red meat, poultry, eggs, and fish. Betaine-rich foods include wheat bran, wheat germ, beets, and riboflavin-containing foods (mushrooms and almonds). B6 sources include fish, poultry, nuts and legumes, and B12 sources are high-quality organic/grass-fed red meat, poultry, and fish.
Table 1. RDA/Adequate Intakes for Folate, Riboflavin, Pyridoxine, Cobalmin, and Choline |
|||
Nutrient |
RDA for Adult Males |
RDA for Adult Females |
RDA for Pregnant Females |
Folate |
400 ìg/day |
400 ìg/day |
600 ìg/day |
Riboflavin (B2) |
1.3 mg/day |
1.1 mg/day |
Not specified |
Pyridoxine (B6) |
19-50 years old = 1.3 mg/day > 50 = 1.7 mg/day |
19-50 years old = 1.3 mg/day > 50 = 1.5 mg/day |
Not specified |
Cobalmin (B12) |
2.4 ìg/day |
2.4 ìg/day |
Not specified |
Choline (adequate intake) |
550 mg/day |
425 mg/day |
Not specified |
Adapted from: Dietary Reference Intakes for Calcium, Phosphorous, Magnesium, Vitamin D, and Fluoride (1997); Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline (1998); Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids (2000); and Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc (2001); Dietary Reference Intakes for Water, Potassium, Sodium, Chloride, and Sulfate (2005); and Dietary Reference Intakes for Calcium and Vitamin D (2011). These reports may be accessed via www.nap.edu. |
|||
The decision of whether to supplement with folate, choline, betaine, riboflavin, B6, and B12 in people with MTHFR mutations is more complicated; there are no data suggesting that supplementation improves disease outcomes.
REFERENCES
- Nazki FH, et al. Folate: Metabolism, genes, polymorphisms and the associated diseases. Gene 2014;533:11-20.
- Anderson OS, et al. Nutrition and epigenetics: An interplay of dietary methyl donors, one-carbon metabolism and DNA methylation. J Nutr Biochem 2012;23:853-859.
- Leclerc D, et al. Molecular Biology of Methylenetetrahydrofolate Reductase (MTHFR) and Overview of Mutations/Polymorphisms. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000. Available at: http://www.ncbi.nlm.nih.gov/books/NBK6561/. Accessed August 10, 2014.
- Frosst P, et al. A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nature Genet 1995;10:111-113.
- Hickey SE, et al. ACMG Practice Guideline: Lack of evidence for MTHFR polymorphism testing. Genet Med 2013;15:153-156.
- Clarke R, et al. Homocysteine and coronary heart disease: Meta-analysis of MTHFR case control studies, avoiding publication bias. PLoS Med 2012;9:e1001177.
- Cohen DA, et al. Laboratory informatics based evaluation of methylene tetrahydrofolate reductase C677T genetic test overutilization. J Pathol Inform 2013;4:33.
- Mudd SH, et al. Homocystinuria associated with decreased methylenetetrahydrofolate reductase activity. Biochem Biophys Res Commun 1972;46:905-912.
- Kang SS, et al. Intermediate homocysteinemia: A thermolabile variant of methylenetetrahydrofolate reductase. Am J Hum Genet 1988;43:414-421.
- Goyette P, et al. Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe MTHFR deficiency. Am J Hum Genet 1995;56:1052-1059.
- Goyette P, et al. Severe and mild mutations in cis for the methylenetetrahydrofolate reductase (MTHFR) gene, and description of 5 novel mutations in MTHFR. Am J Hum Genet 1996;59:1268-1275.
- D’Aco KE, et al. Severe 5,10-Methylenetetrahydrofolate reductase deficiency and two MTHFR variants in an adolescent with progressive myoclonic epilepsy. Pediatr Neurol 2014;51:266-270.
- Ogino S, Wilson RB. Genotype and haplotype distributions of MTHFR677C>T and 1298A>C single nucleotide polymorphisms: A meta-analysis. J Hum Genet 2003;48:1-7.
- Gariglio L, et al. Comparison of homocysteinemia and MTHFR 677CT polymorphism with Framingham Coronary Heart Risk Score. Arch Cardiol Mex 2014;84:71-78.
- Liu TC, et al. [Meta analysis on the association between parental 5,10-methylenetetrahydrofolate reeducates C677T polymorphism and the neural tube defects of their offspring]. Zhonghua Liu Xing Bing Xue Za Zhi 2011:32:60-67.
- Zappacosta B, et al. Homocysteine lowering by folate-rich diet or pharmacological supplementations in subjects with moderate hyperhomocysteinemia. Nutrients 2013;5:1531-1543.
- Marti-Carvajal AJ, et al. Homocysteine-lowering interventions for preventing cardiovascular events. Cochrane Database Syst Rev 2013;1:CD006612.
- Blom HJ, Smulders Y. Overview of homocysteine and folate metabolism. With special references to cardiovascular disease and neural tube defects. J Inherit Metab Dis 2011;34:75-81.
- Strickland KC, et al. Molecular mechanisms underlying the potentially adverse effects of folate. Clin Chem Lab Med 2013;51:607-616.
- Farrell CJ, et al. Red cell or serum folate: What to do in clinical practice? Clin Chem Lab Med 2013;51;555-569.
- Sibani S, et al. Characterization of mutations in severe methylenetetrahydrofolate reductase deficiency reveals an FAD-responsive mutation. Hum Mutat 2003;5:509-520.
- Obeid R. The metabolic burden of methyl donor deficiency with focus on the betaine homocysteine methyltransferase pathway. Nutrients 2013;5:3481-3495.
- Ogier de Baulny H, et al. Remethylation defects: Guidelines for clinical diagnosis and treatment. Eur J Pediatr 1998;157(Suppl2):S77-83.
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