MTHFR and Folate Metabolism

Folate metabolism

Folate refers to a family of complex molecules composed of three moieties: a petering moiety, a p-aminobenzoate moiety and a glutamate moiety (Fig. 2). Different forms of folate vary at three positions of the molecular structure: N5 position of the petering moiety, N10 of the p-aminobenzoate moiety and the number of the glutamate molecules in the glutamate moiety. Dietary folate is normally a mixture of tetrahydrofolate (THF) and derivatives. The majority of the natural folate is composed of 5-methyl THF and 10-formyl THF with up to 8 glutamate residuals.  The polyglutamated folates are preferred substrates for enzymes hence the bioactive form in all the intracellular metabolisms. However, they cannot cross cell membrane. Therefore natural dietary folate, which is always polyglutamated, has to be hydrolyzed to monoglutamated form to be absorbed. Folic acid in fortified food is already in monoglutamate form so is ready to be absorbed. Both the hydrolysis and absorption of dietary folate occur in the intestine. Within intestinal cells, the monoglutamate is converted to the polyglutamated form by the enzyme folypolyglutamate synthase. The polyglutamation reaction maintains a concentration gradient that favors the intake of the monoglutamated folate from the intestine.  It also enables the folate to become suitable substrates for intracellular folate metabolism enzymes. The polyglutamated folate and metabolic intermediates have to be hydrolyzed to monoglutamated form again in order to be transported to blood circulation and up taken by cells in liver and other tissues. Cross membrane transportation of the monoglutamated folates are mostly carried out by folate transporters on the membrane.

Figure 2. Molecular structure of folate, which is a complex of a pterin moiety (colored red), a p-aminobenzoate moiety (colored blue) and a polyglutamate moiety (colored black). The residual R1 at N5 position of the pterin and residual R2 at N10 position of the p-aminobenzoate determine the species of the folate as shown in the table at the right of the molecule structure.  The number (n) of the glutamate residual can be 1 to 8.

Once inside the cell, oxidized forms of folate such as 5-formyl THF, 10-formyle THF are first converted to DHF (dihydrofolate), then to THF (tetrahydrofolate) by the enzyme dihydrofolate reductase. THF can be used directly for purine synthesis or converted to 5’10’-MTHF (5’, 10’-methyltetrahydrofolate) by the enzyme SHMT (serine hydroxymethyltransferase) using vitamin B6 as a cofactor. 5’10’-MTHF is a central compound in folate metabolism.  It can function as a substrate for the conversion of dUMP to dTMP for subsequent DNA synthesis. It can also be converted to 5’-methyltetrahydrofolate (5-MTHF) by MTHFR with vitamin B2 as a cofactor.  5-MTHF is a substrate for the methionine synthase (MS) reaction that generates methionine from homocysteine.  Methionine is converted to SAM (S-adenosylmethionine), which is the substrate for DNA (and many other) methylation reactions, which produces SAH (S-adenosylhomocysteine), which can be converted to homocysteine. The conversion of homocysteine to cysteine involved two steps catalyzed by CBS (cystathionine-beta-synthase) and CL (cystathionine lyase). Both enzymes require vitamin B6 as cofactor. Cysteine can be incorporated into protein or further converted to glutathione, an anti-oxidant molecule in cells. Since folate metabolism is characterized by cellular reactions involving methyl-group transfer, it is also referred to as methyl-group metabolism or one carbon metabolism (Fig. 2).

MTHFR and its polymorphisms

MTHFR catalyze the conversion of 5’10-MTHF to 5’-MTHF (Fig. 2). The human MTHFR gene spans about 21 Kb at the short arm (position 36.3) of chromosome 1. The mature mRNA, joined from 11 exons, encode a 77 kDa MRHFR protein, which function as a dimer.

Among the 485 SNPs recorded in the NCBI SNP database, at least 40 mutations in the MTHFR gene have been identified in people with homocystinuria, an inherited disorder that affects the metabolism of methionine. Most of these mutations change single amino acids in the enzyme. These changes impair the function of the enzyme, and some cause the enzyme to be turned off (inactivated). Other mutations lead to the production of an abnormally small, nonfunctional version of the enzyme (Leclerc et al, 2000). The most common and well characterized mutations are the 677C>T (rs1801133) and 1298 A>C (rs1801131) polymorphisms.

The MTHFR 677C>T mutation result in an alanine to valine substitution at codon 222 (Ala222Val). The MTHFR 1298 A>C mutation leads to a glutamic acid to alanine substitution at codon 429 (Glu429Ala).  The 677T allele encodes a thermoliable enzyme with a reduced specific enzyme activity (25% of the wild type) at normal temperature.  The 1298C allele encodes a thermostable enzyme with a less reduced MTHFR activity (61% of the wild type).

The MTHFR 677C>T polymorphism was first identified in 1995 (Frosst et al, 1995). It represents the most common and the best studied MTHFR polymorphism. The frequency of the minor T allele varies from 13-57% in different populations.  The frequency of the homozygous minor allele genotype 677TT varies from 2-32% world wise (Table 3). In the United States, the frequencies of the homozygous minor allele genotype 677TT are different among ethnic groups, higher in Hispanics (18%) and Caucasians (11%) while lower in Asians (3%) and Africans (1%). Within each ethnic group, the frequency of genotypes also varies greatly.  For example, the 677TT genotype occurs much less in Northern European countries (about 4-8% in Finland, Russian, Norway and the Netherlands) while much higher in Southern Europe (about 12-27% in France, Greek and Italy).  In contract, the 677TT genotype occurs much more frequent in Northern Chinese Han population (20% T allele) when compared to Southern Chinese Han (8%) or Asian population living in the US (3% T allele). The dramatic frequency difference reflects the effect of dietary folate and its selection pressure on successful pregnancy and pre-natal survival (Wilcken et al, 2003).

Table 3. Percentage of alleles and genotypes of the MTHFR 677C>T polymorphisms among different ethnic groups and geographic areas.

The MTHFR 1298A>C polymorphism was first identified in 1998 (van der Pu et al, 1998). The frequency of the minor C allele varies from 17 to 42% and the frequency of the homozygous minor allele genotype 1298CC varies from 2-12% world wise (Table 4). The homologous minor allele genotype 1298CC is more common in Caucasians and relatively less common in other ethnic groups (2-4%).

Table 4. Percentage of alleles and genotypes of the MTHFR 1298A>C polymorphisms.

Both minor alleles predispose the homozygote carriers to an increases risk for folate deficient related diseases.  In addition, functional interaction between the 677C>T and 1298 A>C polymorphisms result in an additive effect on MTHFR enzyme activity.  People carrying the heterozygote of both variants (MTHFR 677CT/1298AC genotype, also called compound heterozygote) have the enzyme activity reduced to 48% of the wide type.

The MTHFR 677C>T and MTHFR 1298 A>C polymorphisms are in a linkage disequilibrium, meaning their genotype distribution is not random but depend up on each other (Table 5). In all nine possible genotype combinations between the 677 and 1298 sites, six genotypes that have two or more combined major alleles (677CC/1298AA, 677CC/1298AC, 677CC/1298CC, 677CT/1298AA, 677CT/1298AC and 677TT/1298AA) account for 99.9% of the population. The two genotypes that have 3 combined minor alleles at both sites, the 677TT/1298AC genotype and the 677CT/1298CC genotype have a prevalence of 0.05% each. The compound homozygous minor allele genotype 677TT/1298CC has never been detected in human population. The rare or non-exist genotypes have been speculated to be partially responsible for many late term miscarriages in pregnant women.

Table 5. Percent of genotypes showing the distribution of MTHFR 677C>T and 1998 A>C polymorphisms and their interaction in a study involving 10,034 Norwegian subjects. The three genotypes (677CC/1298CC, 677CT/1298AC and 677TT/1298AA) that are associated with increased homocysteine level are color shaded. Together, these three genotypes account for 38.6% of the study population (Modified from Ulvik et al, 2007).

MTHFR functions as a dimer. Each monomer is composed of a catalytic domain that binds folate and the vitamin B2 cofactor, and a regulatory domain that binds S-adenosylmethionine. Upon heat treatment and enzyme dilution, a 677T containing dimer dissociates and loses its enzymatic activity.  But high intracellular concentration, folate appears to be able to hold the mutant MTHFR protein in the appropriate and fully functional 3D structure, thus stabilizing the thermoliable form and counteracting the reduction in enzyme activity (Ulvik et al, 2007). Heat does not affect the activity of 1298C containing dimer, but low folate concentration does.  Therefore, high cellular folate concentration compensate the lost of specific activity of the minor allele carriers.

MTHFR polymorphisms and hyperhomocysteinemia

Dysfunction of the folate metabolism pathways due to insufficient dietary folate intake, vitamin B deficiency, certain medical conditions and genetic variations that impairs the activity of enzymes involved in these processes will lead to the reduced conversion of homocysteine to methionine.  Since methionine, an amino acid contained in almost all proteins, can also come from diet and other metabolic pathways, the net result will be the accumulation of homocysteine, which is not only a biomarker of folate metabolism dysfunction, but also an active biohazard molecule itself. Excess homocysteine can covalently link to the cysteine (via a process called S-homocysteinylation) or the lysine (via a process called N-homocysteinylation) residuals of a protein, permanently disrupt cysteine disulfide bonds and modify the function of proteins. The accumulation of homocysteine to 15 mmol/L or more in circulating blood is considered hyperhomocysteinemia, a condition associates with increased risk for many disorders, including vascular and neurodegenerative diseases, auto immune disorders, birth defects, diabetes, renal disease, osteoporosis, neuropsychiatric disorders and cancers (Brustolin et al, 2010). It has been speculated that the pro-atherosclerosis effect of hyperhomocysteinemia was due to the homocysteinylation of fibrinogen and other coagulation factors. Homocysteinylation also disrupts the native structure of the main structural components of the artery collagen, elastin and proteoglycans, leading to increased risk for cardiovascular diseases (Karolczak & Olas, 2009). Hyperhomocysteinemia is classified as moderate (15–30mmol/L), intermediate (31–100mmol/L) or severe (>100mmol/L) (Brustolin et al, 2010).

In a large-scale epidemiological study involving 10,034 middle-aged (50-64 years old) Norwegian subjects (Ulvik et al, 2007), lowest serum folate and highest homocysteine levels was found for the 677TT/1298AA genotype. Among all the six most common genotype combinations between the 677 and 1298 sites (Table 6), the association with homocysteine accumulation (hence the risk for hyperhomocysteinemia) ranked from the strongest to the least in the following order: 677TT/1298AA > 677CT/1298AC > 677CT/1298AA = 677CC/1298CC > 677CC/1298AC > 677CC/1298AA (wild type). Therefore, in this particular population, only 14.8 percent of people (the wild type 677CC/1298AA in Table 6) had a normal MTHFR activity and the rest 85.2% were predisposed for increased risks for hyperhomocysteinemia.

MTHFR polymorphisms and Cancer

Epidemiological studies show that the MTHFR variants modulate risks for cancer in a cancer-type and folate intake dependent manner.  For example, the minor alleles 677T and 1298C decreases the risk for colorectal cancer and acute lymphocytic leukemia while increases the risk for gastric cancer, bladder cancer, cervical cancer, endometrial cancer, and esophageal squamous cell carcinoma (Promthet et al, 2010; Toffoli & De Mattia, 2008 and the references therein). At sufficient folate intake, 677C>T does not correlate with breast cancer.  However, when folate intake is low, the 677TT genotypes have a much increased risk for breast cancer (Gao et al, 2009; Alshatwi, 2010).

It is hypothesized that the mechanisms for cancer association are related to the roles MTHFR play in maintaining the balance between DNA methylation and DNA synthesis (Figure 2). In the situation of reduced MTHFR activity, the synthesis of SAM is limited, leading to decreased DNA methylation and increased DNA synthesis.  For the minor allele carriers, the reduced MTHFR activity combined with high methionine diet leads to increased SAM and increased DNA methylation.  Both DNA hypomethylation (decreased DNA methylation) and hypermethylation (increased DNA methylation) could lead to cancer though altered gene transcription activation. Increased DNA synthesis has been suspected to lead to uracil mis-incorporation into DNA, interfering with DNA damage repair and leading to genetic instability, ultimately leading to cancer development.

MTHFR polymorphisms and miscarriages

Low maternal folate serum levels and hyperhomocysteinemia has been associated with recurrent spontaneous abortions (SA) or miscarriages. Many association studies also reported a positive correlation between MTHFR 677 C>T and 1298 A>C polymorphisms vs. risks for SA. In a case-control study within a perinatal cohort of women, the MTHFR 677TT genotype carriers showed a 5-fold increased risk of SA and the MTHFR 1298AC showed a 5.5-fold increased risk for SA (Rodríguez-Guillén Mdel et al, 2009).  In another case-control study, paternal (father’s) MTHFR 677T allele carriers had a 2.3-fold increased risk for recurrent pregnancy loss of the infant (Govindaiah et al, 2009). In a study of MTHFR C677T and A1298C polymorphisms in DNA samples from SA embryos (fetal death between sixth and twentieth week after conception) and adult controls, an astonished 14.2-fold increased risk was observed for embryos carrying one or more 677T and 1298C alleles vs. the wild types (Zetterberg et al, 2002). All these data point to the important roles of parental and offspring MTHFR genotypes on fetal survival, which is also reflected in the rare frequency of combined 677CT/1298CC, 677TT/1298AC and non-exist of the 677TT/1298CC genotypes in human population. Fortunately, many of the SAs can be avoided by folic acid supplementation during pregnancy.

MTHFR polymorphisms and other disease 

Both 677TT and 1298CC genotypes are associated with increased risks for cardiovascular dysfunctions (Klerk et al, 2002; Huang et al, 2011), hypertension (Huang et al, 2011). The 677 genotype, but not the 1298CC genotype, is also associated with thrombosis (Den Heijer et al, 2005), neuronal tube defects (Botto & Yang, 2000), oral cleft (Mills et al, 1999), Down’s syndrome (Hobbs et al, 2000) and psychiatric disorders (Gilbody et al, 2007). It is also observed that the A1298CC genotype, but not the C677T genotype, is associated with increased risk for male infertility (Shen et al, 2011; Eloualid et al, 2012).

MTHFR polymorphisms and dietary response

Folate, methionine, and alcohol intake are the three dietary factors that show the most interaction with MTHFR polymorphism.

As mentioned in most of the disease associations, low folate exacerbate the adverse effects of the minor alleles and high folate compensate the reduced activity of the enzymes. In the study involving 10,034 Norwegian subjects reported by Ulvik et al (2007), the interaction of blood homocysteine levels and serum folate levels among different genotypes of MTHFR 677C>T and 1298A>C were analyzed.  Serum folate was used as an indicator dietary folate intake in this study. The results showed that among different genotypes involving one or two minor alleles, homocysteine levels were significantly greater in the minor allele carriers with lower serum folate. However, at the highest serum folate quintile, the homocysteine levels were similar among all genotypes.  Many recent studies on the interaction between dietary folate intake and MTHFR polymorphism with regard to cancer prevention have similar conclusions.  For example, at sufficient folate intake, MTHFR 677 genotype did not correlate with breast cancer. But when folate intake was low, the 677TT genotypes had a much increased risk for breast cancer (Alshatwi, 2010). In another study involving 257 gastric cancer cases, MTHFR 677TT carriers with low folate and vitamin B12 intakes had the lowest survival rate in 3 years. Meanwhile, MTHFR 677TT carriers with high intakes of folate and vitamin B12 before diagnosis dramatically decreased the risk for gastric cancer mortality (86% and 77% by folate and B12 respectively) (Galván-Portillo et al, 2009). Therefore, sufficient folate intake is the first line of defense against the associated diseases. Along with folate, other vitamin B family members, especially B2, B6 and B12 that are directly involved in folate metabolism, need to meet sufficient recommended daily values (Curtin et al, 2004).

Methionine is essential for protein synthesis, methylation of DNA and polyamine synthesis. Restriction of methionine is becoming appreciated as an important strategy in cancer growth control. Several studies have reported that many malignant cell lines from different cancers (breast, bladder, colon, glioma, kidney, melanoma, prostate and others) are methionine dependent. Furthermore, methionine dependence has been reported in fresh patient tumors derived from multiple tumor sites and grown in primary cultures. Methionine dependence in cancer may be due to one or a combination of deletions, polymorphisms or alterations in expression of genes in the methionine de novo and salvage pathways. Cancer cells with these defects are unable to regenerate methionine via these pathways. Defects in the metabolism of folate may also contribute to the methionine dependence phenotype in cancer. Cell culture studies demonstrated that culture media deficient in methionine can selectively kill the methionine-dependent cancer cells while maintain the growth of the normal cells in co-culture. Animal studies on methionine restricted diet have reported inhibition of cancer growth and extension of a healthy life-span. In humans, vegan diets, which are typically low in methionine, is a useful nutritional strategy in cancer growth control (Cavuoto & Fenech, 2012). Through experimentation, researchers are starting to see that restricting the content of methionine in the diet of mammals - while leaving the calorie count unchanged - has many of the same results as restricting overall calories (Caro et al, 2009). In terms of the interaction with MTHFR polymorphism, the reduced enzymatic activity of the 677T and 1298C alleles respond to dietary methionine intake less efficiently. For example, post-methionine-load homocysteine concentrations in the 677TT carriers were significantly higher than that of the wild type carriers (Candito et al, 1999). Therefore, for the minor allele carriers, methionine restriction is the second line of defense against cancer and hyperhomocysteinemia associated diseases.

Alcohol is another strong factor interacting with folate metabolism. It has been recognized for about 30 years that folate deficiency is a common clinical sign of chronic alcohol abuse. Studies in human, monkey and pig indicate that chronic alcohol ingestion reduces folate absorption, increases folate excretion, and decreases the hepatic uptake of circulating folate due to reduced expressions of both glutamate carboxypeptidase and reduced folate carrier (Kim, 2007; Halsted et al, 2002). Moderate alcohol intake is beneficial for certain MTHFR genotypes.  Men with the 677 TT/1298 AA genotype and alcohol intake of <20 g/day experienced a 70% reduced risk for colon cancer compared with 677 CC/1298AA and no alcohol intake individuals (Curtin et al, 2004). However, the reduced risk of colorectal cancer of 677TT genotype was abolished among those who drank heavily (Kim, 2007). Considering the overall impact of alcohol intake on folate metabolism and the reduced activity of MTHFR minor alleles, alcohol restriction is recommended for all genotypes.

MTHFR polymorphisms and medicine response

MTHFR 677C>T and MTHFR 1298 A>C influence the side effects of the chemotherapeutic drugs fluoropyrimidines and antifolates due to their differential responses from that of the wild type genotypes.

5-fluorouracil (5-FU) is a member of the fluoropyrimidines that has been widely used in to treat solid malignancies such as colorectal carcinoma. In one in vitro study in human colon cancer and breast cancer cells, the 667 T allele rendered the carriers more sensitive to 5-FU (Sohn et al, 2004). In another study involving 19 human cancer cell lines, while 677T allele did not show effect on 5-FU sensitivity, the 1298C allele did (Etienne et al, 2004). In addition, the 677T allele also associated with a higher risk to develop severe treatment related side effects such as vomiting and nausea (Lu et al, 2004).

Raltitrexed is a folic acid mimic chemotherapy drugs that inhibit dihydrofolate reductase, glycinamide ribonucleotide formyltransferase and thymidine synthase in the folate metabolism pathways.  On patients had received the drug in combination with irinotecan (a DNA topoisomerase I inhibitor) to treat solid tumors, the 677TT genotype is associated with a significantly reduced toxicity. It was hypothesized that increased availability of 5, 10-MTHF, as a result of impaired MTHFR activity, could compete with raltitrexed, leading to a diminished cytotoxicity of the raltitrexed (Toffoli & De Mattia, 2008).

Methotrexate (MTX) is an anti-neoplastic and anti-folate compound that has been used for the treatment of a number of solid tumors and hematologic malignancies.  Due to its potential anti-inflammatory and autoimmune effects, this compound is one of the most commonly employed drugs in rheumatic and other inflammatory conditions. In many studies, not confirmed by some others, the 677T allele was associated with an increased toxicity after treatment with MTX.  The effect of A1298C in response to MTX therapy has been less reported, and the data obtained so far are rather controversial (Toffoli & De Mattia, 2008).

Conclusions

MTHFR 677C>T and MTHFR 1298 A>C are two common polymorphisms that are associated with increased risks for many diseases due to the impaired enzymatic activity of the protein encoded by the minor alleles. Folate intake (along with vitamin B supplement), methionine restriction and alcohol control are the first three dietary approaches to prevent the development of associated diseases. Scientists at GB Lifesciences are working diligently to develop dietary regimens that feature folate rich and methionine depleted natural foods.

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