The Mthfr Gene Product MTHFR

The MTHFR gene product consists of 656 amino acid residues with a predicted molecular mass of 74.6 kDa (3-6) and is termed 5,10-methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20). The amino acid sequence is highly conserved, showing 90% homology with the mouse polypeptide (4). MTHFR consists of two identical subunits of approx 70 kDa (7) and represents a key enzyme in the folate cycle. It reduces 5,10-

methylenetetrahydrofolate to 5-methyltetrahydrofolate, thus catalyzing the only reaction in the cell that ultimately generates 5-methyltetrahydrofolate, the biologically active folate derivative. Interestingly, knockout mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition (8).

Common Polymorphisms in the MTHFR Gene

Three polymorphisms exist in MTHFR that are located at nucleotide position 677 (MTHFR 677C^T) (5), position 1298 (MTHFR 1298A^C) (911), and position 1317 (MTHFR 1317T^C) (10). MTHFR 677C^T occurs in exon 4 at the folate-binding site of 5,10-methylenetetrahydrofolate reductase and changes an alanine into a valine residue (A222V). MTHFR 1298A^C is located in exon 7 within the presumptive regulatory domain changing a glutamic acid into an alanine residue (E429A). The MTHFR 1317T^C is a silent mutation that is also located in exon 7. MTHFR 677C^T, MTHFR 1298A^C, as well as compound heterozygosity for 1298A^C and 677C^T are associated with a reduced enzyme activity of 45%, 68%, and 42%, respectively (12).

Allele Frequencies of MTHFR Polymorphisms

The MTHFR 677C^T polymorphism has a relatively high frequency throughout the world. The 677TT genotype is present in about 12% of the general population and shows a heterogeneous distribution among ethnic groups with an allele frequency ranging from 0.045 in Sri Lanka to 0.3 in Caucasians and Americans (13). MTHFR 1298A^C shows an allele frequency of 0.3 in Canadians, Austrians, and Dutch individuals (10,11,14). The allele frequency of MTHFR 1317T^C is 0.05 in Canadian individuals (10) and 0.059 in a Turkish population (15) and was quite common in a small population of Africans (the allele frequency among nine healthy Africans was 0.39) (10). In the study of Meisel et al., none of 1000 healthy Caucasians and only 1 of 1000 coronary artery disease patients tested positive for MTHFR 1317T^C (16).

Metabolic Effects of MTHFR Polymorphisms

The MTHFR 677C^T mutation is associated with decreased formation of 5-methyltetrahydrofolate and an accumulation of formylated tetrahydrofolate polyglutamates in erythrocytes (17). Furthermore, MTHFR 677TT is frequently associated with significantly higher total homocysteine plasma levels as compared to heterozygotes or people without mutation. This effect is observed in all countries and regions of Europe, indicating a steady impact of the mutation on total homocysteine plasma levels (18).

The MTHFR 1298A^C mutation alone does not influence folate status or total homocysteine concentrations. By contrast, compound heterozygosity for the 677T and the 1298C alleles can be associated with decreased folate plasma concentrations (11,14) and higher total homocysteine concentrations (11).

Disease Associations of MTHFR Polymorphisms

Some of the clinical implications of MTHFR 677C^T are summarized in Chapter 2. The implications of MTHFR 677C^T and MTHFR 1298A^C in cardiovascular disease, cerebrovascular disease, venous thrombosis, longevity, neural tube defects, pregnancy, congenital abnormalities, preclampsia, diabetes, cancer, psychiatry, and renal failure are reviewed in refs. 19 and 20.

More recently, Dekou et al. reported that the frequency of the MTHFR 677T allele of the coronary heart disease high-risk town of Dewsbury was significantly higher than in the coronary heart disease low-risk town of Maidstone and was associated with increased total homocysteine plasma concentrations (21). This effect was seen in men but not in women and was not observed for MTHFR 1298A^C. Furthermore, a higher susceptibility for malignant lymphoma has been observed in individuals with the combined MTHFR 677CC/1298AA genotype, as well as those with the MTR 2756GG genotype (22). Moreover, the MTHFR 677C^T mutation, smoking, and folate status were strong interactive determinants of high-risk adenomas of the colorectum (23). In this study, the risk was particularly high in smokers with low folate and the MTHFR 677CT or the MTHFR 677TT genotype, and in smokers with high folate and the MTHFR 677CC genotype. This risk pattern was also observed for colorectal hyperplastic polyps. Together, these data demonstrate a strong gene-nutrition interaction involving the MTHFR 677C^T polymorphism.

Most recently, Meisel et al. observed no association of the MTHFR 677/ 1298 genotypes with coronary artery disease or total homocysteine plasma levels (16). In another study, the MTHFR 1298C allele was associated with early-onset coronary artery disease, even when total homocysteine levels were not elevated (24).

The MTHFR 677TT and the MTHFR 677CT/1298AC genotype can modulate the efficacy of oral or intravenous folic or folinic acid therapy (25-27). Furthermore, the effect of drugs that interfere with the folate status is suggested to be influenced by the MTHFR 677C^T polymorphism (20).

The MTHFR 1317T^C mutation was not associated with either deep vein thrombosis (15) or with coronary artery disease (16).

Rare Mutations in the MTHFR Gene

Rare mutations exist in MTHFR that are associated with severe enzyme deficiency (enzyme activity less than 20% in fibroblast cultures; an overview is provided in ref. 28). The first mutations in severely MTHFR-defi-cient patients were identified in 1993 by Goyette and coworkers (29). During the following years, evidence accumulated that severe MTHFR deficiency results from heterogeneous mutations associated either with extremely low enzyme activity and early onset of symptoms, or with moderately reduced enzyme activity and later onset of symptoms (30-32). To date, approx 25 different mutations have been reported that may, at least partly, explain the heterogeneous clinical phenotypes (30-34).

THE METHIONINE SYNTHASE GENE (MTR)

The human methionine synthase gene (MTR) is located on chromosome 1q43 (35,36). The entire coding region (GenBank accession number U71285) has a length of 3.795 bp, with a predicted molecular mass of 140 kDa (37,38). Two mRNAs of 7.5 kb and 10 kb are present in human tissues (38). Furthermore, a minor mRNA of 4.4 kb as well as other partially spliced larger mRNAs have been detected (35).

The MTR Gene Product Methionine Synthase

The MTR gene product consists of 1.265 amino acid residues (37,38) and is termed methionine synthase (MS, EC 2.1.1.13, alternative titles: 5-methyltetrahydrofolate-homocysteine S-methyltransferase, tetrahydro-pteroylglutamate methyltransferase). The amino acid sequence shows about 55% and 65% homology with the polypeptide of Escherichia coli and Caenorhabditis elegans, respectively (37,38).

Methionine synthase is a cytoplasmic enzyme that requires methyl-cobalamin for activity and catalyzes the remethylation of homocysteine to methionine. In this reaction, the methyl group of 5-methyltetrahydrofolate is transferred to the enzyme bond cob(I)alamin to generate methylcobalamin followed by the transfer of the methyl group to homocysteine to reform methionine. The enzyme consists of four separate regions referred to as the homocysteine-binding region (residues 2 - 353), the methyltetrahydrofolate-binding region (residues 354 - 649), a region responsible for binding the cobalamin prosthetic group (residues 650 - 896), and an S-adeno-sylmethionine-binding domain (residues 897 - 1227). Importantly, functional integrity of the latter region is required for reductive activation of methionine synthase by the enzyme methionine synthase reductase (EC 2.1.1.135) (36).

Common Polymorphisms in the MTR Gene

In MTR, a polymorphism exists that is located at nucleotide position 2756 (MTR 2756A^G) (35,37,38). MTR 2756A^G occurs at the C-terminal end of the a/p domain of the enzyme and changes an aspartic acid into a glycine residue (D919G). Because MTR 2756A^G is located in a helix between the cobalamin domain and the S-adenosylmethionine-binding domain, the glycine substitution could affect the secondary structure of the enzyme. However, the functional significance of this polymorphism will have to be examined in expression experiments to characterize its impact on the protein.

Allele Frequency of MTR Polymorphism

The MTR 2756A^G has an allele frequency of 0.15 (37) and 0.16 (39), showing differences among study populations (allele frequencies ranging between 0.18 and 0.38) (22,35,40,41).

Metabolic Effects of MTR Polymorphism

The mutation alters formyltetrahydropteroylglutamic acid (H4PteGlu) disposition of erythrocytes (42) in that the MTR 2756AG genotype is associated with more formyl-H4PteGlu, relative to 5-methyl-H4PteGlu, as compared to individuals with wild-type alleles. This relationship is not present in red blood cells of individuals with a neural tube defect (42).

The influence of MTR 2756A^G on total homocysteine plasma levels is a matter of debate. Harmon et al. reported an association with increased total homocysteine levels (43), which has not been confirmed by others (39,40,42,44-46). Furthermore, an association of MTR 2756A^G with low plasma total homocysteine levels has been observed (47,48).

Disease Associations of MTR Polymorphism

An association of MTR 2756A^G with severity of coronary artery disease and dose of lifetime smoking has been demonstrated (49). Furthermore, the MTR 2756AG genotype was associated with a longer event-free survival in coronary artery disease patients as compared to AA genotype patients (50). Other investigators found no association of MTR 2756A^G with birth defects (39,42,46), cerebrovascular and cardiovascular disease (44,51), and early-onset vascular thrombosis (45). A decrease of the odds ratio for neural tube defects has been described for patients with the GG genotype (41). Furthermore, a higher susceptibility for malignant lymphoma has been observed for individuals with the MTR 2756GG genotype (22).

Effect of Combined MTHFR and MTR Genotypes

Only few studies addressed the effect of the combined MTR/MTHFR genotypes on homocysteine-related disorders. A lack of association of the combined MTR 2756A^G and MTHFR 677C^T genotypes with neural tube defects (42) and hyperhomocysteinemia of patients with early-onset vascular thrombosis (45) has been described. The association of the combined genotypes with vascular disease was not clear in the study of Morita et al. (52). No combined effect of MTHFR 677C^T, MTR 2756A^G and of the cystathionine ^-synthase (CBS) polymorphism CBS 844ins68 on fasting or post-methionine-loading total homocysteine levels has been observed among vascular disease patients (48). These findings are in line with the results of the study of Harmon et al. (43), who did not detect an interaction of MTR 2756A^G and MTHFR 677C^T genotypes with respect to total homocysteine levels in healthy subjects.

The MTR 2756A^G polymorphism was equally distributed among healthy individuals as well as subjects with renal insufficiency with extremely high or with extremely low total homocysteine plasma concentrations (53). By contrast, the combination of MTR 2756AG and 2756GG with the MTHFR 677TT/1298AA and the MTHFR 677CT/1298AC genotypes was associated with extremely high total homocysteine plasma concentrations (53).

Rare Mutations in the MTR Gene

Two point mutations, three insertions, and two deletions have been identified among six patients with severe methionine synthase deficiency associated with a defective synthesis of methylcobalamin (referred to as the complementation G type of cobalamin deficiency, cblG type) (37,54-56). Two of these mutations were located in the vincinity of the cobalamin-bind-ing site (37,55). Another mutation is embedded in a sequence that makes direct contact with bond adenosylmethionine, probably disrupting activation of the enzyme (55).

The clinical symptoms of patients with severe methionine synthase deficiency include developmental delay, neurological deteriorations, mental retardation, and megaloblastic anemia, which often leads to diagnosis. Typical laboratory characteristics include hyperhomocysteinemia, homo-cystinuria, hypomethioninemia, and low methylcobalamin levels without methylmalonic aciduria.

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