Complete deficiency of methylenetetrahydrofolate reductase in mice is associated with impaired retinal function and variable mortality, hematological profiles, and reproductive outcomes.

Severe deficiency of methylenetetrahydrofolate reductase (MTHFR) with homocystinuria can result in early demise or later-onset neurological impairment, including developmental delay, motor dysfunction, and seizures. We previously characterized BALB/c Mthfr (-/-)mice as a model for this disorder and have recently backcrossed the disrupted allele onto the C57Bl/6 background to examine the variable phenotypes in MTHFR deficiency. Compared with BALB/c Mthfr (-/-)mice, C57Bl/6 Mthfr (-/-)mice have enhanced survival rates (81% vs 26.5%). Four-day-old BALB/c mutant pups had lower body, brain, and spleen weights relative to their wild-type counterparts compared with C57Bl/6 mutants. Pregnant BALB/c Mthfr (+/-)mice had increased resorptions and embryonic delays compared with wild-type littermates, whereas these outcomes in C57Bl/6 c Mthfr (+/-)mice were similar to those of wild-type C57Bl/6 mice. BALB/c-mutant pups had altered hematological profiles (higher hematocrit, hemoglobin, and white blood cell counts, with lower platelet counts) compared with C57Bl/6 mutants. Mutants of both strains had similar degrees of hepatic steatosis, hepatic activity of betaine:homocysteine methyltransferase, and altered cerebellar histology. Electroretinograms (ERG) in C57Bl/6 Mthfr (-/-)mice revealed decreased amplitude of scotopic and photopic waves in 6-week-old mice, with normalized ERGs at 13 weeks. Plasma homocysteine was modestly higher in C57Bl/6 compared with BALB/c mice. Our results emphasize the variable presentation of MTHFR deficiency in different genetic backgrounds and suggest that plasma homocysteine is not a predictor of severity. In addition, our novel findings of decreased spleen weights, thrombocytopenia, and impaired retinal function warrant investigation in patients with severe MTHFR deficiency or other forms of homocystinuria.

Strain-specific defects in testicular development and sperm epigenetic patterns in 5,10-methylenetetrahydrofolate reductase-deficient mice.

Methylenetetrahydrofolate reductase (MTHFR) is a crucial folate pathway enzyme that contributes to the maintenance of cellular pools of S-adenosylmethionine, the universal methyl donor for several reactions including DNA methylation. Whereas Mthfr(-/-) BALB/c mice show growth retardation, developmental delay, and spermatogenic defects and infertility, C57BL/6 mice appear to have a less severe phenotype. In the present study, we investigated the effects of MTHFR deficiency on early germ cell development in both strains and assessed whether MTHFR deficiency results in DNA methylation abnormalities in sperm. The reproductive phenotype associated with MTHFR deficiency differed strikingly between the two strains, with BALB/c mice showing an early postnatal loss of germ cell number and proliferation that was not evident in the C57BL/6 mice. As a result, the BALB/c MTHFR-deficient mice were infertile, whereas the C57BL/6 mice had decreased sperm numbers and altered testicular histology but showed normal fertility. Imprinted genes and sequences that normally become methylated during spermatogenesis were unaffected by MTHFR deficiency in C57BL/6 mice. In contrast, a genome-wide restriction landmark genomic scanning approach revealed a number of sites of hypo- and hypermethylation in the sperm of this mouse strain. These results showing strain-specific defects in MTHFR-deficient mice may help to explain population differences in infertility among men with common MTHFR polymorphisms.

Methotrexate-induced apoptosis is enhanced by altered expression of methylenetetrahydrofolate reductase.

Folates are essential for DNA synthesis and methylation reactions. The antifolate methotrexate (MTX) is a widely used chemotherapeutic drug which inhibits DNA synthesis and induces apoptosis. Changes in activity of a critical folate-metabolizing enzyme, methylenetetrahydrofolate reductase (MTHFR), might alter the chemosensitivity to MTX, as the MTHFR substrate is required for nucleotide synthesis and its product is used in homocysteine remethylation to methionine. Mild MTHFR deficiency is common in many populations due to a polymorphism at bp 677. We previously showed that altered expression of MTHFR enhanced MTX-induced myelosuppression in mice. To determine the cause of the impaired hematopoietic profile in mice with decreased or increased MTHFR expression, we evaluated MTX-induced apoptosis in the major hemolytic organ, spleen, using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining and caspase-3/7 activity assays, in MTHFR-deficient mice and in MTHFR-overexpressing mice after MTX administration. Decreased or increased expression of MTHFR in mice significantly increased TUNEL-positive cells and caspase-3/7 activities in MTX-treated spleen, compared with that of wild-type littermates. Plasma homocysteine levels correlated with apoptotic index in MTX-treated MTHFR-deficient mice and dUTP/dTTP ratios correlated with apoptotic index in MTX-treated MTHFR-overexpressing mice. The increased apoptosis may therefore relate to hyperhomocysteinemia and deoxyribonucleotide pool imbalances, respectively. Our results suggest that MTHFR underexpression and overexpression enhances MTX-induced apoptosis and myelosuppression, and that genotyping for the MTHFR polymorphism may have therapeutic implications.

Altered expression of methylenetetrahydrofolate reductase modifies response to methotrexate in mice.

OBJECTIVE: Folates provide one-carbon units for nucleotide synthesis and methylation reactions. A common polymorphism (677C-->T) in methylenetetrahydrofolate reductase (MTHFR) encodes an enzyme with reduced activity. Response to the antifolate methotrexate (MTX) may be modified in 677TT individuals because MTHFR converts nonmethylated folates, used for thymidine and purine synthesis, to 5-methyltetrahydrofolate, used in homocysteine remethylation to methionine. To study potential interactions between MTHFR activity and MTX, we examined the impact of decreased and increased MTHFR expression on MTX response in mice. METHODS: Mthfr-deficient (Mthfr and Mthfr) and wild-type (Mthfr) mice were injected with MTX or saline and assessed for hematological parameters (hematocrit, hemoglobin, red, and white blood cell numbers), plasma homocysteine, nephrotoxicity, hepatotoxicity, and splenic 2'-deoxyuridine 5'-triphosphate/2'-deoxythymidine 5'-triphosphate ratios. MTHFR-overexpressing transgenic mice (MTHFR-Tg) were generated, metabolites and folate distributions were measured, and response to MTX was assessed. RESULTS: MTX-treated Mthfr and Mthfr mice displayed hyperhomocysteinemia and decreased hematocrit, hemoglobin, and red blood cell numbers compared with wild-type animals. Mthfr mice also showed increased nephrotoxicity and hepatotoxicity. MTHFR-Tg mice were generated and confirmed to have increased levels of MTHFR with altered distributions of folate and thiols in a tissue-specific manner. After MTX treatment, MTHFR-Tg mice exhibited the same decreases in hematological parameters as Mthfr-deficient mice, and significantly decreased thymidine synthesis (higher 2'-deoxyuridine 5'-triphosphate/2'-deoxythymidine 5'-triphosphate ratios) compared with wild-type mice, but they were protected from MTX-induced hyperhomocysteinemia. CONCLUSION: Underexpression and overexpression of Mthfr/MTHFR increase MTX-induced myelosuppression but have distinct effects on plasma homocysteine and nephrotoxicity. Pharmacogenetic analysis of polymorphisms in folate-dependent enzymes may be useful in optimization of MTX therapy.

Methionine synthase reductase deficiency results in adverse reproductive outcomes and congenital heart defects in mice.

Low dietary folate and polymorphisms in genes of folate metabolism can influence risk for pregnancy complications and birth defects. Methionine synthase reductase (MTRR) is required for activation of methionine synthase, a folate- and vitamin B(12)-dependent enzyme. A polymorphism in MTRR (p.I22M), present in the homozygous state in 25% of many populations, may increase risk for neural tube defects. To examine the impact of MTRR deficiency on early development and congenital heart defects, we used mice harboring a gene-trapped (gt) allele in Mtrr. Female mice (Mtrr(+/+), Mtrr(+/gt), and Mtrr(gt/gt)) were mated with male Mtrr(+/g) mice. Reproductive outcomes and cardiac phenotype (presence of defects and myocardial thickness) were assessed at E14.5. Mtrr-deficient mothers had more resorptions and more delayed embryos per litter (resorptions per litter: 0.29+/-0.13; 1.21+/-0.41; 1.87+/-0.38 and delayed embryos per litter: 0.07+/-0.07; 0.14+/-0.14; 0.60+/-0.24 in Mtrr(+/+), Mtrr(+/gt), and Mtrr(gt/gt) mothers respectively). Placentae of Mtrr(gt/gt) mothers were smaller and their embryos were smaller, with myocardial hypoplasia and a higher incidence of ventricular septal defects (VSD) per litter (0; 0.57+/-0.30; 1.57+/-0.67 in Mtrr(+/+), Mtrr(+/gt), and Mtrr(gt/gt) groups respectively). Embryonic Mtrr(gt/gt) genotype was associated with reduced embryonic length, reduced embryonic and placental weight, and higher incidence of VSD, but did not affect myocardial thickness or embryonic delay. We conclude that Mtrr deficiency adversely impacts reproductive outcomes and cardiac development in mice. These findings may have implications for nutritional prevention of heart defects, particularly in women with the common MTRR polymorphism.

Strain differences in mice highlight the role of DNA damage in neoplasia induced by low dietary folate.

In earlier work, we showed that low dietary folate induced intestinal tumors in BALB/c mice. In this study, our goal was to examine the effect of the same diets on a strain that is more resistant to tumorigenesis (C57Bl/6). We also questioned whether supplementation of the folate-deficient diet (FD) with betaine, an alternate methyl donor, would influence tumor formation. C57Bl/6 mice were fed the same diets [control diet (CD) with 2 mg folate/kg diet and FD with 0.3 mg folate/kg diet] as those in our previous study for 1 y, but they did not develop tumors. We also fed BALB/c mice the FD or FD supplemented with betaine for 1 y, but there was no change in tumor incidence. To determine the relative contributions of DNA damage and altered methylation patterns, we measured intestinal dUTP:dTTP ratios, phosphorylated histone H2AX (p-H2AX) staining, and global DNA methylation in both strains. Only BALB/c mice showed changes due to diet in dUTP:dTTP (from 2.19 +/- 0.20 in CD to 2.77 +/- 0.18 in FD; P = 0.05) and in p-H2AX staining (from 14.10 +/- 3.59% in CD to 22.40 +/- 2.65% in FD; P = 0.054). In BALB/c mice only, FD tended to have less (P = 0.06) global DNA methylation than CD. Although the FD increased plasma homocysteine and the betaine-supplemented FD lowered plasma homocysteine, the latter diet did not reduce tumor incidence. We conclude that plasma homocysteine is not likely to be associated with tumorigenesis in our model. However, DNA damage plays a critical role in initiating tumorigenesis when dietary folate is low and methylation changes may also be contributory.

Genetic and nutritional deficiencies in folate metabolism influence tumorigenicity in Apcmin/+ mice.

Epidemiological studies indicate that adequate dietary folate is protective against colon cancer, although mechanisms remain largely elusive. We investigated the effects of genetic disruptions of folate transport and metabolism and of dietary folate deficiency in a mouse model of colon cancer, the Apc(min/+) mouse. Apc(min/+) mice with heterozygous knockout of the gene for reduced folate carrier 1 (Rfc1(+/-)) developed significantly fewer adenomas compared to Rfc1(+/+)Apc(min/+) mice [30.3+/-4.6 vs. 60.4+/-9.4 on a control diet (CD) and 42.6+/-4.4 vs. 55.8+/-7.6 on a folate-deficient diet, respectively]. Rfc1(+/-)Apc(min/+) mice also carried a lower tumor load, an indicator of tumor size as well as of tumor number. In contrast, there were no differences in adenoma formation between Apc(min/+) mice carrying a knockout allele for methionine synthase (Mtr(+/-)), an enzyme that catalyzes folate-dependent homocysteine remethylation, and Mtr(+/+)Apc(min/+) mice. However, in both Mtr groups of mice, dietary folate deficiency significantly increased adenoma number (from 32.3+/-3.8 on a CD to 48.1+/-4.2 on a folate-deficient diet), increased plasma homocysteine, decreased global DNA methylation in preneoplastic intestines and increased apoptosis in tissues. There were no genotype-associated differences in these parameters in the Rfc1 group, suggesting that the protection conferred by Rfc1 deficiency is carried out through a different mechanism. In conclusion, genetic and nutritional disturbances in folate metabolism can have distinct influences on tumorigenesis in Apc(min/+) mice; altered levels of homocysteine, global DNA methylation and apoptosis may contribute mechanistically to dietary influence.