Human Molecular Genetics, 2001, Vol. 10, No. 5 433-443
© 2001 Oxford University Press
Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition
1Departments of Biology, Human Genetics and Pediatrics, 2Department of Medicine, 6Department of Pathology, McGill University, Montreal, Quebec H3A 1B1, Canada, 3The Jackson Laboratory, Bar Harbor, ME 04609, USA, 4National Center for Toxicological Research, Division of Biochemical Toxicology, Jefferson, AR 72079, USA, 5Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada, 7Institute of Metabolic Disease, Dallas, TX 75226, USA, 8Jean Mayer USDA Human Nutrition Research Center on Ageing, Tufts University, Boston, MA 02111, USA and 9Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario L8S 4K1, Canada
Received 9 November 2000; Revised and Accepted 29 December 2000.
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ABSTRACT |
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Hyperhomocysteinemia, a risk factor for cardiovascular disease, is caused by nutritional and/or genetic disruptions in homocysteine metabolism. The most common genetic cause of hyperhomocysteinemia is the 677C
T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. This variant, with mild enzymatic deficiency, is associated with an increased risk for neural tube defects and pregnancy complications and with a decreased risk for colon cancer and leukemia. Although many studies have reported that this variant is also a risk factor for vascular disease, this area of investigation is still controversial. Severe MTHFR deficiency results in homocystinuria, an inborn error of metabolism with neurological and vascular complications. To investigate the in vivo pathogenetic mechanisms of MTHFR deficiency, we generated mice with a knockout of Mthfr. Plasma total homocysteine levels in heterozygous and homozygous knockout mice are 1.6- and 10-fold higher than those in wild-type littermates, respectively. Both heterozygous and homozygous knockouts have either significantly decreased S-adenosylmethionine levels or significantly increased S-adenosylhomocysteine levels, or both, with global DNA hypomethylation. The heterozygous knockout mice appear normal, whereas the homozygotes are smaller and show developmental retardation with cerebellar pathology. Abnormal lipid deposition in the proximal portion of the aorta was observed in older heterozygotes and homozygotes, alluding to an atherogenic effect of hyperhomocysteinemia in these mice. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Homocysteine is a sulfur-containing amino acid which is derived from methionine and can be remethylated back to methionine as part of the methionine cycle. Homocysteine can also be catabolized through the transsulfuration pathway and ultimately degraded. Hyperhomocysteinemia results from genetic or nutrient-related disturbances in homocysteine metabolism (1,2). Methylenetetrahydrofolate reductase (MTHFR) converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (5-methylTHF), a methyl donor for homocysteine remethylation to methionine. A common variant in MTHFR, 677C
T, is associated with mild hyperhomocysteinemia, particularly when folate status is low (3–5). Individuals homozygous for the mutation (10–15% of Caucasians) who maintain adequate folate levels have normal homocysteine levels, as folate may stabilize the enzyme and allow it to function normally (6). Other more deleterious but less common mutations in MTHFR have been described in patients with homocystinuria, an inborn error of metabolism (2,7–9). The most common feature in these patients is developmental delay; other problems include motor and gait abnormalities, thromboses, psychiatric disturbances, hypotonia and seizures. Several clinical trials for lowering homocysteine to reduce the risk for vascular disease are under way, yet the mechanism by which hyperhomocysteinemia results in vasculopathies and other disorders is unclear. Experiments in vitro and in vivo have demonstrated that homocysteine can lead to endothelial cell injury (10,11). Homocysteine has been demonstrated to induce proliferation of vascular smooth muscle cells in culture (12) and to promote prothrombotic effects through an increase in Factor V and Factor VII activity, or through a decrease in expression of thrombomodulin and activated protein C (13). Increased production of free radicals may also be involved in homocysteine-mediated damage (14). However, few studies have been performed in vivo due to the lack of a suitable animal model for hyperhomocysteinemia.
Most studies on the pathogenicity of homocysteine have focused on the direct effects of the amino acid. However, hyperhomocysteinemia may be an indirect marker for a disturbance in the methionine or methylation cycle. The latter disruption could contribute to the diverse clinical consequences of elevated homocysteine or of mild MTHFR deficiency. In this cycle, methionine is utilized for synthesis of S-adenosylmethionine (SAM), which is converted to S-adenosylhomocysteine (SAH) and homocysteine (15). SAM is the methyl donor for >100 different transmethylation reactions, including methylation of DNA and proteins, phospholipid synthesis and neurotransmitter synthesis (16). Alterations in DNA methylation patterns impact several critical cellular processes including gene expression, X-inactivation, carcinogenesis, aging and development (17,18). Mice deficient in a DNA methyltransferase (Dnmt1) do not survive gestation (19). Mutations in a different DNA methyltransferase (DNMT3B) result in the immunodeficiency, centromere instability and facial anomalies (ICF) syndrome (20,21), and Rett syndrome, a progressive neurodevelopmental disorder, is caused by mutations in the methyl-CpG-binding protein, MeCP2 (22).
Here we show that heterozygous and homozygous Mthfr knockout mice have elevated plasma homocysteine and disrupted SAM and SAH levels that are associated with global DNA hypomethylation in several tissues. Mice with a homozygous disruption of the Mthfr gene have reduced survival or delayed development and cerebellar abnormalities. Older heterozygous and homozygous knockout mice have abnormal lipid deposition in the proximal portion of the aorta. These results are consistent with the proposed role of MTHFR deficiency in vascular disease.
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RESULTS |
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Generation of mice with a targeted disruption of the Mthfr gene
To inactivate the mouse Mthfr gene in embryonic stem (ES) cells, we constructed an insertion type of targeting vector, in which exon 3 of the mouse Mthfr gene was interrupted by the neor gene (Fig. 1A). After transfection of the construct into ES cells, 5 of 150 doubly-resistant ES clones were identified by Southern blot analysis to have undergone the expected homologous recombination event (Fig. 1B). Three of the positive clones were injected into blastocysts and chimeric mice from one cell line successfully transmitted the modified Mthfr gene to the next generation. Mice with a heterozygous disruption of the gene were mated to generate homozygous knockout animals. Southern blotting and PCR were used to distinguish the three possible genotypes (Fig. 1C).
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Confirmation of gene disruption was performed by RT–PCR and enzymatic assays for MTHFR in several tissues. Two sets of primers were used to amplify Mthfr mRNA in brain (Fig. 2A). There was no detectable Mthfr mRNA in homozygous mutant mice. Mthfr mRNA was present in heterozygous mice, but at a reduced level compared with wild-type animals. For MTHFR enzymatic activity, only background levels of the assay were detected in homozygous mutants (Fig. 2B). The specific activity of MTHFR in heterozygotes was
60–70% of that in wild-type mice, suggesting the possibility of feedback upregulation of the enzyme.
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Phenotype of Mthfr-deficient mice
Both heterozygous and homozygous Mthfr-deficient mice were viable. Mating of heterozygous animals fed on standard mouse chow produced offspring with the expected 1:2:1 proportions of wild-type:heterozygous:homozygous mutant (n = 55:108:55) at 6 days of age. The ratios for male and female offspring were similar (n = 27:46:33 for males and n =28:62:22 for females).
2 tests showed no significant differences from expected Mendelian ratios for the entire group (P = 0.99) or for males and females separately (P = 0.28 and 0.38, respectively). These data suggest that there was no significant fetal loss of homozygous mutant mice during gestation. The heterozygotes grew normally and appeared healthy. Mortality of heterozygotes was similar to that of wild-type mice. Litter size resulting from matings of heterozygotes was similar to that resulting from matings of wild-type mice (data not shown). Homozygous knockout mice showed a variable phenotype with reduced survival (76.4% at 5 weeks of age). Mice who died in the first few weeks had motor and gait abnormalities or severe tremors. After the initial critical period of a few weeks, survival was relatively stable, suggesting that Mthfr may play an important role in early development.
Body weights of pups were measured from 1 to 5 weeks of age (Fig. 3). There was no significant difference in body weight between wild-type and heterozygous mice, whereas body weights of homozygous knockout mice were significantly less than those of wild-type animals at all time points studied. The tail lengths of homozygous mutant mice were also shorter than wild-type animals at 5 weeks of age (5.4 ± 0.3 cm versus 6.3 ± 0.3 cm; P < 0.001). Preliminary analysis of the skeleton suggested that this was not due to different numbers of vertebrae. The shape of the tail was abnormal in some of the homozygous mutant animals who died before weaning; it had the appearance of a kinked tail (Fig. 4), which is commonly observed in a mouse model of neural tube defects, the curly tail (ct) mouse (23). Homozygous mutants developed more slowly than their heterozygous and wild-type littermates in several areas. Fur appeared 5 days late and was sparser compared with normal mice. This feature facilitated the identification of homozygous mutant pups at a very early age. Homozygous mutants showed delayed maturation of external genitalia, but the majority were fertile. Homozygous mutants died within the first 2 weeks of their lives and had clear facial abnormalities and protrusive eyes. Kyphosis was observed occasionally (Fig. 4).
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Pathology of Mthfr-deficient mice
Since severe MTHFR deficiency is associated primarily with neuropathology (2), extensive examinations were performed for brain. Examination of brain sections of 3- and 5-week-old homozygous knockout animals revealed no gross abnormalities in overall structure. However, a dramatic reduction in cerebellar size was observed in sagittal sections of homozygotes. Regions of the inner granule layer (IGL) appeared more diffuse in the rostral cerebellum (Fig. 5A and B). To more closely examine the trilaminar structure of the homozygous mutant cerebellum, immunohistochemical analysis of Purkinje cells was performed on near-midline sections (Fig. 5C and D). In contrast to the distinct one cell-thick layer of Purkinje cells directly overlying the granule cell layer in wild-type mice, homozygous mutant mice had clusters of Purkinje cells intermingled with granule cells. These laminar structure defects were only apparent in the anterior region of the cerebellum and were more severe in the vermis than in the hemispheres.
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Gross pathologic changes were not observed after brief inspection of several other tissues of 5-week-old heterozygous and homozygous Mthfr-deficient mice. However, some degree of microvesicular steatosis was noticed in livers of homozygotes.
To determine whether hyperhomocysteinemia is atherogenic in mice, oil red O-stained serial aortic sections from older mice were examined by light microscopy. Some lipid staining was first observed in the aortic valve region of 10-month-old homozygous knockout mice, but not in their wild-type littermates; heterozygous knockout mice of the same age were not examined. However, significant lipid deposition was identified in both heterozygous and homozygous knockout mice at 14 months of age (Fig. 6A and B). The lipid accumulation was patchy and restricted to the initial portion of the aorta at the aortic valve region of the heart, and did not extend beyond the end of the aortic sinus in both heterozygous and homozygous knockout mice. The lesions were flat and did not contain foam cells. Luminal staining was not observed. Further analysis of the nature of these lesions is in progress.
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Biochemical disruptions in Mthfr-deficient mice
Plasma total homocysteine levels were significantly increased in both heterozygotes and homozygotes compared with their wild-type littermates (Table 1). The values were
1.6- and 10-fold higher than wild-type in heterozygotes and homozygous mutant animals, respectively. To rule out the possibility of genetic background influence, plasma total homocysteine was measured in inbred 129/SvJ mice, inbred BALB/cAnNCrlBR mice and wild-type mice from matings of Mthfr+/– mice. No difference was observed between the three different genetic background groups of the same age [analysis of variance (ANOVA), P = 0.94 (data not shown)].
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There were no significant differences in total folate content between the three genotypes in liver and brain (data not shown). Since MTHFR synthesizes 5-methylTHF, the distribution of folate derivatives was also examined. Table 1 shows the proportion of folate that was 5-methylTHF in liver and brain as percentage of total folate. The differences between genotype groups were significant by ANOVA (P < 0.05). Pairwise comparison with the wild-type group indicated depletion of 5-methylTHF in the liver of heterozygotes and homozygous mutant mice; in brain, the decrease was seen only in homozygous mutant mice. The percentage of 5-methylTHF in brain was greater than that in liver in all three genotype groups (Table 1). However, the percentage of 5-methylTHF in brain relative to that in liver increased substantially, from 1.4 to 2.3 to 4.6, with the increase in degree of disruption of the Mthfr gene (from +/+ to +/– to –/–, respectively).
Table 2 shows the changes in SAM and SAH levels in several tissues of Mthfr-deficient mice (liver, brain, testes and ovaries). Single-factor ANOVA was performed to assess the variance between the three different genotype groups. All groups except SAH in the testes showed significant differences between the three genotypes (P < 0.005). Pairwise comparison with wild-type mice showed that mean SAM levels were decreased in all tissues of homozygous mutant animals, although the decrease in liver was not statistically significant; SAH levels were significantly increased in all tissues in these animals. In heterozygous knockout mice, each tissue showed either a significant decrease in SAM or a significant increase in SAH. These results suggest that the disrupted balance between SAM and SAH could be due to either a decrease in SAM or an increase in SAH, or possibly both, with some degree of tissue specificity. Furthermore, the effect is dependent on the number of disrupted Mthfr genes.
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To determine whether the effect on SAM and SAH levels resulted in a detectable decrease in methylation, potential global DNA methylation levels were measured in these tissues using the cytosine-extension assay (24). In Table 2, the relative increase in the incorporation of [3H]dCTP in the heterozygous and homozygous Mthfr-deficient mice was directly proportional to the increase in hypomethylated sites in DNA. Single-factor ANOVA showed significant differences between the three genotypes for brain, testes and ovaries, but not for liver (P < 0.02). Pairwise comparison with wild-type mice showed that the increase in DNA hypomethylation was statistically significant for both heterozygous and homozygous mutant animals in brain and ovaries, with heterozygotes exhibiting values intermediate between wild-type and homozygous mutants. An increase in hypomethylated sites was also observed in testes and liver DNA; however, most likely because of low sample numbers, the increase did not reach statistical significance in these tissues.
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DISCUSSION |
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Mouse models with a disruption of enzymes of homocysteine metabolism would be very useful in studying the consequences of hyperhomocysteinemia in vivo. Mice with a disruption of cystathionine-ß-synthase (CBS), the first enzyme in the transsulfuration pathway for homocysteine metabolism, were generated several years ago and reported to have high plasma homocysteine, but these mice die within the first 5 weeks of life (25). Severe deficiency of CBS in humans is associated with homocystinuria, but common variants resulting in mild hyperhomocysteinemia and cardiovascular disease have not been reported.
The common variant in MTHFR is associated with decreased enzyme activity (35–40% of control levels in homozygous mutant individuals). This mild deficiency is similar to that observed in the heterozygous Mthfr knockout mice. Homocysteine levels in individuals homozygous for the polymorphism are increased from 50 to 100% (3–5), depending on folate status, similar to the increase seen in the heterozygous knockout mice. The mutant genotype in humans has been associated with increased risk for cardiovascular disease in several studies (26–28) or it may modify risk in individuals with other risk factors (29,30). Several studies have not observed an increased risk, but these reports did not examine folate status (31). As previously mentioned, the mutant genotype may only affect risk in individuals with low folate. The availability of Mthfr-deficient mice will allow us to address this issue. Thus far we have not observed obvious vascular pathology in young animals. However, abnormal lipid deposition in the aorta was observed in older heterozygous and homozygous knockout mice, but not in wild-type animals, although the lipid-staining lesions in the aortas of 14-month-old mutant mice were not typical of the advanced atherosclerotic lesions reported in other mouse models for atherosclerosis, such as the apoE knockout mouse. In the apoE knockout mouse, the lipid aggregates are larger and appear to protrude into the lumen of the vessel (32). The lipid accumulation seen in the Mthfr-deficient mice may be a reflection of hyperhomocysteinemia at the strategic site of the aorta, or it could simply be an early stage of the atherosclerotic process in these animals. The examined mice are offspring of heterozygous Mthfr-deficient mice, which were backcrossed two generations from (129/Sv x BALB/c) F1 heterozygotes to BALB/c. Since the BALB/c strain is considered to be relatively resistant to aortic atherosclerotic lesion development, it may be necessary to backcross Mthfr-deficient mice to a more susceptible strain, such as C57BL/6. To mimic the human condition, dietary manipulation may also be required to enhance the atherogenic process. The mice have been maintained thus far on standard mouse chow, which contains a relatively high level of folate. Low folate diets may be required to increase homocysteine levels even further to assess vascular changes and alterations in DNA methylation.
Homocystinuric patients with undetectable or very low levels of MTHFR activity have a variable phenotype, with some patients not surviving beyond the first year of life (8,9); Mthfr-deficient mice also exhibit this variability, without an obvious explanation. Other patients (7–9), with equally low levels of enzyme activity, exhibit severe neurological problems, including seizures, hypotonia, incoordination and developmental delay, similar to the Mthfr-deficient mice. Histological analysis of homozygous mutant mice revealed defects in the size and trilaminar structure of the cerebellum, suggesting that this gene may play a role in migration and/or proliferation of neuronal precursors in the developing cerebellum. Interestingly, laminar structure abnormalities were confined to the anterior region of the cerebellar cortex. This result indicates that the developmental processes underlying certain regions of the cerebellum are sensitive to the loss of MTHFR, whereas others are not, and may reflect the inherent compartmentation of the cerebellum. Evidence for cerebellar regionalization has been established by the expression of numerous molecules in specific subsets of Purkinje or granule cells (33,34). Furthermore, this compartmentation has been genetically demonstrated in mutant strains of mice in which abnormal cell migration or specific cell loss results in aberrant cerebellar development primarily in the rostral region of the cerebellum (35,36). The mechanisms by which MTHFR deficiency affects cerebellar development are unclear at this time and require further study, but it is possible that the enzyme affects the migration process of specific neurons through an alteration in methylation reactions.
Our results for the proportion of 5-methylTHF in total folate (Table 1) suggest that 5-methylTHF levels may be maintained in the brain, at the expense of the liver, in heterozygotes but not homozygotes. This type of protective mechanism would ensure adequate synthesis of methionine and SAM. Consistent with this reasoning, SAM levels (Table 2) were maintained in brains of heterozygous mice. The liver, on the other hand, can also use betaine as a methyl donor for synthesis of methionine/SAM from homocysteine. The enzyme that catalyzes this reaction, betaine homocysteine methyltransferase, is specific to the liver and kidney; it is not present in the brain (37).
MTHFR is the only enzyme that can synthesize 5-methylTHF. The low levels of 5-methylTHF in homozygous mutant mice may have come from maternal milk or from bacteria in the colon (38,39). Folate transporters in the colon (40) and at the blood–brain barrier (41) would serve to provide some 5-methylTHF to various tissues, including the brain.
With the transfer of its methyl group, SAM is converted to SAH, the sole precursor for homocysteine production via the SAH hydrolase reaction. Because 5-methylTHF provides a methyl group for conversion of homocysteine to methionine and consequently SAM, a decrease in 5-methylTHF due to MTHFR deficiency might be expected to decrease SAM levels; this expectation was borne out in the Mthfr-deficient mice. Increased SAH might also be observed since SAH can also be generated from homocysteine by reversal of the S-adenosylhomocysteine hydrolase reaction, particularly when homocysteine levels are high (42). A significant increase in SAH was demonstrated in both heterozygous and homozygous mutant mice in several tissues. A chronic elevation in SAH should result in feedback inhibition of SAM-dependent methyltransferase reactions (15) and reduce methylation capacity, as demonstrated both in vitro and in vivo (43,44).
Although many genes have been demonstrated to cause neural tube defects (NTDs) in mice (45), the polymorphism at 677 bp in MTHFR is the first identified genetic risk factor for NTDs in humans (46). We carefully examined the pups born from heterozygote or homozygote matings but no severe NTDs, such as exencephaly or spina bifida, were observed. However, as previously noted, some of the homozygotes had kyphosis and their tails were not straight. Several had tails that were quite similar to the kinked tail seen in curly tail (ct) mice, which is considered to be a good model for human NTDs (23). The cerebellar pathology observed in Mthfr-deficient mice supports a role for Mthfr in central nervous system (CNS) development, but the exact role is still unclear. Although the possibility that the inserted neor gene affects the phenotype cannot formally be excluded, the similarity between Mthfr-deficient mice and homocystinuric patients makes this highly unlikely.
The common MTHFR variant has been reported to be associated with several other complex traits, including pregnancy complications (pre-eclampsia, recurrent pregnancy loss and placental abruption) (47), neuropsychiatric disturbances such as schizophrenia (48), Down syndrome (49), inflammatory bowel disease (50) and retinal occlusion (51). Many of these reports still require confirmation. However, since mild MTHFR deficiency in the heterozygous knockout mice is associated with a disruption of the methionine cycle, it is possible that mild hyperhomocysteinemia is an indirect biomarker for disturbances in cellular methylation reactions (52). Methylation reactions are required for synthesis of membrane phospholipids, myelin basic protein and neurotransmitters, three important pathways in the CNS. The maintenance of normal DNA methylation patterns is essential for normal gene expression, genomic imprinting and cellular differentiation (17,18). Alterations in DNA methylation also play a role in the carcinogenic process (53). One such study, which crossed the Dnmt1 heterozygous knockout with a mouse model for intestinal neoplasia (the Min mouse), demonstrated the suppression of intestinal neoplasia by DNA hypomethylation (54). The Mthfr-deficient mouse represents another genetically modified mouse model to study consequences of DNA hypomethylation. This mouse model is distinct from the former since it targets the balance between SAM and SAH, an established predictor of cellular methylation potential. Relevant to this discussion is the finding that the MTHFR polymorphism has been reported to reduce the risk of colorectal cancer (55) and of leukemia in man (56); crossing of the Mthfr heterozygous knockout with the Min mouse should provide additional information to understand the role of MTHFR in DNA hypomethylation and carcinogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of Mthfr-deficient mice
Using a 1.27 kb human MTHFR cDNA as a probe, we isolated a genomic clone by screening a
DASH II 129/Sv mouse genomic library (a gift from J. Rossant, University of Toronto). The clone contained 15 kb of the 5' end of the mouse Mthfr gene, including exons 1–4. Using this genomic clone and the pPNT plasmid (57), an insertion type of targeting vector was constructed (Fig. 1A). Exponentially growing R1 ES cells were electroporated (Bio-Rad Gene Pulser at 240 V, 500 µF) with NotI-linearized targeting vector DNA and selected in a medium containing 350 µg/ml G418[Geneticin] (Life Technologies) and 0.2 µmol/l 1-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl)-5-iodouracil (FIAU) (Moravek Biochemical). Resistant ES clones were picked 9–10 days later and cell lines were established. Five positive ES clones were identified by Southern blot analysis using XbaI- or BamHI-digested genomic DNA isolated from 150 doubly-resistant ES cell clones and hybridization with a 5' external probe (Fig. 1A). Three positive clones were microinjected into 3.5-day-old blastocysts from BALB/c females and transferred into BDF pseudopregnant mice to generate chimeric mice. Chimeric mice produced from one of the ES clones demonstrated germline transmission. Heterozygous mice were generated through breeding of chimeric mice with BALB/cAnNCrlBR mice (Charles River Canada). Homozygous Mthfr-deficient mice were obtained by breeding of heterozygotes. All experiments were conducted in accordance with the guidelines of the Canadian Council on Animal Care. Animals used in this report were offspring from the breeding second generation of BALB/c backcrossed heterozygous knockout mice. All animals were maintained on standard rodent chow, Laboratory rodent diet 5001 (Agribrands Purina Canada), which contains 23.4% protein (including 0.43% methionine) and 5.9 mg folic acid/kg diet. Wild-type littermates were used as normal controls in all experiments.
Determination of genotypes of Mthfr-deficient mice
Mice were genotyped either by Southern blotting or by PCR. For Southern blot analysis, mouse genomic DNA was digested with XbaI and hybridized with the 5' probe (Fig. 1A). The three primers used in PCR analysis are: sense primer 1 (5'-GAA GCA GAG GGA AGG AGG CTT CAG-3') in exon 3, sense primer 2 (5'-AGC CTG AAG AAC GAG ATC AGC AGC-3') in the neor gene and antisense primer 3 (5'-GAC TAG CTG GCT ATC CTC TCA TCC-3') in intron 3. The expected sizes of wild-type and targeted alleles are shown in Figure 1.
RT–PCR
Mouse brain RNA was isolated using the Atlas Pure Total RNA Isolation kit (Clontech). After DNase I treatment, the A260/A280 ratio of isolated total RNA was between 1.9 and 2.1. The 40 µl RT reaction contained 4 µg of total RNA, 160 ng of random hexamers, 500 µM each of dATP, dCTP, dGTP and dTTP, 10 mM dithiothreitol and 200 U of SuperScript II Reverse Transcriptase (Life Technologies), in 1x First-Strand Buffer supplied by the manufacturer. The RNA, primers and dNTP were preheated at 65°C for 5 min and quick-chilled on ice before incubation with the other components, except SuperScript II, at 25°C for 10 min. The reverse transcription then proceeded at 42°C for 50 min after addition of SuperScript II.
PCRs were then performed using various primers. Two sets of primers were used to amplify mouse Mthfr mRNA. Ex1-3 amplified a 326 bp product including part of exon 1, exon 2 and part of exon 3, 5' to the neor insertion site (sense, 5'-ATG GAC TCT GGT GAC AAG TGG-3'; antisense, 5'-AGT GGT CAC CTA CAG GGT CTC C-3'). Ex2-5 amplified a 400 bp product covering exons 2–5 (sense, 5'-ACA GGC CAT CTG CAC AGA GCC AA-3'; antisense, 5'-CAG TTT TAC AAG CTG CCG AAG GGA-3'). Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control (sense, 5'-CAG GAG CGA GAC CCC ACT AAC AT-3'; antisense, 5'-GTC AGA TCC ACG ACG GAC ACA TT-3').
Assays of MTHFR enzyme activity
Tissues (brain, kidney, liver, ovaries and testes) were isolated from 5-week-old mice, frozen immediately on dry ice and stored at –70°C until used. Frozen tissues were thawed in homogenization buffer [50 mM potassium phosphate pH 7.2, 1 mM EDTA and 50 mM sucrose, with protease inhibitors (0.1 mg/ml phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin and 2 µg/ml aprotonin)] (Roche Diagnostics), and homogenized in a Polytron (Brinkmann Instruments). Supernatants were collected after centrifugation at 18 000 g for 30 min at 4°C. Protein concentrations were determined with the Bio-Rad protein assay. The 14C-labeled methyltetrahydrofolate-menadione oxidoreductase assay was used to measure MTHFR activity as described (8), with 100 µg of protein extract for each assay.
Histological examination
Mice at 5 weeks of age were anesthetized and perfused. Organs were excised, further fixed in 4% paraformaldehyde in sodium phosphate buffer pH 7.4 and embedded in paraffin. Five micrometer sections were stained with hematoxylin/eosin. Examined organs were brain, liver, spleen, kidney, pancreas, heart, stomach, lung, small intestine, large intestine, limbs, spinal cord, tongue and salivary glands.
Lipid deposition in the aorta was examined by oil red O staining (58). Briefly, perfused heart and ascending aorta were removed and further fixed in 10% buffered formalin, embedded sequentially in 5, 10 and 25% gelatin, grossly cut through the ventricles parallel to the atria and frozen in optimal cutting temperatuee (OCT) compound (Miles Laboratories). Every second 10 µm section was placed on gelatin-coated slides, stained with 0.24% oil red O, counterstained with 2.4% hematoxylin and 0.25% fast green (all from Sigma-Aldrich Canada) and then examined by light microscopy.
Measurements of metabolites
Plasma total homocysteine.
Mice were decapitated and blood samples were collected in Vacutainer tubes containing EDTA (Becton Dickinson). Plasma was collected after centrifugation and stored at –70°C. Total homocysteine was determined by a high pressure liquid chromatography (HPLC) method (59).
Tissue folate.
Total folate content and the distribution of different folates in brain and liver were determined by an affinity/HPLC method using electrochemical (coulometric) detection (60).
Tissue SAM and SAH.
Concentrations of SAM and SAH in various tissues were determined by HPLC with electrochemical (coulometric) detection (61).
Evaluation of global DNA methylation
The cytosine-extension assay was used to evaluate global DNA hypomethylation as described by Pogribny et al. (24). Briefly, 1 µg of genomic DNA was digested for 16–18 h with 20 U of HpaII. A second DNA aliquot served as a background control and was similarly incubated without addition of restriction enzyme. The single nucleotide extension reaction was performed in a 25 µl reaction mixture containing 0.5 µg of DNA, 1x PCR buffer II, 1.0 mM MgCl2, 0.25 U of AmpliTaq DNA polymerase (Perkin Elmer) and 0.1 µl of [3H]dCTP (57.4 Ci/mmol; NEN) and incubated at 56°C for 1 h, then placed on ice. Duplicate 10 µl aliquots from each reaction were applied to a Whatman DE-81 ion-exchange filter and washed three times. Dried filters were processed for scintillation counting. Background radioactivity in untreated samples was subtracted from enzyme-treated samples. The results were expressed as [3H]dCTP incorporation/0.5 µg DNA. MspI digestion was used routinely to verify that the disintegrations per minute (d.p.m.) values obtained were significantly higher than those obtained with HpaII.
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ACKNOWLEDGEMENTS |
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We thank B. He, L.J. Fortin, E. Arning and F. Hiou-Tim for technical assistance, S.A. Huber for providing the histological analysis protocol for aorta, and R. Gravel and J. Trasler for helpful comments. This work was supported by the Medical Research Council of Canada Group Grant in Medical Genetics (R.R. and A.C.K.) and the Canadian Genetic Diseases Network (M.A.R. and R.R.). Z.C. was supported by a Studentship from the Medical Research Council of Canada.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: McGill University—Montreal Children’s Hospital, 4060 Sainte Catherine West, Room 242, Montreal H3Z 2Z3, Canada. Tel: +1 514 934 4358; Fax: +1 514 934 4331; Email: mbjh@musica.mcgill.ca
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 . Mudd, S.H., Levy, H.L. and Skovby, F. (1995) Disorders of transsulfuration. In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), The Metabolic Basis of Inherited Disease. McGraw-Hill, New York, NY, pp. 1279–1327.
2 . Rosenblatt, D.S. (1995) Inherited disorders of folate transport and metabolism. In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), The Metabolic Basis of Inherited Disease. McGraw-Hill, New York, NY, pp. 3111–3128.
3 . Frosst, P., Blom, H.J., Milos, R., Goyette, P., Sheppard, C.A., Matthews, R.G., Boers, G.J.H., den Heiher, M., Kluijtmans, L.A.J., van den Heuvel, L.P. and Rozen, R. (1995) A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genet., 10, 111–113.[Web of Science][Medline]
4 . Ma, J., Stampfer, M.J., Hennekens, C.H., Frosst, P., Selhub, J., Horsford, J., Malinow, M.R., Willett, W.C. and Rozen, R. (1996) Methylenetetrahydrofolate reductase polymorphism, plasma folate, homocysteine, and risk of myocardial infarction in US physicians. Circulation, 94, 2410–2416.
5 . Jacques, P.F., Bostom, A.G., Williams, R.R., Ellison, R.C., Eckfeldt, J.H., Rosenberg, I.H., Selhub, J. and Rozen, R. (1996) Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation, 93, 7–9.
6 . Guenther, B.D., Sheppard, C.A., Tran, P., Rozen, R., Matthews, R.G. and Ludwig, M.L. (1999) The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia. Nature Struct. Biol., 6, 359–365.[Web of Science][Medline]
7 . Goyette, P., Sumner, J.S., Milos, R., Duncan, A.M.V., Rosenblatt, D.S., Matthews, R.G. and Rozen, R. (1994) Human methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification. Nature Genet., 7, 195–200.[Web of Science][Medline]
8 . Goyette, P., Christensen, B., Rosenblatt, D.S. and Rozen, R. (1996) Severe and mild mutations in cis for the methylenetetrahydrofolate reductase (MTHFR) gene, and description of five novel mutations in MTHFR. Am. J. Hum. Genet., 59, 1268–1275.[Web of Science][Medline]
9 . Goyette, P., Frosst, P., Rosenblatt, D.S. and Rozen, R. (1995) Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency. Am. J. Hum. Genet., 56, 1052–1059.[Web of Science][Medline]
10 . Wall, R.T., Harlan, J.M., Harker, L.A. and Striker, G.E. (1980) Homocysteine-induced endothelial cell injury in vitro: a model for the study of vascular injury. Thromb. Res., 18, 113–121.[Web of Science][Medline]
11 Harker, L.A., Ross, R., Slichter, S.J. and Scott, C.R. (1976) Homocysteine-induced arteriosclerosis. The role of endothelial cell injury and platelet response in its genesis. J. Clin. Invest., 58, 731–741.
12 . Tsai, J.C., Wang, H., Perrella, M.A., Yoshizumi, M., Sibinga, N.E., Tan, L.C., Haber, E., Chang, T.H., Schlegel, R. and Lee, M.E. (1996) Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells. J. Clin. Invest., 97, 146–153.[Web of Science][Medline]
13 . Rees, M.M. and Rodgers, G.M. (1993) Homocysteinemia: association of a metabolic disorder with vascular disease and thrombosis. Thromb. Res., 71, 337–359.[Web of Science][Medline]
14 . Loscalzo, J. (1996) The oxidant stress of hyperhomocysteinaemia. J. Clin. Invest., 98, 5–7.[Web of Science][Medline]
15 . Ueland, P.M. (1982) Pharmacological and biochemical aspects of S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase. Pharmacol. Rev., 34, 223–253.[Web of Science][Medline]
16 . Chiang, P.K., Gordon, R.K., Tal, J., Zeng, G.C., Doctor, B.P., Pardhasaradhi, K. and MaCann, P.P. (1996) S-adenosylmethionine and methylation. FASEB J., 10, 471–480.[Abstract]
17 . Singal, R. and Ginder, G.D. (1999) DNA methylation. Blood, 93, 4059–4070.
18 . Reik, W. and Walter, J. (1998) Imprinting mechanisms in mammals. Curr. Opin. Genet. Dev., 8, 154–164.[Web of Science][Medline]
19 . Li, E., Bestor, T.H. and Jaenisch, R. (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell, 69, 915–926.[Web of Science][Medline]
20 . Okano, M., Bell, D.W., Haber, D.A. and Li, E. (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 99, 247–257.[Web of Science][Medline]
21 . Xu, G.L., Bestor, T.H., Bourc’his, D., Hsieh, C.L., Tommerup, N., Bugge, M., Hulten, M., Qu, X., Russo, J.J. and Viegas-Pequignot, E. (1999) Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature, 402, 187–190.[Medline]
22 . Amir, R.E., van der Veyver, I.B., Wan, M., Tran, C.Q., Francke, U. and Zoghbi, H.Y. (1999) Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature Genet., 23, 185–188.[Web of Science][Medline]
23 . Seller, M.J. and Adinolfi, M. (1981) The curly-tail mouse: an experimental model for human neural tube defects. Life Sci., 29, 1607–1615.[Web of Science][Medline]
24 . Pogribny, I., Yi, P. and James, S.J. (1999) A sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands. Biochem. Biophys. Res. Commun., 262, 624–628.[Web of Science][Medline]
25 . Watanabe, M., Osada, J., Aratani, Y., Kluckman, K., Reddick, R., Malinow, M.R. and Maeda, N. (1995) Mice deficient in cystathionine ß-synthase: animal models for mild and severe homocyst(e)inemia. Proc. Natl Acad. Sci. USA, 92, 1585–1589.
26 . Kluijtmans, L.A.J., van den Heuvel, L.P., Boers, G.H.J., Frosst, P., Stevens, E.M.B., van Oost, B.A., den Heijer, M., Trijbels, F.J.M., Rozen, R. and Blom, H.J. (1996) Molecular genetic analysis in mild hyperhomocysteinemia: a common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease. Am. J. Hum. Genet., 58, 35–41.[Web of Science][Medline]
27 . Morita, H., Taguchi, J., Kurihara, H., Kitaoka, M., Kaneda, H., Kurihara, Y., Maemura, K., Shindo, T., Minamino, T., Ohno, M. et al. (1997) Genetic polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR) as a risk factor for coronary artery disease. Circulation, 95, 2032–2036.
28 . Morita, H., Kurihara, H., Tsubaki, S., Sugiyama, T., Hamada, C., Kurihara, Y., Shindo, T., Oh-hashi, Y., Kitamura, K. and Yazaki, Y. (1998) Methylenetetrahydrofolate reductase gene polymorphism and ischemic stroke in Japanese. Arterioscler. Thromb. Vasc. Biol., 18, 1465–1469.
29 . Cattaneo, M., Tsai, M.Y., Bucciarelli, P., Taioli, E., Zighetti, M.L., Bignell, M. and Mannucci, P.M. (1997) A common mutation in the methylenetetrahydrofolate reductase gene (C677T) increases the risk for deep-vein thrombosis in patients with mutant factor V (factor V:Q506). Arterioscler. Thromb. Vasc. Biol., 17, 1662–1666.
30 . Gardemann, A., Weidemann, H., Philipp, M., Katz, N., Tillmanns, H., Hehrlein, F.W. and Haberbosch, W. (1999) The TT genotype of the methylenetetrahydrofolate reductase C677T gene polymorphism is associated with the extent of coronary atherosclerosis in patients at high risk for coronary artery disease. Eur. Heart J., 20, 584–592.
31 . Fletcher, O. and Kessling, A.M. (1998) MTHFR association with arteriosclerotic vascular disease? Hum. Genet., 103, 11–21.[Web of Science][Medline]
32 . Qiao, J.-H., Xie, P.-Z., Fishbein, M.C., Kreuzer, J., Drake, T.A., Demer, L.L. and Lusis, A.J. (1994) Pathology of athermatous lesions in inbred and genetically engineered mice. Arterioscler. Thromb. Vasc. Biol., 14, 1480–1497.
33 . Hawkes, R. and Eisenman, L.M. (1997) Stripes and zones: the origins of regionalization of the adult cerebellum. Perspect. Dev. Neurobiol., 5, 95–105.[Web of Science][Medline]
34 . Oberdick, J., Baader, S.L. and Schilling, K. (1998) From zebra stripes to postal zones: deciphering patterns of gene expression in the cerebellum. Trends Neurosci., 21, 383–390.[Web of Science][Medline]
35 . Ackerman, S.L., Kozak, L.P., Przyborski, S.A., Rund, L.A., Boyer, B.B. and Knowles, B.B. (1997) The mouse rostral cerebellar malformation gene encodes an UNC-5-like protein. Nature, 386, 838–842.[Medline]
36 . Eisenman, L.M. and Brothers, R. (1998) Rostral cerebellar malformation (rcm/rcm): a murine mutant to study regionalization of the cerebellum. J. Comp. Neurol., 394, 106–117.[Web of Science][Medline]
37 . McKeever, M.P., Weir, D.G., Molloy, A. and Scott, J.M. (1999) Betaine-homocysteine methyltransferase: organ distribution in man, pig and rat and subcellular distribution in rat. Clin. Sci., 81, 551–556.
38 . Camilo, E., Zimmerman, J., Mason, J.B., Golner, B., Russell, R., Selhub, J. and Rosenberg, I.H. (1996) Folate synthesized by bacteria in the human upper small intestine is assimilated by the host. Gastroenterology, 110, 991–998.[Web of Science][Medline]
39 . Rong, N., Selhub, J., Goldin, B.R. and Rosenberg, I.H. (1991) Bacterially synthesized folate in rat large intestine is incorporated into host tissue folyl polyglutamates. J. Nutr., 121, 1955–1999.
40 . Dudeja, P.K., Torania, S.A. and Said, H.M. (1997) Evidence for the existence of a carrier-mediated folate uptake mechanism in human colonic luminal membranes. Am. J. Physiol., 272, G1408–G1415.
41 . Wu, D. and Pardridge, V.M. (1999) Blood–brain barrier transport of reduced folic acid. Pharm. Res., 16, 415–419.[Web of Science][Medline]
42 . De La Haba, G. and Cantoni, G.L. (1959) The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine. J. Biol. Chem., 234, 603–608.
43 . Barbes, C., Sanchez, J., Yebra, M.J., Robert-Gero, M. and Hardsson, C. (1990) Effects of sinefungin and S-adenosylhomocysteine on DNA and protein methyltransferases from Streptomyces and other bacteria. FEMS Microbiol. Lett., 69, 239–244.
44 . De Cabo, S.F., Santos, J. and Fernandez-Piqueras, J. (1995) Molecular and cytological evidence of S-adenosyl-L-homocysteine as an innocuous undermethylation agent in vivo. Cytogenet. Cell Genet., 71, 187–192.[Web of Science][Medline]
45 . Linder, C. and Davisson, M.T. (1994) Neurology News: Mouse models for studying neural tube defects.. The Jackson Laboratory, Bar Harbor, ME.
46 . van der Put, N.M.J., Steegers-Theunissen, R.P.M., Frosst, P., Trijbels, F.J.M., Eskes, T.K.A.B., van den Heuvel, L.P., Mariman, E.C.M., den Heyer, M., Rozen, R. and Blom, H.J. (1995) Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida. Lancet, 346, 1070–1071.[Web of Science][Medline]
47 . Kupferminc, M.J., Eldor, A., Steinman, N., Many, A., Bar-Am, A., Jaffa, A., Fait, G. and Lessing, J.B. (1999) Increased frequency of genetic thrombophilia in women with complications of pregnancy. N. Engl. J. Med., 340, 9–13.
48 . Regland, B., Germgard, T., Gottfries, C.G., Grenfeldt, B. and Koch-Schmidt, A.C. (1997) Homozygous thermolabile methylenetetrahydrofolate reductase in schizophrenia-like psychosis. J. Neural Transm., 104, 931–941.
49 . James, S.J., Pogribna, M., Pogribny, I.I., Melnyk, S., Hine, R.J., Gibson, J.B., Yi, P., Tafoya, D.L., Swenson, D.H., Wilson, V.L. and Gaylor, D.W. (1999) Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome. Am. J. Clin. Nutr., 70, 495–501.
50 . Mahmud, N., Molloy, A., McPartlin, J., Corbally, R., Whitehead, A.S., Scott, J.M. and Weir, D.G. (1999) Increased prevalence of methylenetetrahydrofolate reductase C677T variant in patients with inflammatory bowel disease, and its clinical implications. Gut, 45, 389–394.
51 . Loewenstein, A., Winder, A., Goldstein, M., Lazar, M. and Eldor, A. (1997) Bilateral retinal vein occlusion associated with 5,10-methylenetetrahydrofolate reductase mutation. Am. J. Ophthalmol., 124, 840–841.[Web of Science][Medline]
52 . Yi, P., Melnyk, S., Pogribna, M., Pogribny, I.P., Hine, R.J. and James, S.J. (2000) Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation. J. Biol. Chem., 275, 29318–29323.
53 . Baylin, S.B., Herman, J.G., Graff, J.R., Vertino, P.M. and Issa, J.P. (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv. Cancer Res., 72, 141–196.[Web of Science][Medline]
54 . Laird, P.W., Jackson-Grusby, L., Fazeli, A., Dickinson, S.L., Jung, W.E., Li, E., Weinberg, R.A. and Jaenisch, R. (1995) Suppression of intestinal neoplasia by DNA hypomethylation. Cell, 81, 197–205.[Web of Science][Medline]
55 . Ma, J., Stampfer, M.J., Giovannucci, E., Artigas, C., Hunter, D., Fuchs, C., Willett, W., Selhub, J., Hennekens, C.H. and Rozen, R. (1997) Methylenetetrahydrofolate reductase polymorphism, reduced risk of colorectal cancer and dietary interactions. Cancer Res., 57, 1098–1102.
56 . Skibola, C.F., Smith, M.T., Kane, E., Roman, E., Rollinson, S., Cartwright, R.A. and Morgan, G. (1999) Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults. Proc. Natl Acad. Sci. USA, 96, 12810–12815.
57 . Tybulewicz, V.L., Crawford, C.E., Jackson, P.K., Bronson, R.T. and Mulligan, R.C. (1991) Neonatal lethality and lymphopenia in mice with a homozygous disruption of c-abl proto-oncogene. Cell, 65, 1153–1163.[Web of Science][Medline]
58 . Plump, A., Scott, C. and Breslow, J. (1994) Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse. Proc. Natl Acad. Sci. USA, 91, 9607–9611.
59 . Durand, P., Fortin, L.J., Lussier-Cacan, S., Davignon, J. and Blache, D. (1996) Hyperhomocysteinemia induced by folic acid deficiency and methionine load—applications of a modified HPLC method. Clin. Chim. Acta, 252, 83–93.[Web of Science][Medline]
60 . Bagley, P.J. and Selhub, J. (1998) A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proc. Natl Acad. Sci. USA, 95, 13217–13220.
61 . Melnyk, S., Pogribna, M., Pogribny, I.P., Yi, P. and James, S.J. (2000) Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulometric electrochemical detection: alterations with plasma homocysteine and pyridoxal 5'-phosphate concentrations. Clin. Chem., 46, 265–272.
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|
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 D. Li, L. Pickell, Y. Liu, Q. Wu, J. S Cohn, and R. Rozen Maternal methylenetetrahydrofolate reductase deficiency and low dietary folate lead to adverse reproductive outcomes and congenital heart defects in mice Am. J. Clinical Nutrition, July 1, 2005; 82(1): 188 - 195. [Abstract] [Full Text] [PDF]
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 S. Dayal and S. R Lentz ADMA and hyperhomocysteinemia Vascular Medicine, July 1, 2005; 10(1_suppl): S27 - S33. [Abstract] [PDF]
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S. Dayal and S. R Lentz ADMA and hyperhomocysteinemia Vascular Medicine, May 1, 2005; 10(2_suppl): S27 - S33. [Abstract] [PDF]
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C. L. Ulrey, L. Liu, L. G. Andrews, and T. O. Tollefsbol The impact of metabolism on DNA methylation Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R139 - R147. [Abstract] [Full Text] [PDF]
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 T. L.J. Kelly, O. R. Neaga, B. C. Schwahn, R. Rozen, and J. M. Trasler Infertility in 5,10-Methylenetetrahydrofolate Reductase (MTHFR)-Deficient Male Mice Is Partially Alleviated by Lifetime Dietary Betaine Supplementation Biol Reprod, March 1, 2005; 72(3): 667 - 677. [Abstract] [Full Text] [PDF]
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S. Zaina, M. W. Lindholm, and G. Lund Nutrition and Aberrant DNA Methylation Patterns in Atherosclerosis: More than Just Hyperhomocysteinemia? J. Nutr., January 1, 2005; 135(1): 5 - 8. [Abstract] [Full Text] [PDF]
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H. Ghandour, Z. Chen, J. Selhub, and R. Rozen Mice Deficient in Methylenetetrahydrofolate Reductase Exhibit Tissue-Specific Distribution of Folates J. Nutr., November 1, 2004; 134(11): 2975 - 2978. [Abstract] [Full Text] [PDF]
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S. Narayanan, J. McConnell, J. Little, L. Sharp, C. J. Piyathilake, H. Powers, G. Basten, and S. J. Duthie Associations between Two Common Variants C677T and A1298C in the Methylenetetrahydrofolate Reductase Gene and Measures of Folate Metabolism and DNA Stability (Strand Breaks, Misincorporated Uracil, and DNA Methylation Status) in Human Lymphocytes In vivo Cancer Epidemiol. Biomarkers Prev., September 1, 2004; 13(9): 1436 - 1443. [Abstract] [Full Text] [PDF]
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G. Lund, L. Andersson, M. Lauria, M. Lindholm, M. F. Fraga, A. Villar-Garea, E. Ballestar, M. Esteller, and S. Zaina DNA Methylation Polymorphisms Precede Any Histological Sign of Atherosclerosis in Mice Lacking Apolipoprotein E J. Biol. Chem., July 9, 2004; 279(28): 29147 - 29154. [Abstract] [Full Text] [PDF]
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 E. J. Corwin The Concept of Epigenetics and Its Role in the Development of Cardiovascular Disease: Commentary on "New and Emerging Theories of Cardiovascular Disease" Biol Res Nurs, July 1, 2004; 6(1): 11 - 16. [PDF]
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F. Bieswal, S. M. Hay, C. McKinnon, B. Reusens, M. Cuignet, W. D. Rees, and C. Remacle Prenatal Protein Restriction Does Not Affect the Proliferation and Differentiation of Rat Preadipocytes J. Nutr., June 1, 2004; 134(6): 1493 - 1499. [Abstract] [Full Text] [PDF]
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 A. M. Devlin, E. Arning, T. Bottiglieri, F. M. Faraci, R. Rozen, and S. R. Lentz Effect of Mthfr genotype on diet-induced hyperhomocysteinemia and vascular function in mice Blood, April 1, 2004; 103(7): 2624 - 2629. [Abstract] [Full Text] [PDF]
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 K.-J. Sohn, R. Croxford, Z. Yates, M. Lucock, and Y.-I. Kim Effect of the Methylenetetrahydrofolate Reductase C677T Polymorphism on Chemosensitivity of Colon and Breast Cancer Cells to 5-Fluorouracil and Methotrexate J Natl Cancer Inst, January 21, 2004; 96(2): 134 - 144. [Abstract] [Full Text] [PDF]
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H. Refsum, A. D. Smith, P. M. Ueland, E. Nexo, R. Clarke, J. McPartlin, C. Johnston, F. Engbaek, J. Schneede, C. McPartlin, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion Clin. Chem., January 1, 2004; 50(1): 3 - 32. [Abstract] [Full Text] [PDF]
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C. N. Craciunescu, E. C. Brown, M.-H. Mar, C. D. Albright, M. R. Nadeau, and S. H. Zeisel Folic Acid Deficiency During Late Gestation Decreases Progenitor Cell Proliferation and Increases Apoptosis in Fetal Mouse Brain J. Nutr., January 1, 2004; 134(1): 162 - 166. [Abstract] [Full Text] [PDF]
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 A. M. Troen, E. Lutgens, D. E. Smith, I. H. Rosenberg, and J. Selhub The atherogenic effect of excess methionine intake PNAS, December 9, 2003; 100(25): 15089 - 15094. [Abstract] [Full Text] [PDF]
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C. N. Craciunescu, C. D. Albright, M.-H. Mar, J. Song, and S. H. Zeisel Choline Availability During Embryonic Development Alters Progenitor Cell Mitosis in Developing Mouse Hippocampus J. Nutr., November 1, 2003; 133(11): 3614 - 3618. [Abstract] [Full Text] [PDF]
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A. Virdis, M. Iglarz, M. F. Neves, F. Amiri, R. M. Touyz, R. Rozen, and E. L. Schiffrin Effect of Hyperhomocystinemia and Hypertension on Endothelial Function in Methylenetetrahydrofolate Reductase-Deficient Mice Arterioscler. Thromb. Vasc. Biol., August 1, 2003; 23(8): 1352 - 1357. [Abstract] [Full Text] [PDF]
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A. A. Noga, L. M. Stead, Y. Zhao, M. E. Brosnan, J. T. Brosnan, and D. E. Vance Plasma Homocysteine Is Regulated by Phospholipid Methylation J. Biol. Chem., February 14, 2003; 278(8): 5952 - 5955. [Abstract] [Full Text] [PDF]
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 P Ranganathan, S Eisen, W M Yokoyama, and H L McLeod Will pharmacogenetics allow better prediction of methotrexate toxicity and efficacy in patients with rheumatoid arthritis? Ann Rheum Dis, January 1, 2003; 62(1): 4 - 9. [Abstract] [Full Text] [PDF]
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T. Bottiglieri S-Adenosyl-L-methionine (SAMe): from the bench to the bedside--molecular basis of a pleiotrophic molecule Am. J. Clinical Nutrition, November 1, 2002; 76 (5): 1151S - 1157S. [Abstract] [Full Text] [PDF]
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A. Reyes-Engel, E. Munoz, M.J. Gaitan, E. Fabre, M. Gallo, J.L. Dieguez, M. Ruiz, and M. Morell Implications on human fertility of the 677C->T and 1298A->C polymorphisms of the MTHFR gene: consequences of a possible genetic selection Mol. Hum. Reprod., October 1, 2002; 8(10): 952 - 957. [Abstract] [Full Text] [PDF]
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M. F. Paz, S. Avila, M. F. Fraga, M. Pollan, G. Capella, M. A. Peinado, M. Sanchez-Cespedes, J. G. Herman, and M. Esteller Germ-Line Variants in Methyl-Group Metabolism Genes and Susceptibility to DNA Methylation in Normal Tissues and Human Primary Tumors Cancer Res., August 1, 2002; 62(15): 4519 - 4524. [Abstract] [Full Text] [PDF]
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S. F. Choumenkovitch, J. Selhub, P. J. Bagley, N. Maeda, M. R. Nadeau, D. E. Smith, and S.-W. Choi In the Cystathionine {beta}-Synthase Knockout Mouse, Elevations in Total Plasma Homocysteine Increase Tissue S-Adenosylhomocysteine, but Responses of S-Adenosylmethionine and DNA Methylation Are Tissue Specific J. Nutr., August 1, 2002; 132(8): 2157 - 2160. [Abstract] [Full Text] [PDF]
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S. J. James, S. Melnyk, M. Pogribna, I. P. Pogribny, and M. A. Caudill Elevation in S-Adenosylhomocysteine and DNA Hypomethylation: Potential Epigenetic Mechanism for Homocysteine-Related Pathology J. Nutr., August 1, 2002; 132(8): 2361S - 2366. [Abstract] [Full Text] [PDF]
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C. Dong, W. Yoon, and P. J. Goldschmidt-Clermont DNA Methylation and Atherosclerosis J. Nutr., August 1, 2002; 132(8): 2406S - 2409. [Abstract] [Full Text] [PDF]
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S. J. Duthie, S. Narayanan, G. M. Brand, L. Pirie, and G. Grant Impact of Folate Deficiency on DNA Stability J. Nutr., August 1, 2002; 132(8): 2444S - 2449. [Abstract] [Full Text] [PDF]
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R. H. Finnell, O. Spiegelstein, B. Wlodarczyk, A. Triplett, I. P. Pogribny, S. Melnyk, and J. S. James DNA Methylation in Folbp1 Knockout Mice Supplemented with Folic Acid during Gestation J. Nutr., August 1, 2002; 132(8): 2457S - 2461. [Abstract] [Full Text] [PDF]
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 M. P. Mattson, S. L. Chan, and W. Duan Modification of Brain Aging and Neurodegenerative Disorders by Genes, Diet, and Behavior Physiol Rev, July 1, 2002; 82(3): 637 - 672. [Abstract] [Full Text] [PDF]
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 E. Di Pietro, J. Sirois, M. L. Tremblay, and R. E. MacKenzie Mitochondrial NAD-Dependent Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase Is Essential for Embryonic Development Mol. Cell. Biol., June 15, 2002; 22(12): 4158 - 4166. [Abstract] [Full Text] [PDF]
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K. Yamada, Z. Chen, R. Rozen, and R. G. Matthews Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase PNAS, December 6, 2001; (2001) 261469998. [Abstract] [Full Text] [PDF]
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K. Yamada, Z. Chen, R. Rozen, and R. G. Matthews Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase PNAS, December 18, 2001; 98(26): 14853 - 14858. [Abstract] [Full Text] [PDF]
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S. Dayal, T. Bottiglieri, E. Arning, N. Maeda, M. R. Malinow, C. D. Sigmund, D. D. Heistad, F. M. Faraci, and S. R. Lentz Endothelial Dysfunction and Elevation of S-Adenosylhomocysteine in Cystathionine {beta}-Synthase-Deficient Mice Circ. Res., June 8, 2001; 88(11): 1203 - 1209. [Abstract] [Full Text] [PDF]
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