Background/Objectives: The MTHFR677C>T gene variant results in a thermolabile MTHFR enzyme associated with elevated plasma homocysteine in TT individuals. Health risks associated with the TT genotype may be modified by dietary and supplemental folate intake. Supplementation with methyltetrahydrofolate (methylTHF) may be preferable to folic acid because it is the MTHFR product, and does not require reduction by DHFR to enter one-carbon folate metabolism. In the Mthfr677C>T mouse model for this variant, female 677TT (TT) mice have an increased incidence of hepatic steatosis. The objective of this study was to compare the effects of methylTHF and folic acid supplementation on hepatic steatosis and one-carbon metabolism in this model. Methods: Male and female C57BL/6J 677CC (CC) and TT mice were fed control (CD), 5xmethylTHF-supplemented (MFSD), or 5xfolic-acid-supplemented (FASD) diets for 4 months. Liver sections were assessed for steatosis by Oil Red O staining. One-carbon metabolites were measured in the liver and plasma. MTHFR protein expression was evaluated in the liver. Results: MFSD had no significant effect on plasma homocysteine, liver SAM/SAH ratios, or hepatic steatosis in males or females as compared to CD. MTHFR protein increased in MFSD TT female liver, but remained <50% of the CC. FASD had no effect on plasma homocysteine but it decreased the liver MTHFR protein and SAM/SAH ratios, and increased hepatic steatosis in CC females. Conclusions: MethylTHF and folic acid supplementation had limited benefits for TT mice, while folic acid supplementation had negative effects on CC females. Further investigation is required to determine if these effects are relevant in humans.

Scope Disturbances in one‐carbon metabolism contribute to nonalcoholic fatty liver disease (NAFLD) which encompasses steatosis, steatohepatitis, fibrosis, and cirrhosis. The goal is to examine impact of folate deficiency and the Mthfr 677C >T variant on NAFLD. Methods and results This study uses the new Mthfr 677C >T mouse model for the human MTHFR 677C >T variant. Mthfr 677CC and Mthfr 677TT mice were fed control diet (CD) or folate‐deficient (FD) diets for 4 months. FD and Mthfr 677TT alter choline/methyl metabolites in liver and/or plasma (decreased S‐adenosylmethionine (SAM):S‐adenosylhomocysteine (SAH) ratio, methyltetrahydrofolate, and betaine; increased homocysteine [Hcy]). FD, with contribution from Mthfr 677TT , provokes fibrosis in males. Studies of normal livers reveal alterations in plasma markers and gene expression that suggest an underlying predisposition to fibrosis induced by FD and/or Mthfr 677TT in males. These changes are absent or reverse in females, consistent with the sex disparity of fibrosis. Sex‐based differences in methylation potential, betaine, sphingomyelin, and trimethylamine‐ N ‐oxide (TMAO) levels may prevent fibrogenesis in females. In contrast, Mthfr 677TT alters choline metabolism, dysregulates expression of lipid metabolism genes, and promotes steatosis in females. Conclusion This study suggests that folate deficiency predisposes males to fibrosis, which is exacerbated by Mthfr 677TT , whereas Mthfr 677TT predisposes females to steatosis, and reveal novel contributory mechanisms for these NAFLD‐related disorders.

PDF file - 663K, Supplementary Figure S1: Confirmation of the quality of RNA in microarray experiments by denaturing gel electrophoresis in 1% agarose. Supplementary Figure S2: Representative pyrograms for PDK4, SPRR2A, SPRR1A, NR1H4 and PYCARD. Supplementary Figure S3: Identification of individual genes and functional gene categories with significant expression changes between BALB/c Mthfr+/- FD and BALB/c Mthfr+/+ CD mice. Supplementary Figure S4: Effect of diet and Mthfr genotype on expression of eight genes in normal intestine of BALB/c mice. Supplementary Figure S5: Correlation of methylation results between quantitative bisulfite-pyrosequencing and the Infinium methylation array. Supplementary Figure S6: Confirmation of DNA methylation differences in normal colon between control subjects and individuals with CRC. Supplementary Table S1: Description of control subjects. Supplementary Table S2: Description of cancer patients. Supplementary Table S3: Primer pairs for qRT-PCR for the 13 genes in Figs S1 and S2, and the internal control, Gapdh. Supplementary Table S4: Taqman primers and probes for qRT-PCR of human genes. Supplementary Table S5: Oligonucleotides used for pyrosequencing (PCR primers and sequencing primers). Supplementary Table S6: List of probe sets showing significant changes comparing Mthfr+/- FD and Mthfr+/+ CD BALB/c mice. Supplementary Table S7: Relative mRNA levels for four genes related to PPAR activation (Bcmo1, Aldh1a1), oxidative stress sensitivity (Sprr2a) or oxidative response (Ugt2b36). Supplementary Table S8: Relative expression of PPAR responsive genes.

Food fortification and increased vitamin intake have led to higher folic acid (FA) consumption by many pregnant women. We showed that FA-supplemented diet in pregnant mice (fivefold higher FA than the recommended level (5xFASD)) led to hyperactivity-like behavior and memory impairment in pups. Disturbed choline/methyl metabolism and altered placental gene expression were identified. The aim of this study was to examine the impact of 5xFASD on the brain at two developmental stages, postnatal day (P) 30 and embryonic day (E) 17.5. Female C57BL/6 mice were fed a control diet or 5xFASD for 1 month before mating. Diets were maintained throughout the pregnancy and lactation until P30 or during pregnancy until E17.5. The 5xFASD led to sex-specific transcription changes in a P30 cerebral cortex and E17.5 cerebrum, with microarrays showing a total of 1003 and 623 changes, respectively. Enhanced mRNA degradation was observed in E17.5 cerebrum. Expression changes of genes involved in neurotransmission, neuronal growth and development, and angiogenesis were verified by qRT-PCR; 12 and 15 genes were verified at P30 and E17.5, respectively. Hippocampal collagen staining suggested decreased vessel density in FASD male embryos. This study provides insight into the mechanisms of neurobehavioral alterations and highlights potential deleterious consequences of moderate folate oversupplementation during pregnancy.

SCOPE:Many pregnant women have higher folic acid (FA) intake due to food fortification and increased vitamin use. It is reported that diets containing five-fold higher FA than recommended for mice (5xFASD) during pregnancy resulted in methylenetetrahydrofolate reductase (MTHFR) deficiency and altered choline/methyl metabolism, with neurobehavioral abnormalities in newborns. The goal is to determine whether these changes have their origins in the placenta during embryonic development.METHODS AND RESULTS:Female mice are fed control diet or 5xFASD for a month before mating and maintained on these diets until embryonic day 17.5. 5xFASD led to pseudo-MTHFR deficiency in maternal liver and altered choline/methyl metabolites in maternal plasma (increased methyltetrahydrofolate and decreased betaine). Methylation potential (S-adenosylmethionine:S-adenosylhomocysteine ratio) and glycerophosphocholine are decreased in placenta and embryonic liver. Folic acid supplemented diet results in sex-specific transcriptome profiles in placenta, with validation of dietary expression changes of 29 genes involved in angiogenesis, receptor biology or neurodevelopment, and altered methylation of the serotonin receptor 2A gene.CONCLUSION:Moderate increases in folate intake during pregnancy result in placental metabolic and gene expression changes, particularly in angiogenesis, which may contribute to abnormal behavior in pups. These results are relevant for determining a safe upper limit for folate intake during pregnancy.

Fifteen to 20% of pregnant women may exceed the recommended intake of folic acid (FA) by more than four-fold. This excess could compromise neurocognitive and motor development in offspring. Here, we explored the impact of an FA-supplemented diet (5× FASD, containing five-fold higher FA than recommended) during pregnancy on brain function in murine offspring, and elucidated mechanistic changes. We placed female C57BL/6 mice for one month on control diets or 5× FASD before mating. Diets were maintained throughout pregnancy and lactation. Behavioural tests were conducted on 3-week-old pups. Pups and mothers were sacrificed at weaning. Brains and livers were collected to examine choline/methyl metabolites and immunoreactive methylenetetrahydrofolate reductase (MTHFR). 5× FASD led to hyperactivity-like behavior and memory impairment in 3-week-old pups of both sexes. Reduced MTHFR protein in the livers of FASD mothers and male pups resulted in choline/methyl metabolite disruptions in offspring liver (decreased betaine) and brain (decreased glycerophosphocholine and sphingomyelin in male pups, and decreased phosphatidylcholine in both sexes). These results indicate that moderate folate supplementation downregulates MTHFR and alters choline/methyl metabolism, contributing to neurobehavioral alterations. Our findings support the negative impact of high FA on brain development, and may lead to improved guidelines on optimal folate levels during pregnancy.

Food fortification with folic acid and increased use of vitamin supplements have raised concerns about high folic acid intake. We previously showed that high folic acid intake was associated with hepatic degeneration, decreased levels of methylenetetrahydrofolate reductase (MTHFR), lower methylation potential, and perturbations of lipid metabolism. MTHFR synthesizes the folate derivative for methylation reactions. In this study, we assessed the possibility that high folic acid diets, fed to wild-type and Mthfr+/− mice, could alter DNA methylation and/or deregulate hepatic cholesterol homeostasis. Digital restriction enzyme analysis of methylation in liver revealed DNA hypomethylation of a CpG in the lipolysis-stimulated lipoprotein receptor (Lsr) gene, which is involved in hepatic uptake of cholesterol. Pyrosequencing confirmed this methylation change and identified hypomethylation of several neighboring CpG dinucleotides. Lsr expression was increased and correlated negatively with DNA methylation and plasma cholesterol. A putative binding site for E2F1 was identified. ChIP-qPCR confirmed reduced E2F1 binding when methylation at this site was altered, suggesting that it could be involved in increasing Lsr expression. Expression of genes in cholesterol synthesis, transport or turnover (Abcg5, Abcg8, Abcc2, Cyp46a1, and Hmgcs1) was perturbed by high folic acid intake. We also observed increased hepatic cholesterol and increased expression of genes such as Sirt1, which might be involved in a rescue response to restore cholesterol homeostasis. Our work suggests that high folic acid consumption disturbs cholesterol homeostasis in liver. This finding may have particular relevance for MTHFR-deficient individuals, who represent ~10% of many populations.

ScopeDietary and genetic folate disturbances can lead to nonalcoholic fatty liver disease (NAFLD). A common variant in methylenetetrahydrofolate reductase (MTHFR 677C→T) causes mild MTHFR deficiency with lower 5‐methyltetrahydrofolate for methylation reactions. The goal is to determine whether mild murine MTHFR deficiency contributes to NAFLD‐related effects.Methods and resultsWild‐type and Mthfr+/− mice, a model for the human variant, are fed control (CD) or high‐fat (HFAT) diets for 8 weeks. On both diets, MTHFR deficiency results in decreased S‐adenosylmethionine, increased S‐adenosylhomocysteine, and decreased betaine with reduced methylation capacity, and changes in expression of several inflammatory or anti‐inflammatory mediators (Saa1, Apoa1, and Pon1). On CD, MTHFR deficiency leads to microvesicular steatosis with expression changes in lipid regulators Xbp1s and Cyp7a1. The combination of MTHFR deficiency and HFAT exacerbates changes in inflammatory mediators and introduces additional effects on inflammation (Saa2) and lipid metabolism (Nr1h4, Srebf1c, Ppara, and Crot). These effects are consistent with increased expression of pro‐inflammatory HDL precursors and greater lipid accumulation. MTHFR deficiency may enhance liver injury through alterations in methylation capacity, inflammatory response, and lipid metabolism.ConclusionIndividuals with the MTHFR variant may be at increased risk for liver disease and related complications, particularly when consuming high‐fat diets.

Supplementation with high doses of folic acid, an important mediator of one-carbon transfers for DNA methylation, is used clinically to improve sperm parameters in infertile men. We recently detected an unexpected loss of DNA methylation in the sperm of idiopathic infertile men after 6 months of daily supplementation with 5 mg folic acid (>10× the daily recommended intake-DRI), exacerbated in men homozygous for a common variant in the gene encoding an important enzyme in folate metabolism, methylenetetrahydrofolate reductase (MTHFR 677C>T). To investigate the epigenomic impact and mechanism underlying effects of folic acid on male germ cells, wild-type and heterozygote mice for a targeted inactivation of the Mthfr gene were fed high-dose folic acid (10× the DRI) or control diets (CDs) for 6 months. No changes were detected in general health, sperm counts or methylation of imprinted genes. Reduced representation bisulfite sequencing revealed sperm DNA hypomethylation in Mthfr+/- mice on the 10× diets. Wild-type mice demonstrated sperm hypomethylation only with a very high dose (20×) of folic acid for 12 months. Testicular MTHFR protein levels decreased significantly in wild-type mice on the 20× diet but not in those on the 10× diet, suggesting a possible role for MTHFR deficiency in sperm DNA hypomethylation. In-depth analysis of the folic acid-exposed sperm DNA methylome suggested mouse/human susceptibility of sequences with potential importance to germ cell and embryo development. Our data provide evidence for a similar cross-species response to high dose folic acid supplementation, of sperm DNA hypomethylation, and implicate MTHFR downregulation as a possible mechanism.

Folate is an important B vitamin required for methylation reactions, nucleotide and neurotransmitter synthesis, and maintenance of homocysteine at nontoxic levels. Its metabolism is tightly linked to that of choline, a precursor to acetylcholine and membrane phospholipids. Low folate intake and genetic variants in folate metabolism, such as the methylenetetrahydrofolate reductase (MTHFR) 677 C>T polymorphism, have been suggested to impact brain function and increase the risk for cognitive decline and late-onset Alzheimer’s disease. Our study aimed to assess the impact of genetic and nutritional disturbances in folate metabolism, and their potential interaction, on features of cognitive decline and brain biochemistry in a mouse model. Wild-type and Mthfr+/− mice, a model for the MTHFR 677 C>T polymorphism, were fed control or folate-deficient diets from weaning until 8 and 10 months of age. We observed short-term memory impairment measured by the novel object paradigm, altered transcriptional levels of synaptic markers and epigenetic enzymes, as well as impaired choline metabolism due to the Mthfr+/− genotype in cortex or hippocampus. We also detected changes in mRNA levels of Presenillin-1, neurotrophic factors, one-carbon metabolic and epigenetic enzymes, as well as reduced levels of S-adenosylmethionine and acetylcholine, due to the folate-deficient diet. These findings shed further insights into the mechanisms by which genetic and dietary folate metabolic disturbances increase the risk for cognitive decline and suggest that these mechanisms are distinct.

BACKGROUND:Cancer cells maintain high rates of glycolysis. Pyruvate dehydrogenase kinases (PDK) contribute to this phenomenon, which favours apoptosis resistance and cellular transformation. We previously reported upregulation of PDK4 in normal mucosa of colorectal cancer (CRC) patients compared with controls and in preneoplastic intestine of our mouse model. Decreased methylation of four consecutive PDK4 CpGs was observed in normal mucosa of patients. Although other members of the PDK family have been investigated for transformation potential, PDK4 has not been extensively studied.METHODS:PDK4 methylation in blood of CRC patients and controls was evaluated by pyrosequencing. PDK4 expression in human colon carcinoma cells was down-regulated by RNAi. Cellular migration and invasion, apoptosis and qRT-PCR of key genes were assessed.RESULTS:Pyrosequencing revealed decreased methylation of the same four consecutive CpGs in the blood of patients compared with controls. Cellular migration and invasion were reduced and apoptosis was increased following transient or stable inhibition of PDK4. Expression of vimentin, HIF-1 and VEGFA was reduced.CONCLUSIONS:These studies demonstrate the involvement of PDK4 in transformation. Methylation assessment of PDK4 in the blood may be useful for non-invasive CRC detection. PDK4 should be considered as a target for development of anticancer strategies and therapies.

Polymorphisms and decreased activity of methylenetetrahydrofolate reductase (MTHFR) are linked to disease, including cancer. However, epigenetic regulation has not been thoroughly studied. Our goal was to generate DNA methylation profiles of murine/human MTHFR gene regions and examine methylation in brain and liver tumors. Pyrosequencing in four murine tissues revealed minimal DNA methylation in the CpG island. Higher methylation was seen in liver or intestine in the CpG island shore 5′ to the upstream translational start site or in another region 3′ to the downstream start site. In the latter region, there was negative correlation between expression and methylation. Three orthologous regions were investigated in human MTHFR, as well as a fourth region between the two translation start sites. We found significantly increased methylation in three regions (not the CpG island) in pediatric astrocytomas compared with control brain, with decreased expression in tumors. Methylation in hepatic carcinomas was also increased in the three regions compared with normal liver, but the difference was significant for only one CpG. This work, the first overview of the Mthfr/MTHFR epigenetic landscape, suggests regulation through methylation in some regions, demonstrates increased methylation/decreased expression in pediatric astrocytomas, and should serve as a resource for future epigenetic studies.

Colorectal cancer risk is increased when dietary folate intake is low, with or without a deficiency in methylenetetrahydrofolate reductase (MTHFR). We have observed that intestinal tumors are induced in mice fed low-folate diets, and that tumor incidence is increased when these mice also have MTHFR deficiency. This study was undertaken to identify differentially expressed proteins in conditions favoring initial steps of murine carcinogenesis in normal preneoplastic intestine. We compared the proteome of BALB/c normal intestine in Mthfr(+/+) mice fed control diets for 1 year (low susceptibility to tumorigenesis) with the proteome of Mthfr(+/-) animals fed low folate diets (higher tumor susceptibility). Our data suggest that the NuRD complex, KRAS-related proteins, the protein synthetic machinery, and fatty acid-related metabolic proteins are upregulated in the early stages of tumorigenesis. These proteins may serve as biomarkers or targets for colorectal cancer diagnosis or therapy.

IMPORTANCE Hereditary spastic paraplegia is a highly heterogeneous group of neurogenetic disorders with pure and complicated clinical phenotypes. No treatment is available for these disorders. We identified 2 unrelated families, each with 2 siblings with severe methylenetetrahydrofolate reductase (MTHFR) deficiency manifesting a complicated form of adult-onset hereditary spastic paraparesis partially responsive to betaine therapy.OBSERVATIONS Both pairs of siblings presented with a similar combination of progressive spastic paraparesis and polyneuropathy, variably associated with behavioral changes, cognitive impairment, psychosis, seizures, and leukoencephalopathy, beginning between the ages of 29 and 50 years. By the time of diagnosis a decade later, 3 patients were ambulatory and 1 was bedridden. Investigations have revealed severe hyperhomocysteinemia and hypomethioninemia, reduced fibroblast MTHFR enzymatic activity (18%-52% of control participants), and 3 novel pathogenic MTHFR mutations, 2 as compound heterozygotes in one family and 1 as a homozygous mutation in the other family. Treatment with betaine produced a rapid decline of homocysteine by 50% to 70% in all 4 patients and. over 9 to 15 years, improved the conditions of the 3 ambulatory patients.CONCLUSIONS AND RELEVANCE Although severe MTHFR deficiency is a rare cause of complicated spastic paraparesis in adults, it should be considered in select patients because of the potential therapeutic benefit of betaine supplementation.

BACKGROUND:β,β-Carotene 15,15'-monooxygenase (BCMO1) converts β-carotene to retinaldehyde. Increased β-carotene consumption is linked to antitumor effects. Retinoic acid reduces the invasiveness in cancer, through inhibition of matrix metalloproteinases (MMPs). In our studies of a mouse model that develops intestinal tumors after low dietary folate, we found reduced BCMO1 expression in normal preneoplastic intestine of folate-deficient tumor-prone mice.OBJECTIVE:Our goal was to determine whether BCMO1 expression could influence transformation potential in human colorectal carcinoma cells, by examining the effect of BCMO1 modulation on cellular migration and invasion, and on expression of MMPs.DESIGN:LoVo colon carcinoma cells were transfected with BCMO1 small interfering RNA (siRNA) or scrambled siRNA. Migration and invasion were measured, and the expression of BCMO1, MMP7, and MMP28 was assessed by quantitative reverse-transcriptase polymerase chain reaction. These variables were also measured after treatment of cells with retinoic acid, 5-aza-2'-deoxycytidine, folate-depleted/high-methionine medium, and β-carotene.RESULTS:Retinoic acid decreased the migration, invasion, and expression of MMP28 mRNA. Transfection of cells with BCMO1 siRNA inhibited BCMO1 expression, enhanced migration and invasion, and increased expression of MMP7 and MMP28. 5-Aza-2'-deoxycytidine decreased, whereas folate-depleted/high-methionine medium increased invasiveness. β-Carotene increased BCMO1 expression and reduced invasiveness with a decrease in expression of MMP7 and MMP28.CONCLUSIONS:Inhibition of BCMO1 expression is associated with increased invasiveness of colon cancer cells and increased expression of MMP7 and MMP28. β-Carotene can upregulate BCMO1 and reverse these effects. These novel associations suggest a critical role for BCMO1 in cancer and provide a mechanism for the proposed antitumor effects of β-carotene.

Background: b-Carotene 15,15#-monooxygenase 1 (BCMO1) converts b-carotene to retinaldehyde. Increased b-carotene consumption is linked to antitumor effects. Retinoic acid reduces the invasiveness in cancer, through inhibition of matrix metalloproteinases (MMPs). In our studies of a mouse model that develops intestinal tumors after low dietary folate, we found reduced BCMO1 expression in normal preneoplastic intestine of folate-deficient

Abstract An understanding of early genetic/epigenetic changes in colorectal cancer would aid in diagnosis and prognosis. To identify these changes in human preneoplastic tissue, we first studied our mouse model in which Mthfr+/− BALB/c mice fed folate-deficient diets develop intestinal tumors in contrast to Mthfr+/+ BALB/c mice fed control diets. Transcriptome profiling was performed in normal intestine from mice with low or high tumor susceptibility. We identified 12 upregulated and 51 downregulated genes in tumor-prone mice. Affected pathways included retinoid acid synthesis, lipid and glucose metabolism, apoptosis and inflammation. We compared murine candidates from this microarray analysis, and murine candidates from an earlier strain-based comparison, with a set of human genes that we had identified in previous methylome profiling of normal human colonic mucosa, from colorectal cancer patients and controls. From the extensive list of human methylome candidates, our approach uncovered five orthologous genes that had shown changes in murine expression profiles (PDK4, SPRR1A, SPRR2A, NR1H4, and PYCARD). The human orthologs were assayed by bisulfite-pyrosequencing for methylation at 14 CpGs. All CpGs exhibited significant methylation differences in normal mucosa between colorectal cancer patients and controls; expression differences for these genes were also observed. PYCARD and NR1H4 methylation differences showed promise as markers for presence of polyps in controls. We conclude that common pathways are disturbed in preneoplastic intestine in our animal model and morphologically normal mucosa of patients with colorectal cancer, and present an initial version of a DNA methylation-based signature for human preneoplastic colon. Cancer Prev Res; 6(11); 1171–81. ©2013 AACR.

An understanding of early genetic/epigenetic changes in colorectal cancer would aid in diagnosis and prognosis. To identify these changes in human preneoplastic tissue, we first studied our mouse model in which Mthfrþ/ BALB/c mice fed folate-deficient diets develop intestinal tumors in contrast to Mthfrþ/þ BALB/c mice fed control diets. Transcriptome profiling was performed in normal intestine from mice with lowor high tumor susceptibility.We identified 12 upregulated and 51 downregulated genes in tumor-prone mice. Affected pathways included retinoid acid synthesis, lipid and glucose metabolism, apoptosis and inflammation.We comparedmurine candidates from thismicroarray analysis, andmurine candidates from an earlier strain-based comparison, with a set of human genes that we had identified in previousmethylome profiling of normal humancolonicmucosa, fromcolorectal cancer patients and controls. Fromthe extensive list of human methylome candidates, our approach uncovered five orthologous genes that had shown changes in murine expression profiles (PDK4, SPRR1A, SPRR2A, NR1H4, and PYCARD). The human orthologs were assayed by bisulfite-pyrosequencing for methylation at 14 CpGs. All CpGs exhibited significant methylation differences in normal mucosa between colorectal cancer patients and controls; expression differences for these genes were also observed. PYCARD and NR1H4 methylation differences showed promise as markers for presence of polyps in controls. We conclude that common pathways are disturbed in preneoplastic intestine in our animal model and morphologically normal mucosa of patients with colorectal cancer, and present an initial version of a DNA methylation-based signature for human preneoplastic colon. Cancer Prev Res; 6(11); 1–11. 2013 AACR.