Methylenetetrahydrofolate reductase catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a carbon donor for the remethylation of homocysteine to methionine. Patients with severe MTHFR deficiency have $<$20% residual enzyme activity, moderate hyperhomocysteinemia, vascular lesions and neurological dysfunction. Mildly-deficient individuals with a thermolabile enzyme are at increased risk for developing cardiovascular disease. / Two MTHFR sequence changes were identified. The first was a C to T transition at bp 764 altering a proline to a leucine codon; this change was found in one severely-deficient patient. The second was a C to T transition at bp 677, substituting a valine for a highly-conserved alanine codon. The $ rm A to V$ substitution was identified on 35-40% of chromosomes. Expression of the $ rm A to V$ mutation in prokaryotic cells revealed increased thermolability over the wild-type enzyme. Genotyping for the $ rm A to V$ mutation in three vascular disease studies showed that it was associated with mild hyperhomocysteinemia, a risk factor for vascular disease. / The preventative effects of folate supplementation on the occurrence and recurrence of neural tube defects (NTDs) have been repeatedly demonstrated. The curly-tail (ct) mouse model for NTDs was used to investigate the involvement of MTHFR in these defects. Ct mice had significantly increased homocysteine levels although differences in MTHFR activity were not demonstrated. The mouse MTHFR gene was mapped to distal chromosome 4, close to the major gene for NTDs in ct. MTHFR is suggested as a candidate locus for the ct defect.

Statement of problem: We have shown that the C677T 5,10 methylenetetrahydrofolate reductase (MTHFR) variant is associated with elevated maternal serum α-fetoprotein (MSAFP), the most common screening test for neural tube defects (NTD). Therefore, past contradictory studies of NTDs and C677T MTHFR may have been biased because of changes in case populations after prenatal diagnosis and termination of pregnancy. Further, an unexplained elevation of MSAFP is known to increase the risk for later pregnancy complications. Is the C677T MTHFR variant a predisposing genetic variant for both NTDs and later complications of pregnancy? Methods: A retrospective study of women with pregnancies resulting in NTD outcome and women with unexplained elevations of MSAFP was undertaken. Women and their partners were genotyped for the C677T MTHFR allele. Couples with a pregnancy resulting in a NTD outcome were compared to couples whose pregnancy outcome did not involve. Couples with unexplained elevations of MSAFP who did and did not have later complications of pregnancy were also compared. Allele frequencies for all groups were then compared against the previously established Manitoba population allele frequency (based on 977 consecutive newborn metabolic screening bloodspots). A review of all studies of NTDs and association with the C677T MTHFR variant was undertaken to determine if the association between the variant and MSAFP is a source of bias. NTD incidence was examined before and after folic acid food fortification introduced in Canada in 1999. Results: There is an increase in the allele frequency of the C677T MTHFR variant in parents with an unexplained elevated MSAFP followed by later complications of pregnancy. The C677T MTHFR variant is also a contributing genetic factor to NTDs worldwide. The incidence of NTDs in Manitoba has decreased by 37% since food fortification with folic acid was introduced. Conclusions: The C677T MTHFR variant is a contributing genetic factor to both later complications of pregnancy after an unexplained elevation of MSAFP and to NTDs. This variant is folate sensitive and folic acid fortification has reduced the incidence of NTDs.

Statement of problem: We have shown that the C677T 5,10 methylenetetrahydrofolate reductase (MTHFR) variant is associated with elevated maternal serum α-fetoprotein (MSAFP), the most common screening test for neural tube defects (NTD). Therefore, past contradictory studies of NTDs and C677T MTHFR may have been biased because of changes in case populations after prenatal diagnosis and termination of pregnancy. Further, an unexplained elevation of MSAFP is known to increase the risk for later pregnancy complications. Is the C677T MTHFR variant a predisposing genetic variant for both NTDs and later complications of pregnancy? Methods: A retrospective study of women with pregnancies resulting in NTD outcome and women with unexplained elevations of MSAFP was undertaken. Women and their partners were genotyped for the C677T MTHFR allele. Couples with a pregnancy resulting in a NTD outcome were compared to couples whose pregnancy outcome did not involve. Couples with unexplained elevations of MSAFP who did and did not have later complications of pregnancy were also compared. Allele frequencies for all groups were then compared against the previously established Manitoba population allele frequency (based on 977 consecutive newborn metabolic screening bloodspots). A review of all studies of NTDs and association with the C677T MTHFR variant was undertaken to determine if the association between the variant and MSAFP is a source of bias. NTD incidence was examined before and after folic acid food fortification introduced in Canada in 1999. Results: There is an increase in the allele frequency of the C677T MTHFR variant in parents with an unexplained elevated MSAFP followed by later complications of pregnancy. The C677T MTHFR variant is also a contributing genetic factor to NTDs worldwide. The incidence of NTDs in Manitoba has decreased by 37% since food fortification with folic acid was introduced. Conclusions: The C677T MTHFR variant is a contributing genetic factor to both later complications of pregnancy after an unexplained elevation of MSAFP and to NTDs. This variant is folate sensitive and folic acid fortification has reduced the incidence of NTDs.

Elevated total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease and neurocognitive disease such as dementia. The B vitamins folate and B12 are the main de terminants of tHcy. tHcy concentration can also be affected by mutations in genes coding for receptors, enzymes and transporters important in the metabolism of Hcy. This thesis focuses on mutations in the genes for folate receptor-alpha and methylenetetrahydrofolate reductase (MTHFR) and the effect they have on tHcy concentrations. Six novel mutations in the gene for folate receptor-alpha were described in Paper I. Taken together they exist in a population with a prevalence of approximately 1% and thus are not unusual. There may be an association of –69dupA and –18C&gt;T to tHcy but for the 25-bp deletion, –856C&gt;T, –921T&gt;C and –1043G&gt;A there is probably no association to tHcy. Mutation screening was continued and four additional mutations, 1314G&gt;A, 1816delC, 1841G&gt;A and 1928C&gt;T, were described in Paper II. The prevalences for the heterozygotes were between 0.5% and 13% in an elderly population. There was no significant difference in prevalence between the elderly subjects and patients with dementia. The 1816(–)-allele and the 1841A-allele were in complete linkage and the haplotype 1816(–)-1841A may possibly have a tHcy raising effect. The 1314G&gt;A and 1928C&gt;T mutations had no association to tHcy. The genotype prevalences and haplotype frequencies of the MTHFR 677C&gt;T, 1298A&gt;C and 1793G&gt;A polymorphisms were determined in a population sample of Swedish children and adolescents (Paper III). The MTHFR 677T-allele was associated with increased tHcy concentrations in both children and adolescents. A small elevating effect of the 1298C-allele and a small lowering effect of the 1793A-allele could be shown. In an epidemiological sample of adults from the Canary Islands, Spain, data for serum folate and vitamin B12 were used for a broader study of the nutrigenetic impact on tHcy (Paper IV). The 677T-allele had a significant tHcy increasing effect in men but not in women. The 1298C-allele had a minor elevating effect on tHcy in men with the 677CT genotype. It was not possible to document any effect of the 1793A-allele on tHcy due to its low prevalence. A slightly superior explanatory power for the genetic impact was obtained using the MTHFR haplotypes in the analysis compared to the MTHFR 677C&gt;T genotype-based approach in both the Swedish children and adolescents and in the Spanish adults. Therefore MTHFR haplotypes should be considered when analysing the impact of the MTHFR 677C&gt;T, 1298A&gt;C and 1793G&gt;A polymorphisms on tHcy. Notwithstanding the large geographical distance between our study populations the haplotype composition is quite similar. The MTHFR 677T-allele is slightly more prevalent in Spain compared to Sweden but it has only an effect on tHcy in the Spanish men. Age, gender and factors linked to the ethnicity of the studied subjects, seem to be able to override the nutrigenetic impact of tHcy-raising genotypes or haplotypes in particular settings, such as in the Spanish women in our study. Gene-nutrient interactions on plasma tHcy levels thus may or may not exist in a certain population. The transferability of nutrigenetic findings may therefore be limited, and must be re-evaluated for each particular setting of age-gender-ethnicity.

The global DNA methylation pattern is erased and re-established during oogenesis and again in preimplantation (PI) embryo development. Understanding where these methyl groups come from and how the process of methylation is regulated is important, as disruptions could result in detrimental effects. The methionine cycle that produces the cellular methyl pool is linked to the folate cycle. The key enzyme linking theses cycles is Methylenetetrahydrofolate Reductase (MTHFR) which converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Mthfr RNA and protein are present throughout mouse oocyte and PI embryo development, including the germinal vesicle, MII egg, 1-cell embryo, 2-cell embryo, morula and blastocysts. In MII eggs the protein appears to be heavier than in any other stage. This was reversed by treatment with Lambda Protein Phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. MTHFR was progressively phosphorylated beginning shortly after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing after egg activation using strontium chloride. Potential kinases responsible for the phosphorylation of MTHFR have been identified however not in oocytes or PI embryos. DYRK1A/1 and GSK3A/B have both been suggested to mediate the phosphorylation, however when inhibited showed no effect on the oocyte sample. An LC-MS/MS assay was attempted to measure the activity of MTHFR in wildtype and knockout mouse liver samples, however unsuccessful in the amounts needed to be used for comparison to oocytes. Overall, MTHFR is present in the developing stages of interest and is mediated in some capacity by phosphorylation modifications around the MII stage of development.

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Epidemiological evidence linking dietary folate deficiency and risk for colorectal cancer is conflicting. Studies using animal models indicate that timing, dose and presence of pre-malignant lesions will influence whether folate deficiency prevents or promotes tumor formation. In this thesis a new model of spontaneous tumor formation due to long-term dietary folate deficiency alone, in non-transgenic mice and without carcinogen induction, is developed. The mechanisms by which folate deficiency might influence cancer risk are also examined. / BALB/c mice, with or without a null allele in a key folate-metabolizing enzyme, Methylenetetrahydrofolate reductase (Mthfr ), develop intestinal tumors due to dietary folate deficiency alone. On folate-deficient (FD) diets, 12.5% of Mthfr+/+ mice and 28.1% of Mthfr+/- mice developed tumors; mice on control diet (CD) did not. C57B1/6 mice (a strain resistant to other methods of tumor induction) placed on the same diets for the same amount of time did not develop any tumors. To investigate possible mechanisms the levels of DNA damage (dUTP/dTTP ratio and p-H2AX staining) and DNA methylation (thin layer chromatography) were examined. FD BALB/c, but not C57B1/6 mice, had a trend towards increased dUTP/dTTP and DNA double-strand breaks and decreased global DNA methylation compared to CD mice. To determine why the FD diet affects the BALB/c and not the C57Bl/6 strain, the expression of genes involved in folate metabolism was examined. Several changes in gene expression were observed. In particular, BALB/c mice had increased Mthfr expression and MTHFR activity compared to C57Bl/6 mice. Increased MTHFR activity may deplete 5,10-methylenetetrahydrofolate supplies for the dTMP synthesis, increasing the dUMP levels and, possibly, DNA damage. The levels of several DNA repair genes were also examined. Two genes involved in base excision repair, Thymine DNA glycosylase (Tdg) and Apurinic/apyrimidinic endonuclease 1 (Apex1), were increased in FD C57B1/6 compared to FD BALB/c mice suggesting increased DNA repair capacity. / These results support the evidence that dietary folate deficiency promotes intestinal tumor formation possibly through increased DNA damage, with subsequent defects in DNA repair.

Disruptions in folate metabolism are known to increase the risk for neural tube defects (NTD) and this is preventable by folic acid supplementation. However, the relationship between folate metabolism and cardiac development remains unclear. The interaction between other folate pathway nutrients, choline and riboflavin, and folate metabolism was studied in a murine model of methylenetetrahydrofolate reductase (MTHFR) deficiency. Maternal choline deficiency, riboflavin deficiency and MTHFR deficiency adversely affected embryonic or heart development. The promoters of MTHFR were also examined for interactions with GATA-4, TBX5, MEF2A and NKX-2.5, known transcription factors of cardiac development. Upstream promoter activity was increased in the presence of GATA-4 and this interaction was further enhanced upon the addition of MEF2A. TBX5 appeared to decrease upstream promoter activity. GATA-4 modestly increased downstream promoter activity. These results highlight the importance of adequate nutrient intake during pregnancy and provide a link between folate metabolism and cardiac development.

Folate deficiency, a prevalent vitamin deficiency in America, can stem from environmental and/or genetic causes. The most common inborn error of folate metabolism is deficiency of methylenetetrahydrofolate reductase (MTHFR), which catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Severe MTHFR deficiency results in hyperhomocysteinemia and homocystinuria; patients present with developmental delay, and various neurological and vascular disorders. This thesis describes three mutations identified in the MTHFR locus in patients with severe deficiency: 1025T&rarr;C (M&rarr;T), 1027T&rarr;G (W&rarr;G), and 1768G&rarr;A (E&rarr;K). Genotype-phenotype correlations are described, along with biochemical characterization of three mutations (983A&rarr;G (N&rarr;S), 1025T&rarr;C, 1027T&rarr;G). All three mutations exert their effect by decreasing Vmax without changing the enzyme's affinity for its substrate, 5-methyltetrahydrofolate. The 983A&rarr;G variant also conferred decreased affinity for FAD, a cofactor. / The more common and mild deficiency observed in the general healthy population is probably due in part to insufficient dietary intake of folate. Folate deficiency has been associated with increased risk for colon cancer. In a pilot study presented here, the impact of altered folate intake on tumor multiplicity in the Min mouse, a model for multiple intestinal neoplasia, was assessed. Folate deficient diets did not produce a consistent change in tumor numbers. However, a linear correlation between S-adenosylmethionine and S-adenosylhomocysteine content of preneoplastic tissue and tumor multiplicity was identified. / This thesis contributes to our understanding of the impact of genetic- and/or dietary-induced folate deficiency on cellular and organismal functions.