Methylenetetrahydrofolate reductase (MTHFR) catalyses the conversion of 5, 10-methylenctetrahydrofolate to 5-methyltetrahydrofolate, co-substrate of methionine synthesis. Two types of deficiency have been described for MTHFR. Severe MTHFR deficiency, associated with severe hyperhomocysteinemia and homocystinuria, shows levels of MTHFR activity below 20% of control values. This deficiency has a variable age of onset and shows a wide range of neurological and vascular defects. Mild MTHFR deficiency, with ≈50% enzyme activity and marked enzyme thermolability, has been proposed as a genetic factor in the development of mild hyperhomocysteinemia, a condition associated with neural tube defects and premature vascular disease. / The goal of this thesis was to determine the molecular basis for severe MTHFR deficiency. In order to study this, I isolated a 1.2 Kb partial cDNA encoding human MTHFR I determined that its primary amino acid sequence is homologous to the bacterial enzyme, and encodes the N-terminal catalytic domain of MTHFR. The cDNA was used to isolate a full length 2.27 Kb cDNA, to map the locus to chromosome position 1p36.3, and to isolate genomic clones for human and mouse MTHFR. I characterized the mouse cDNA sequence, as well as the gene structure for both human and mouse genes. I observed 90% identity at the amino acid level, almost identical sizes of exons and location of introns, and similar sizes of introns. The exon sizes ranged from 102bp to 432bp, and intron sizes varied from 250bp to 4.2Kb. / I identified 13 mutations in severe MTHFR deficiency: 10 missense mutations, 1 nonsense mutation, and 2 splicing defects. I determined that a previously-identified common variant (an Ala →Val mutation) was causative of thermolability in severe MTHFR deficiency. I showed a correlation between genotype, residual activity and phenotype in this disease. I also correlated the presence of another genetic defect, Factor V Leiden mutation, with the possible development of thrombo-embolic events in MTHFR deficiency patients. Finally, I analyzed 8 MTHFR mutations in a bacterial expression system. I determined that 4 of these caused significant reduction of activity (below 20% of control). / This thesis contains the first reports of genetic defects in folate metabolism, and a review of available data in severe MTHFR deficiency.

Breast cancer is the most common malignancy affecting women in developed countries. The BRCA1 and BRCA2 germline mutations predispose carriers to breast and ovarian cancers and account for approximately 10% of all breast cancer cases. Diets rich in micronutrients, such as carotenoids, folate, vitamin C and E are associated with reduced breast cancer risk. / Folate functions in one-carbon metabolism, and is an important factor in DNA-synthesis, DNA repair as well as DNA methylation. Moderate folate deficiency induces global DNA hypomethylation, gene promoter CpG hypermethylation and excessive uracil incorporation into DNA, causing genome instability and successively increasing cancer risk. Genome instabilities such as chromosome 17 mal-segregation and Her2 amplification are frequently observed in human breast tumours. / Methionine, another key factor in one-carbon metabolism, is the sole methy-donor for DNA methylation. Many human tumours are methionine-dependent, a phenotype characterised by the inability of cells to grow when methionine is replaced by its precursor homocysteine, causing elevated homocysteine levels, global DNA hypomethylation when methionine is restricted. In addition, genetic polymorphisms may affect enzyme activity and modulate cancer risk. / This thesis describes a study on the impact of selected nutrients, growth hormones and in vivo genome stability on breast cancer risk in BRCA1 or BRCA2 germline mutation carriers. Peripheral blood lymphocyes of BRCA germline mutation carriers and healthy non-carrier controls were studied for the impact of folic acid deficiency on genome damage and the methionine-dependence phenotype (MDP; in combination with common polymorphisms in one-carbon metabolism) on breast cancer risk, respectively. Plasma IGF-1 and IGFBP-3 were determined and chromosome 17 aneuploidy and Her2 amplification were assessed in mononucleated lymphocytes to establish the association of these markers on breast cancer risk in BRCA germline mutation carriers, independently or in combination with plasma folate, vitamin B12, homocysteine, selenium and common gene variants in the one-carbon metabolism, DNA repair genes or glutathionine S-transferase. / Results indicated that folic acid deficiency was a much more important factor affecting chromosome instability than carrying a BRCA1 or a BRCA2 germline mutation. In addition, MDP was associated with development of breast cancer in BRCA1 germline mutation carriers and appeared to be affected by common polymorphisms in methyltetrahydrofolate reductase. The methionine synthase (MTR) A2756G polymorphisms was associated with elevated cell growth when methionine is present in excess and was the only polymorphism studied that was associated with breast cancer risk in BRCA1 and BRCA2 germline mutation carriers. In addition, chromosome 17 aneuploidy, Her2 amplification and plasma IGF-1 and IGFBP-3 did not directly affect breast cancer risk; however, these biomarkers were significantly correlated with each other and MTR A2756G, suggesting a common mechanism for their inter-relationship. Plasma folate, vitamin B12, homocysteine and selenium of BRCA1 and BRCA2 germline mutation carriers were not associated with breast cancer risk and did not differ from non-carrier relatives. Further research with larger study populations are required to confirm these findings. / Thesis (PhDPharmacy)--University of South Australia, 2006.

Breast cancer is the most common malignancy affecting women in developed countries. The BRCA1 and BRCA2 germline mutations predispose carriers to breast and ovarian cancers and account for approximately 10% of all breast cancer cases. Diets rich in micronutrients, such as carotenoids, folate, vitamin C and E are associated with reduced breast cancer risk. / Folate functions in one-carbon metabolism, and is an important factor in DNA-synthesis, DNA repair as well as DNA methylation. Moderate folate deficiency induces global DNA hypomethylation, gene promoter CpG hypermethylation and excessive uracil incorporation into DNA, causing genome instability and successively increasing cancer risk. Genome instabilities such as chromosome 17 mal-segregation and Her2 amplification are frequently observed in human breast tumours. / Methionine, another key factor in one-carbon metabolism, is the sole methy-donor for DNA methylation. Many human tumours are methionine-dependent, a phenotype characterised by the inability of cells to grow when methionine is replaced by its precursor homocysteine, causing elevated homocysteine levels, global DNA hypomethylation when methionine is restricted. In addition, genetic polymorphisms may affect enzyme activity and modulate cancer risk. / This thesis describes a study on the impact of selected nutrients, growth hormones and in vivo genome stability on breast cancer risk in BRCA1 or BRCA2 germline mutation carriers. Peripheral blood lymphocyes of BRCA germline mutation carriers and healthy non-carrier controls were studied for the impact of folic acid deficiency on genome damage and the methionine-dependence phenotype (MDP; in combination with common polymorphisms in one-carbon metabolism) on breast cancer risk, respectively. Plasma IGF-1 and IGFBP-3 were determined and chromosome 17 aneuploidy and Her2 amplification were assessed in mononucleated lymphocytes to establish the association of these markers on breast cancer risk in BRCA germline mutation carriers, independently or in combination with plasma folate, vitamin B12, homocysteine, selenium and common gene variants in the one-carbon metabolism, DNA repair genes or glutathionine S-transferase. / Results indicated that folic acid deficiency was a much more important factor affecting chromosome instability than carrying a BRCA1 or a BRCA2 germline mutation. In addition, MDP was associated with development of breast cancer in BRCA1 germline mutation carriers and appeared to be affected by common polymorphisms in methyltetrahydrofolate reductase. The methionine synthase (MTR) A2756G polymorphisms was associated with elevated cell growth when methionine is present in excess and was the only polymorphism studied that was associated with breast cancer risk in BRCA1 and BRCA2 germline mutation carriers. In addition, chromosome 17 aneuploidy, Her2 amplification and plasma IGF-1 and IGFBP-3 did not directly affect breast cancer risk; however, these biomarkers were significantly correlated with each other and MTR A2756G, suggesting a common mechanism for their inter-relationship. Plasma folate, vitamin B12, homocysteine and selenium of BRCA1 and BRCA2 germline mutation carriers were not associated with breast cancer risk and did not differ from non-carrier relatives. Further research with larger study populations are required to confirm these findings. / Thesis (PhDPharmacy)--University of South Australia, 2006.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2000. / Errata pasted onto t.p. verso. Bibliography: leaves 129-148.

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Pharmacology and Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 219-238.

Thesis (M.S.)--University of Wisconsin--Madison, 1954. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 67-77).

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