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.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37524 |
| Date | January 1997 |
| Creators | Goyette, Philippe. |
| Contributors | Rozen, Rima (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001615783, proquestno: NQ44442, Theses scanned by UMI/ProQuest. |
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