It is now well established that elevated total plasma homocysteine is an independent risk factor for cardiovascular disease and that low folate shows association with increased risk for having a child with neural tube defects. Extrapolating from the central role played by a polymorphism of MTHFR in this process, we set out to clone genes and identify polymorphisms to determine if other genes involved in the metabolism of homocysteine would show such associations. A major outcome was the discovery of a novel enzyme methionine synthase reductase (gene and enzyme symbol, MTRR) required for the activation of cobalamin-dependent methionine synthase (MS). Through these experiments we detected a polymorphism, 66A>G (122M), present at a remarkable - aboutO.5 frequency, that appears to be associated with premature coronary artery disease, neural tube defects and Down syndrome. Significant progress included identification of mutations in severely affected patients with MTRR deficiency, modeling the 122M variant in the orthologous E. coli flavodoxin, and development of a mouse model of MTRR deficiency (founder heterozygotes confirmed). We hypothesize that the association of the 122M polymorphism with disease derives from structural and functional disturbances in the variant enzyme with impact on biochemical homeostasis and development. Accordingly, we propose the following specific aims to characterize the mechanism and physiological role of this novel enzyme and polymorphism: (1) Determine the biochemical impact of the 122M mutation in E. coil flavodoxin and humanMTRR. These studies will include biochemical characterization of 122 and M22 MTRR; assessment of FMN binding and domain interactions by 122M flavodoxin; and comparative crystal structure of 122M flavodoxin. (2) Characterize the phenotype of homozygous and heterozygous MTRR knock-out mice to gain understanding of the impact of severe and mild MTRR deficiency on physiology and development. We will examine the expression patterns of MS and MTRR during embryogenesis and in adult tissues by in-situ hybridization, Western blot and tissue immuno-fluorescence; evaluate levels of folate and methionine cycle metabolites in heterozygous and homozygous mutant mice, characterize the general pathology and behavioral phenotype of affected mice; evaluate vascular pathology and function; and explore nutrition-based treatment of homozygous mutant mice. In summary, our study will give a biochemical and physiological face to the impact of 122M on enzyme function, development and disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL058955-05
Application #
6438897
Study Section
Special Emphasis Panel (ZRG1-SSS-2 (03))
Program Officer
Ershow, Abby
Project Start
1997-08-01
Project End
2006-11-30
Budget Start
2001-12-15
Budget End
2002-11-30
Support Year
5
Fiscal Year
2002
Total Cost
$283,725
Indirect Cost
Name
University of Calgary
Department
Type
DUNS #
207663915
City
Calgary
State
AB
Country
Canada
Zip Code
T2 1-N4
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Elmore, C Lee; Wu, Xuchu; Leclerc, Daniel et al. (2007) Metabolic derangement of methionine and folate metabolism in mice deficient in methionine synthase reductase. Mol Genet Metab 91:85-97
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Morin, Isabelle; Platt, Robert; Weisberg, Ilan et al. (2003) Common variant in betaine-homocysteine methyltransferase (BHMT) and risk for spina bifida. Am J Med Genet A 119A:172-6
Breksa 3rd, Andrew P; Garrow, Timothy A (2002) Random mutagenesis of the zinc-binding motif of betaine-homocysteine methyltransferase reveals that Gly 214 is essential. Arch Biochem Biophys 399:73-80
Hall, D A; Vander Kooi, C W; Stasik, C N et al. (2001) Mapping the interactions between flavodoxin and its physiological partners flavodoxin reductase and cobalamin-dependent methionine synthase. Proc Natl Acad Sci U S A 98:9521-6
Brown, C A; McKinney, K Q; Kaufman, J S et al. (2000) A common polymorphism in methionine synthase reductase increases risk of premature coronary artery disease. J Cardiovasc Risk 7:197-200

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