Molecular cloning, expression and physical mapping of the human methionine synthase reductase gene.
ABSTRACT Methionine synthase reductase (EC 220.127.116.11) is a flavoprotein essential for maintenance of methionine synthase in an active state. We characterized the human gene for methionine synthase reductase (MTRR). The gene is approximately 34kb and comprises 15 exons, varying in size from 43 to 1213bp, and 14 introns whose sizes vary from 108bp to 5kb. The positions of several junctions are conserved between the MTRR gene and the C. elegans ortholog, as well as with the rat cytochrome P450 reductase gene. A 1.3kb CpG island encompasses the 5'-flanking region and exon 1 and extends into intron 1. A short region including the transcription start site is sufficient to confer promoter activity, with a better outcome when accompanied by intron 1. The promoter region contains putative binding sites for Sp1, AP-1, AP-2 as well as CAAT motifs, but no consensus TATA box. Primer extension analysis revealed a single major transcription start site, located 137bp upstream of the previously reported initiator ATG. An alternative splicing event involving a portion of exon 1 predicts that translation can potentially be initiated at two different ATG codons. The gene was physically assigned to a narrow area between markers WI1755 and D5S1957.
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ABSTRACT: The cblE type of homocystinuria is a rare autosomal recessive disorder caused by impaired reductive activation of methionine synthase. Although earlier biochemical studies proposed that the methionine synthase enzyme might be activated by two different reducing systems, mutations were reported in only the methionine synthase reductase gene (MTRR) in cblE patients. The pathogenicity of MTRR mutations, however, has not yet been tested functionally. We report on nine patients of European origin affected by the cblE type of homocystinuria. They presented between 2 weeks and 3 years of age (median age 4 weeks) with anemia, which was macrocytic in only three patients, and with neurological involvement in all but two cases. Bone marrow examination performed in seven patients showed megaloblastic changes in all but one of them. All patients exhibited moderate to severe hyperhomocysteinemia (median plasma total homocysteine [Hcy] 92 mumol/L, range 44-169), while clearly reduced methionine was observed only in four cases. Pathogenic mutations were identified in both parental alleles of the MTRR gene in all patients. Five known (c.903+469T>C, c.1361C>T, c.1459G>A, c.1557-4_1557+3del7, and c.1622_1623dupTA) and three novel mutations (c.7A>T, c.1573C>T, and c.1953-6_1953-2del5) were detected. Importantly, transfection of fibroblasts of cblE patients with a wild-type MTRR minigene expression construct resulted in a significant approximately four-fold increase of methionine synthesis, indicating correction of the enzyme defect. Our study shows a link between a milder predominantly hematological presentation and homozygosity for the c.1361C>T mutation, but no other obvious genotype-phenotype correlation. The identification of mutations in the MTRR gene, together with restoration of methionine synthesis following MTRR minigene expression in cblE cells confirms that this disease is caused by defects in the MTRR gene.Human Mutation 03/2005; 25(3):239-47. DOI:10.1002/humu.20131 · 5.05 Impact Factor
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ABSTRACT: This study is directed towards investigating the ability of various forms of vitamin B12 to augment intracellular gluthathione (GSH) level as well as studying their possible protective effect against the thimerosal-inuced oxidative stress. SH-SY5Y human neuroblastoma cell culture system was used as a model for this study. Cells were acutely (60 minutes) treated with 3 different vitamin B12 derivatives (methyl-, hydroxy- and cyanocobalamin) in the presence and absence of thimerosal, vaccine antimicrobial agent. The results revealed that only methylcobalamin augmented the GSH level, as compared to other forms (hydrorxy- and cyanocobalamin). Also methylcobalamin was the only derivative that showed a protective effect against the thimerosal-induced depletion of GSH. Our findings provide evidence that thimerosal induces oxidative stress in human neuronal cells by depleting GSH and only methylcobalamin protects the cells against such insult and therfore improving the antioxidant intracellular capacity against oxidative stress, a common cause for the pathogenesis of many diseases including, autism.
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ABSTRACT: A revision about the role of hyperhomocysteinemia in the developmentof preeclampsia is presented, which summarises our experiencein different biochemical and genetic points in relation to this possibleassociation. Plasma total homocysteine concentrations (tHcy) during pregnancywere significantly lower than those of non-pregnant women: 2nd trimester(median, 5.3 ìmol/l; range, 3.1-10.0 μmol/l); 3rd trimester(median, 6.3 μmol/l; range, 3.2-13.0 ìmol/l). Hyperhomocysteinemia(tHcy > P95) was established as values higher than 7.7 ìmol/l in the2nd trimester, and as values higher than 10.5 ìmol/l in the 3rd trimesterof pregnancy. We found an association between hyperhomocysteinemia and preeclampsia:tHcy values were significantly higher in the preeclampticgroup than in uncomplicated pregnancies; the OR for preeclampsia inhyperhomocysteinemic patients was 7.7 (CI 95%, 1.7-34.8). The otheramino acid concentrations were also higher in preeclamptic women.The negative correlation observed between homocysteine andfolate in the control group, was not present in preeclamptic women.Medicina Clínica 01/2003; 121(9):350-355. DOI:10.1157/13050826 · 1.25 Impact Factor