Project

Discipline

vitamin B12; cobalamin; homocysteine; methylmalonic acid; 5-methyl-tetrahydrofolate; S-adenosylmethionine; methionine synthase deficiency; methylmalonyl-CoA mutase deficiency; organic acidurias; cobalamin complementation

Lead
Lay summary
Large elevations of homocysteine, an amino acid derived from the protein amino acid methionine, and methylmalonic acid (MMA), an intermediary metabolite in the breakdown of several substances, are found in rare inborn errors of intracellular vitamin B12 (cobalamin) metabolism and in vitamin B12 deficiency. These elevations can lead to severe neurological damage. A milder increase of homocysteine is linked as risk factor or marker to premature arteriosclerosis. An important player in homocysteine regulation is cobalamin. Understanding the role of severe and mild disturbances of homocysteine and cobalamin metabolism requires knowledge of the basic metabolic steps involved. After cobalamin enters the cell it has to be converted to two active coenzymes, one is adenosylcobalamin, and the other methylcobalamin. This takes place in two different compartments in the cell, the cytosol and mitochondria and the conversion requires a number of steps that are defined biochemically and by genetic complementation as cblA-G. We have shown that the cblD defect affects both mitochondrial and cytosolic cobalamin metabolism indicating that the cblD protein plays a key role in channelling of cobalamin within the cell. Using the microcell mediated chromosome transfer technique we have identified the gene (MMADHC) responsible for the CblD defect. The cblC gene (MMACHC) is thought to encode a cobalamin transporter. We collaborated in the discovery of the the cblF gene (LMBRD1) that encodes a lysosomal membrane protein. We have recently discovered a further step in the pathway, cblJ which is closely related to cblF. The discovery of these genes and mutations responsible for the cblC, cblD, cblF and cblJ are important advances in understanding these processes. Although many open questions remain the knowledge we have gained underpins the concept of special molecule channeling systems which protect essential substances present in very low concentrations in the cell, in this case cobalamin and which direct them to their targets. Further, our studies will provide insight into how the different components of the channelling system interact and especially how the intriguing cblD protein acts as a molecular switch in trafficking of this micronutrient in different cellular compartments. Identification of genes will provide the basis for the discovery of common polymorphisms which may lead to mild disturbances of homocysteine metabolism in common disease such as loss of cognitive function as well as macular degeneration in the elderly, and sub clinical cobalamin deficiency in childhood. Knowledge gained may lead to improved treatment of patients with inherited defects of cobalamin and folate metabolism as well as of patients with milder abnormalities of homocysteine and methylmalonic acid metabolism.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

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Background: Inborn errors of methionine metabolism which result in a large elevation of homocysteine are associated with severe vascular abnormalities. Milder increases of plasma homocysteine are associated with cardiovascular events. In man homocysteine is formed from methionine, then either catabolised via transsulphuration, involving vitamin B6, or remethylated to methionine involving several co-factors including vitamin B12 (cobalamin) and 5-methyltetrahydrofolate. Key players in homocysteine regulation are cobalamin, 5- methyltetrahydrofolate and adenosylmethionine. Understanding the role of severe and mild disturbances of metabolism of these compounds in causing disease requires knowledge of the basic metabolic steps involved.Many abnormalities of metabolism of methylmalonic acid are known resulting in disease of widely varying severity. In man only 2 reactions are known that require a cobalamin (Cbl) coenzyme for activity: adenosylcobalamin is the coenzyme for the mutase which catalyses the conversion of methylmalonyl-CoA to succinyl-CoA; methionine synthase which remethylates homocysteine to methionine requires methylcobalamin as well as 5-methyltetrahydrofolate which is formed by methylenetetrahydrofolate reductase (MTHFR). Polymorphisms of this reductase have been implicated as a risk factor in common diseases such as cardiovascular disease, cancer and neural tube defects. The intracellular conversion of OHCbl to the active coenzymes adenosylcobalamin in the mitochondrion and methylcobalamin in the cytosol requires a number of molecular steps associated with mutant classes that are defined biochemically and by genetic complementation as cblA-G. We have shown that the cblD defect affects both cytosolic and mitochondrial pathways indicating that the cblD protein must play a key role in channelling of Cbl within the cell. Our recent discovery of the gene and mutations responsible for the cblD, cblF and the novel cblX defects are important steps in understanding these processes but many open questions remain. The goals of this project are to elucidate the molecular mechanisms and interactions of the intriguing cblD protein and investigate its role in cobalamin trafficking. Also the function of the novel cblX protein including detailed studies of the expression of normal and mutant genes will be investigated. Furthermore, we will attempt to answer open questions regarding clinical and pathophysiological aspects of methylmalonic aciduria and the cblC disorder and also study novel therapeutic approaches. Finally, we will continue our studies of functional consequences of mutant alleles in patients with MTHFR deficiency.Working Hypothesis: The overall goals of this project are to understand intracellular metabolism of cobalamin and folate as determinants of homocysteine and methylmalonic acid in health and disease. Elucidation of the cblD and cblX defects in human mutants and the function of the cblD protein will increase understanding of cobalamin trafficking within the cell. Specific Aims and Experimental Design: 1. Detailed classification of cell lines from patients with disorders of homocysteine (Hcy) and methylmalonic acid (MMA) metabolism.2. Elucidation of molecular mechanisms and interactions of the cblD gene product and its role in intracellular cobalamin trafficking by:a.testing of functional significance of mutant MMADHC alleles detected in patients with the cblD defectb.characterization of functional domains of the cblD proteinc.investigation of the intracellular localisation of the cblD proteind.identification of potential cblD binding partners e.purification and crystallography of the cblD protein in collaboration with another group3. Elucidation of molecular mechanisms of the cblX gene product and its role in intracellular cobalamin trafficking by:a.testing of functional significance of mutant ABCD4 alleles detected in patients with the cblX defectb.investigation of the intracellular localisation of the cblX proteinc.studies of the function of ABCD4 and identification of potential binding partners4. Studies of the rare severe inherited disorders of Hcy and MMA metabolism:a.investigations of patients with variant severe forms of MTHFR deficiencyb.search for a specific enzyme deficiency among patients with unexplained hyperhomocysteinaemia and unexplained methylmalonic aciduriac.Study of the effect of PTC124 (Ataluren) on read-through of nonsense mutations in patients with isolated MMAuria 5. Prospective study on diagnostic, clinical and therapeutic aspects and long-tem outcome in patients with isolated MMAurias and the cblC defect as part of the European registry and network for Intoxication type Metabolic Disorders (E-IMD) Expected value of the proposed project: Knowledge gained in the proposed studies will provide insight into the so far incompletely understood cellular and molecular biology of intracellular cobalamin processing, especially the role of the novel cblX protein and the intriguing cblD protein with its ability to act as a molecular switch in trafficking of this micronutrient in different cellular compartments. Characterization of the genes may lead to discovery of common polymorphisms which cause mild disturbances of Hcy metabolism in common disease such as loss of cognitive function as well as macular degeneration in the elderly, and sub clinical Cbl deficiency in childhood. Knowledge gained may lead to improved treatment of patients with inherited defects of Cbl and folate metabolism as well as to a better understanding of milder abnormalities of Hcy and MMA metabolism.