Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type.

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

Transcobalamin in cultured fibroblasts from patients with inborn errors of vitamin B12 metabolism.

Derivatives of vitamin B(12) (cobalamin, Cbl) are required for activity of the mitochondrial enzyme L-methylmalonyl-CoA mutase and the cytoplasmic enzyme methionine synthase in human cells. We recently described a putative novel Cbl-binding protein in crude mitochondrial fractions isolated from cultured fibroblasts. The amount of Cbl bound to this protein varied in fibroblasts from patients with different genetic defects affecting cobalamin metabolism. We have now identified this protein as the cobalamin transport protein transcobalamin (TC) by its binding to anti-TC antibodies and mass spectrometry, and suggest that its presence in crude mitochondrial fractions was the result of lysosomal contamination. Increased Cbl bound TC levels were confirmed in whole cell extracts in at least one cell line from both the cblB and mut classes of inborn errors of cobalamin metabolism.

Homocysteine levels in A/J and C57BL/6J mice: genetic, diet, gender, and parental effects.

Increased levels of homocysteine in the blood have been associated with various birth defects and adult diseases. However, the extent to which genetic factors control homocysteine levels in healthy individuals is unclear. Laboratory mice are valuable models for dissecting the genetic and environmental controls of total homocysteine (tHcy) levels. We assessed the inheritance of tHcy levels in two inbred strains, A/J and C57BL/6J (B6), under controlled physiological conditions and assessed the relative importance of genetic, diet, gender, and parental effects. Diet affected mean tHcy levels, whereas gender affected both the mean and variance of tHcy levels. Moreover, gender of the parents influenced mean tHcy levels in reciprocal F1 hybrids, suggesting maternal effects. Finally, gene-diet interactions affected heritability of mean tHcy levels. These studies showed that each of these factors contributes to tHcy levels and provided important clues to understanding homocysteine homeostasis in humans.

Parallel changes in metabolite and expression profiles in crooked-tail mutant and folate-reduced wild-type mice.

Anomalies in homocysteine (HCY) and folate metabolism are associated with common birth defects and adult diseases, several of which can be suppressed with dietary folate supplementation. Although supplementation reduces the occurrence and severity of neural tube defects (NTDs), many cases are resistant to these beneficial effects. The basis for variable response and biomarkers that predict responsiveness are unknown. Crooked-tail (Cd) mutant mice are an important model of folate-responsive NTDs. To identify features that are diagnostic for responsiveness versus resistance to dietary folate supplementation, we surveyed metabolite and expression levels in liver samples from folate-supplemented, folate-reduced and control diets in Cd mutant and wild-type adult females. Cd homozygotes had normal total homocysteine (tHcy) levels suggesting that folate suppresses NTDs through a mechanism that does not involve modulating serum tHcy levels. Instead, parallel changes in metabolite and expression profiles in folate-supplemented Cd/Cd homozygotes and folate-reduced+/+and Cd/+mice suggest that Crooked-tail homozygotes have a defect in the utilization of intracellular folate. Then, by combining these expression and metabolite profile results with published results for other models and their controls, two clusters were found, one of which included several folate-responsive NTD models and the other previously untested and presumably folate-resistant models. The predictive value of these profiles was verified by demonstrating that NTDs of Ski-/-mutant mice, whose profile suggested resistance to folate supplementation, were not suppressed with dietary folate supplementation. These results raise the possibility of using metabolite and expression profiles to distinguish folate-responsive and resistance adult females who are at risk for bearing fetuses with an NTD.

Nutrigenes, functional genomics and systems biology.

Traditionally, the classic reductionist approach attributes functions to individual genes. For instance, this has involved the analysis of motifs or the amino acid sequences of single gene products. It is unclear how the products of particular collections genes act together to provide higher order functionality in health and disease. To address this higher order problem, the function of collections of genes, as opposed to "one gene at a time" has to be studied. Accordingly, a model system is needed to test systems biology. In our studies, we used the homocysteine-folate metabolism as a model system.

Genetic and molecular control of folate-homocysteine metabolism in mutant mice.

Hyperhomocysteinemia adversely affects fundamental aspects of fetal development, adulthood, and aging, but the role of elevated homocysteine levels in these birth defects and adult diseases remains unclear. Mouse models are valuable for investigating the causes and consequences of hyperhomocysteinemia. We used a phenotype-based approach to identify mouse mutants for studying the relation between single gene mutations, homocysteine levels as a measure of the status of homocysteine metabolism, and gene expression profiles as a way to assess the impact of protein deficiency in mutant mice on steady-state transcription levels of genes in the folate-homocysteine pathways. These mutants were selected based on their propensity to produce phenotypes that are reminiscent of those associated with anomalies in folate-homocysteine metabolism in humans. We report identification of new, single-gene mouse models of homocysteinemia and characterization of their molecular and physiological impact on folate-homocysteine metabolism. Mutations in several genes involved in the hedgehog and WNT signal transduction pathways, as well as a gene involved in lipid metabolism, resulted in elevated homocysteine levels and altered expression profiles of folate-homocysteine metabolism genes. These results begin to unravel the complex relations between elevation of a single amino acid in the blood and the diverse birth defects and adult diseases associated with hyperhomocysteinemia.

Hyperhomocysteinemia due to methionine synthase deficiency, cblG: structure of the MTR gene, genotype diversity, and recognition of a common mutation, P1173L.

Mutations in the MTR gene, which encodes methionine synthase on human chromosome 1p43, result in the methylcobalamin deficiency G (cblG) disorder, which is characterized by homocystinuria, hyperhomocysteinemia, and hypomethioninemia. To investigate the molecular basis of the disorder, we have characterized the structure of the MTR gene, thereby identifying exon-intron boundaries. This enabled amplification of each of the 33 exons of the gene, from genomic DNA from a panel of 21 patients with cblG. Thirteen novel mutations were identified. These included five deletions (c.12-13delGC, c.381delA, c.2101delT, c.2669-2670delTG, and c.2796-2800delAAGTC) and two nonsense mutations (R585X and E1204X) that would result in synthesis of truncated proteins that lack portions critical for enzyme function. One mutation was identified that resulted in conversion of A to C of the invariant A of the 3' splice site of intron 9. Five missense mutations (A410P, S437Y, S450H, H595P, and I804T) were identified. The latter mutations, as well as the splice-site mutation, were not detected in a panel of 50 anonymous DNA samples, suggesting that these sequence changes are not polymorphisms present in the general population. In addition, a previously described missense mutation, P1173L, was detected in 16 patients in an expanded panel of 24 patients with cblG. Analysis of haplotypes constructed using sequence polymorphisms identified within the MTR gene demonstrated that this mutation, a C-->T transition in a CpG island, has occurred on at least two separate genetic backgrounds.