David Coelho

Gene identification for the cblD defect of vitamin B12 metabolism

BACKGROUND Vitamin B12 (cobalamin) is an essential cofactor in several metabolic pathways. Intracellular conversion of cobalamin to its two coenzymes, adenosylcobalamin in mitochondria and methylcobalamin in the cytoplasm, is necessary for the homeostasis of methylmalonic acid and homocysteine. Nine defects of intracellular cobalamin metabolism have been defined by means of somatic complementation analysis. One of these defects, the cblD defect, can cause isolated methylmalonic aciduria, isolated homocystinuria, or both. Affected persons present with multisystem clinical abnormalities, including developmental, hematologic, neurologic, and metabolic findings. The gene responsible for the cblD defect has not been identified. METHODS We studied seven patients with the cblD defect, and skin fibroblasts from each were investigated in cell culture. Microcell-mediated chromosome transfer and refined genetic mapping were used to localize the responsible gene. This gene was transfected into cblD fibroblasts to test for the rescue of adenosylcobalamin and methylcobalamin synthesis. RESULTS The cblD gene was localized to human chromosome 2q23.2, and a candidate gene, designated MMADHC (methylmalonic aciduria, cblD type, and homocystinuria), was identified in this region. Transfection of wild-type MMADHC rescued the cellular phenotype, and the functional importance of mutant alleles was shown by means of transfection with mutant constructs. The predicted MMADHC protein has sequence homology with a bacterial ATP-binding cassette transporter and contains a putative cobalamin binding motif and a putative mitochondrial targeting sequence. CONCLUSIONS Mutations in a gene we designated MMADHC are responsible for the cblD defect in vitamin B12 metabolism. Various mutations are associated with each of the three biochemical phenotypes of the disorder.

Spectrum of mutations in MMACHC, allelic expression, and evidence for genotype-phenotype correlations.

Methylmalonic aciduria and homocystinuria, cblC type, is a rare disorder of intracellular vitamin B12 (cobalamin [Cbl]) metabolism caused by mutations in the MMACHC gene. MMACHC was sequenced from the gDNA of 118 cblC individuals. Eleven novel mutations were identified, as well as 23 mutations that were observed previously. Six sequence variants capture haplotype diversity in individuals across the MMACHC interval. Genotype–phenotype correlations of common mutations were apparent; individuals with c.394C>T tend to present with late‐onset disease whereas patients with c.331C>T and c.271dupA tend to present in infancy. Other missense variants were also associated with late‐ or early‐onset disease. Allelic expression analysis was carried out on human cblC fibroblasts compound heterozygous for different combinations of mutations including c.271dupA, c.331C>T, c.394C>T, and c.482G>A. The early‐onset c.271dupA mutation was consistently underexpressed when compared to control alleles and the late‐onset c.394C>T and c.482G>A mutations. The early‐onset c.331C>T mutation was also underexpressed when compared to control alleles and the c.394C>T mutation. Levels of MMACHC mRNA transcript in cell lines homozygous for c.271dupA, c.331C>T, and c.394C>T were assessed using quantitative real‐time RT‐PCR. Cell lines homozygous for the late onset c.394C>T mutation had significantly higher levels of transcript when compared to cell lines homozygous for the early‐onset mutations. Differential or preferential MMACHC transcript levels may provide a clue as to why individuals carrying c.394C>T generally present later in life. Hum Mutat 30:1–10, 2009.

The cblD Defect Causes Either Isolated or Combined Deficiency of Methylcobalamin and Adenosylcobalamin Synthesis

Intracellular cobalamin is converted to adenosylcobalamin, coenzyme for methylmalonyl-CoA mutase and to methylcobalamin, coenzyme for methionine synthase, in an incompletely understood sequence of reactions. Genetic defects of these steps are defined as cbl complementation groups of which cblC, cblD (described in only two siblings), and cblF are associated with combined homocystinuria and methylmalonic aciduria. Here we describe three unrelated patients belonging to the cblD complementation group but with distinct biochemical phenotypes different from that described in the original cblD siblings. Two patients presented with isolated homocystinuria and reduced formation of methionine and methylcobalamin in cultured fibroblasts, defined as cblD-variant 1, and one patient with isolated methylmalonic aciduria and deficient adenosylcobalamin synthesis in fibroblasts, defined as cblD-variant 2. Cell lines from the cblD-variant 1 patients clearly complemented reference lines with the same biochemical phenotype, i.e. cblE and cblG, and the cblD-variant 2 cell line complemented cells from the mutant classes with isolated deficiency of adenosylcobalamin synthesis, i.e. cblA and cblB. Also, no pathogenic sequence changes in the coding regions of genes associated with the respective biochemical phenotypes were found. These findings indicate heterogeneity within the previously defined cblD mutant class and point to further complexity of intracellular cobalamin metabolism.

Clinical and Molecular Heterogeneity in Patients with the CblD Inborn Error of Cobalamin Metabolism

OBJECTIVES To describe 3 patients with the cblD disorder, a rare inborn error of cobalamin metabolism caused by mutations in the MMADHC gene that can result in isolated homocystinuria, isolated methylmalonic aciduria, or combined homocystinuria and methylmalonic aciduria. STUDY DESIGN Patient clinical records were reviewed. Biochemical and somatic cell genetic studies were performed on cultured fibroblasts. Sequence analysis of the MMADHC gene was performed on patient DNA. RESULTS Patient 1 presented with isolated methylmalonic aciduria, patient 3 with isolated homocystinuria, and patient 2 with combined methylmalonic aciduria and homocystinuria. Studies of cultured fibroblasts confirmed decreased synthesis of adenosylcobalamin in patient 1, decreased synthesis of methylcobalamin in patient 3, and decreased synthesis of both cobalamin derivatives in patient 2. The diagnosis of cblD was established in each patient by complementation analysis. Mutations in the MMADHC gene were identified in all patients. CONCLUSIONS The results emphasize the heterogeneous clinical, cellular and molecular phenotype of the cblD disorder. The results of molecular analysis of the MMADHC gene are consistent with the hypothesis that mutations affecting the N terminus of the MMADHC protein are associated with methylmalonic aciduria, and mutations affecting the C terminus are associated with homocystinuria.

Molecular mechanisms leading to three different phenotypes in the cblD defect of intracellular cobalamin metabolism

The cblD defect of intracellular vitamin B(12) metabolism can lead to isolated methylmalonic aciduria (cblD-MMA) or homocystinuria (cblD-HC), or combined methylmalonic aciduria and homocystinuria (cblD-MMA/HC). We studied the mechanism whereby MMADHC mutations can lead to three phenotypes. The effect of various expression vectors containing MMADHC modified to contain an enhanced mitochondrial leader sequence or mutations changing possible downstream sites of reinitiation of translation or mutations introducing stop codons on rescue of adenosyl- and methylcobalamin (MeCbl) formation was studied. The constructs were transfected into cell lines derived from various cblD patients fibroblasts. Expression of 10 mutant alleles from 15 cblD patients confirmed that the nature and location of the mutations correlate with the biochemical phenotype. In cblD-MMA/HC cells, improving mitochondrial targeting of MMADHC clearly increased the formation of adenosylcobalamin (AdoCbl) with a concomitant decrease in MeCbl formation. In cblD-MMA cells, this effect was dependent on the mutation and showed a negative correlation with endogenous MMADHC mRNA levels. These findings support the hypothesis that a single protein exists with two different functional domains that interact with either cytosolic or mitochondrial targets. Also a delicate balance exists between cytosolic MeCbl and mitochondrial AdoCbl synthesis, supporting the role of cblD protein as a branch point in intracellular cobalamin trafficking. Furthermore, our data indicate that the sequence after Met116 is sufficient for MeCbl synthesis, whereas the additional sequence between Met62 and Met116 is required for AdoCbl synthesis. Accordingly, western blot studies reveal proteins of the size expected from the stop codon position with subsequent reinitiation of translation.

Nutritional models of foetal programming and nutrigenomic and epigenomic dysregulations of fatty acid metabolism in the liver and heart

Barker’s concept of ‘foetal programming’ proposes that intrauterine growth restriction (IUGR) predicts complex metabolic diseases through relationships that may be further modified by the postnatal environment. Dietary restriction and deficit in methyl donors, folate, vitamin B12, and choline are used as experimental conditions of foetal programming as they lead to IUGR and decreased birth weight. Overfeeding and deficit in methyl donors increase central fat mass and lead to a dramatic increase of plasma free fatty acids (FFA) in offspring. Conversely, supplementing the mothers under protein restriction with folic acid reverses metabolic and epigenomic phenotypes of offspring. High-fat diet or methyl donor deficiency (MDD) during pregnancy and lactation produce liver steatosis and myocardium hypertrophy that result from increased import of FFA and impaired fatty acid β-oxidation, respectively. The underlying molecular mechanisms show dysregulations related with similar decreased expression and activity of sirtuin 1 (SIRT1) and hyperacetylation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α). High-fat diet and overfeeding impair AMPK-dependent phosphorylation of PGC-1α, while MDD decreases PGC-1α methylation through decreased expression of PRMT1 and cellular level of S-adenosyl methionine. The visceral manifestations of metabolic syndrome are under the influence of endoplasmic reticulum (ER) stress in overnourished animal models. These mechanisms should also deserve attention in the foetal programming effects of MDD since vitamin B12 influences ER stress through impaired SIRT1 deacetylation of HSF1. Taken together, similarities and synergies of high-fat diet and MDD suggest, therefore, considering their consecutive or contemporary influence in the mechanisms of complex metabolic diseases.

Inherited disorders of cobalamin metabolism disrupt nucleocytoplasmic transport of mRNA through impaired methylation/phosphorylation of ELAVL1/HuR

Abstract The molecular mechanisms that underlie the neurological manifestations of patients with inherited diseases of vitamin B12 (cobalamin) metabolism remain to date obscure. We observed transcriptomic changes of genes involved in RNA metabolism and endoplasmic reticulum stress in a neuronal cell model with impaired cobalamin metabolism. These changes were related to the subcellular mislocalization of several RNA binding proteins, including the ELAVL1/HuR protein implicated in neuronal stress, in this cell model and in patient fibroblasts with inborn errors of cobalamin metabolism and Cd320 knockout mice. The decreased interaction of ELAVL1/HuR with the CRM1/exportin protein of the nuclear pore complex and its subsequent mislocalization resulted from hypomethylation at R-217 produced by decreased S-adenosylmethionine and protein methyl transferase CARM1 and dephosphorylation at S221 by increased protein phosphatase PP2A. The mislocalization of ELAVL1/HuR triggered the decreased expression of SIRT1 deacetylase and genes involved in brain development, neuroplasticity, myelin formation, and brain aging. The mislocalization was reversible upon treatment with siPpp2ca, cobalamin, S-adenosylmethionine, or PP2A inhibitor okadaic acid. In conclusion, our data highlight the key role of the disruption of ELAVL1/HuR nuclear export, with genomic changes consistent with the effects of inborn errors of Cbl metabolisms on brain development, neuroplasticity and myelin formation.