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.     

Inherited disorders of vitamin B12 utilization

Inborn errors of vitamin B12 (cobalamin) metabolism are associated with homocystinuria and methylmalonic aciduria, either alone or in combination. A number of these disorders have provided the first evidence for the existence of important steps in the transport or metabolism of cobalamin in eukaryotic cells. Eight complementation classes have been defined on the basis of somatic cell hybridization studies. Although the majority of patients present in infancy or early childhood, some are not diagnosed until adolescence or later. For some of these disorders, prenatal diagnosis and therapy with cobalamin during pregnancy has been attempted. Although only males have been described with cblE disease, all of these disorders are presumed to be autosomal recessive in inheritance. The clinical and laboratory aspects of the different complementation classes (cblA-cblG) are reviewed here.     

Two cases of benign methylmalonic aciduria detected during a pilot study of neonatal urine screening

Two cases of benign methylmalonic aciduria (MMAuria) were found among 9780 neonatal screenings using the previously described screening method consisting of urease digestion, ethanol deproteinization and gas chromatography-mass spectrometry. Combining this screening method with the stable isotope dilution technique showed very specific and sensitive measurements of methylmalonic acid in urine. The concentrations of urinary methylmalonic acid were measured at several ages. The levels of urinary methylmalonic acid in two patients varied from 0.27 to 3.04 mol/mol creatinine (control<0.01 mol/mol creatinine). Methylcitrate and homocystine were not increased in the patient's urine or blood. Blood propionylcarnitine was also at normal levels. The urinary methylmalonate excretions were decreased to the levels of about 50% of the start point after vitamin B12 treatment in one patient, but the other patient showed no change. No clinical abnormalities were observed during these periods.     

A knock-out mouse model for methylmalonic aciduria resulting in neonatal lethality

Methylmalonic aciduria is a human autosomal recessive disorder of organic acid metabolism resulting from a functional defect in the activity of the enzyme methylmalonyl-CoA mutase. Based upon the homology of the human mutase locus with the mouse locus, we have chosen to disrupt the mouse mutase locus within the critical CoA binding domain using gene-targeting techniques to create a mouse model of methylmalonic aciduria. The phenotype of homozygous knock-out mice (mut-/-) is one of early neonatal lethality. Mice appear phenotypically normal at birth and are indistinguishable from littermates. By 15 h of age, they develop reduced movement and suckle less. This is followed by the development of abnormal breathing, and all of the mice with a null phenotype die by 24 h of age. Urinary levels of methylmalonic and methylcitric acids are grossly increased. Measurement of acylcarnitines in blood shows elevation of propionylcarnitine with no change in the levels of acetylcarnitine and free carnitine. Incorporation of [14C]propionate in primary fibroblast cultures from mut-/- mice is reduced to approximately 6% of normal level, whereas there is no detectable synthesis of mut mRNA in the liver. This is the first mouse model that recapitulates the key phenotypic features of mut0 methylmalonic aciduria.     

Methylmalonic acidemia controlled with oral administration of vitamin B12.

A 3-month-old male infant had two episodes of fever, projectile vomiting, dehydration, generalized fine tremors and gross metabloic ketoacidosis. Methylmalonic acid was found in high concentration in both serum and urine, although the concentration of serum vitamin B12 was normal. A therapeutic trial of vitamin B12, administered parenterally, reduced greatly the methylmalonic aciduria. The patient has since been given vitamin B12 supplements continuously, initially 1 mg intramuscularly every other day, then 15 mg/d orally, and the protein in his diet was subsequently restricted. The most effected control of the methylmalonic aciduria was achieved with the combined regimen of oral vitamin therapy and dietary protein restriction. His physical and intellectual development have progressed normally and he has survived several acute respiratory tract infections without recurrence of metabolic acidosis.     

Phenotype of disease in three patients with identical mutations in methylmalonyl CoA mutase

Summary We have previously identified a mutation in the gene for methylmalonyl CoA mutase in a patient with the mut^- phenotype of methylmalonic aciduria. This mutation (G717V) interferes with the binding of the deoxyadenosylcobalamin cofactor to the apoenzyme producing a mutant holoenzyme that is defective, but not completely inactive, in vitro. This report describes the clinical phenotype associated with this mutation in the original patient and two additional patients who are homozygous for this allele. All three patients presented in the first years of life with multiple episodes of life-threatening organic acidosis and hyperammonemia. None had evidence of disease in the perinatal period, and all three have low-normal intelligence. These three children exhibit a distinctive phenotype of disease that is intermediate between the fulminant and benign forms of methylmalonic aciduria. These data suggest that this phenotype is the specific consequence of the G717V mutation, and that the degree of residual enzyme activity associated with the G717V mutation is close to the threshold required in vivo for maintaining metabolic homeostasis.     

Studies on the urinary acidic metabolites from three patients with methylmalonic aciduria

By gas chromatographic mass spectrometric analysis, 2-methyl-3-oxovaleric acid, 3-oxovaleric acid, 3-hydroxyvaleric acid and 2-methyl-3-hydroxybutyric acid were identified in the urine from three patients with methylmalonic aciduria. The level of 2-methyl-3-oxovaleric acid increased with the increase of methylmalonic acid concentration and the level of 3-oxovaleric acid increased with the increase of ketone bodies. It was demonstrated that these metabolites are detectable from patients with propionic acidemia and methylmalonic aciduria when propionyl CoA accumulates in the cells of the patients either primarily or secondarily.     

Screening urine of 3-week-old newborns: transient methylmalonic and hydroxyphenyllactic aciduria.

Urinary methylmalonic acid (MMA) and 4-hydroxyphenyllactic acid (HPL) have been determined in 3345 and 2498 3-week-old newborns, respectively. Urine was collected onto filter paper and assayed by a rapid gas chromatography-mass spectrometry method. Forty-six infants (1.7%) had elevated MMA levels (greater than 58.5 micrograms/mg creatinine, means + 5 SD) and 31 infants (1.2%) had elevated levels of HPL (greater than 87.7 micrograms/mg creatinine, means + 5 SD). Fifteen infants with elevated values of MMA were retested from one to several months after the first test. In 12 infants the MMA levels normalized, while in the remaining three, elevated methylmalonic acid persisted. Nine infants with elevated values of HPL were retested, and in all except one, HPL levels normalized. No access to clinical evaluation of the infants was available. Transient methylmalonic aciduria and transient tyrosyluria affect a substantial number of infants and the clinical significance of this phenomenon has yet to be determined.     

Loss of allostery and coenzyme B12 delivery by a pathogenic mutation in adenosyltransferase

ATP-dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional protein: an enzyme that catalyzes the adenosylation of cob(I)alamin and an escort that delivers the product, adenosylcobalamin (AdoCbl or coenzyme B(12)), to methylmalonyl-CoA mutase (MCM), resulting in holoenzyme formation. Failure to assemble holo-MCM leads to methylmalonic aciduria. We have previously demonstrated that only 2 equiv of AdoCbl bind per homotrimer of ATR and that binding of ATP to the vacant active site triggers ejection of 1 equiv of AdoCbl from an adjacent site. In this study, we have mimicked in the Methylobacterium extorquens ATR, a C-terminal truncation mutation, D180X, described in a patient with methylmalonic aciduria, and characterized the associated biochemical penalties. We demonstrate that while k(cat) and K(M)(Cob(I)) for D180X ATR are only modestly decreased (by 3- and 2-fold, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold), and the negative cooperativity associated with its binding is lost. We also demonstrate that the D180X mutation corrupts ATP-dependent cofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM. These results suggest that the pathogenicity of the corresponding human truncation mutant results from its inability to sequester AdoCbl for direct transfer to MCM. Instead, cofactor release into solution is predicted to reduce the capacity for holo-MCM formation, leading to disease.     

Mechanisms for intragenic complementation at the human argininosuccinate lyase locus.

Argininosuccinate lyase (ASL) is a homotetrameric enzyme that catalyzes the reversible cleavage of argininosuccinate to arginine and fumarate. Deficiencies in the enzyme result in the autosomal, recessive disorder argininosuccinic aciduria. Considerable clinical and genetic heterogeneity is associated with this disorder, which is thought to be a consequence of the extensive intragenic complementation identified in patient strains. Our ability to predict genotype-phenotype relationships is hampered by the current lack of understanding of the mechanisms by which complementation can occur. The 3-dimensional structure of wild-type ASL has enabled us to propose that the complementation between two ASL active site mutant subunits, Q286R and D87G, occurs through a regeneration of functional active sites in the heteromutant protein. We have reconstructed this complementation event, both in vivo and in vitro, using recombinant proteins and have confirmed this hypothesis. The complementation events between Q286R and two nonactive site mutants, M360T and A398D, have also been characterized. The M360T and A398D substitutions have adverse effects on the thermodynamic stability of the protein. Complementation between either the M360T or the A398D mutant and the stable Q286R mutant occurs through the formation of a more stable heteromeric protein with partial recovery of catalytic activity. The detection and characterization of a novel complementation event between the A398D and D87G mutants has shown how complementation in patients with argininosuccinic aciduria may correlate with the clinical phenotype.     

Carglumic acid: an additional therapy in the treatment of organic acidurias with hyperammonemia?

Background

Hyperammonemia in patients with methylmalonic aciduria (MMA) and propionic aciduria (PA) is caused by accumulation of propionyl-CoA which decreases the synthesis of N-acetyl-glutamate, the natural activator of carbamyl phosphate synthetase 1. A treatment approach with carglumic acid, the structural analogue of N-acetyl-glutamate, has been proposed to decrease high ammonia levels encountered in MMA and PA crises.

Case presentation

We described two patients (one with MMA and one with PA) with hyperammonemia at diagnosis. Carglumic acid, when associated with standard treatment of organic acidurias, may be helpful in normalizing the ammonia level.

Conclusion

Even though the usual treatment which decreases toxic metabolites remains the standard, carglumic acid could be helpful in lowering plasma ammonia levels over 400 micromol/L more rapidly.     

Neurodegeneration in methylmalonic aciduria involves inhibition of complex II and the tricarboxylic acid cycle, and synergistically acting excitotoxicity

Methylmalonic acidurias are biochemically characterized by an accumulation of methylmalonate (MMA) and alternative metabolites. There is growing evidence for basal ganglia degeneration in these patients. The pathomechanisms involved are still unknown, a contribution of toxic organic acids, in particular MMA, has been suggested. Here we report that MMA induces neuronal damage in cultures of embryonic rat striatal cells at a concentration range encountered in affected patients. MMA-induced cell damage was reduced by ionotropic glutamate receptor antagonists, antioxidants, and succinate. These results suggest the involvement of secondary excitotoxic mechanisms in MMA-induced cell damage. MMA has been implicated in inhibition of respiratory chain complex II. However, MMA failed to inhibit complex II activity in submitochondrial particles from bovine heart. To unravel the mechanism underlying neuronal MMA toxicity, we investigated the formation of intracellular metabolites in MMA-loaded striatal neurons. There was a time-dependent intracellular increase in malonate, an inhibitor of complex II, and 2-methylcitrate, a compound with multiple inhibitory effects on the tricarboxylic acid cycle, suggesting their putative implication in MMA neurotoxicity. We propose that neuropathogenesis of methylmalonic aciduria may involve an inhibition of complex II and the tricarboxylic acid cycle by accumulating toxic organic acids, and synergistic secondary excitotoxic mechanisms.     

Enrichment of human heterokaryons by Ficoll gradient for complementation analysis of iduronate sulfatase deficiency.

Ficoll gradients have been used to enrich for heterokaryons in cultures of human skin fibroblasts following polyethylene glycol (PEG) induced fusion. These gradients provide a simple and consistent method for obtaining populations of multinucleated cells, at least twofold greater than those resulting from fusion alone. Formation of glucose-6-phosphate dehydrogenase (G6PD) heteropolymers has been used as a functional assay for the presence of heterokaryons. Analysis of cell populations enriched for multinucleated cells has revealed complementation leading to iduronate sulfatase activity in heterokaryons derived from iduronate sulfatase-deficient fibroblasts expressing the Hunter and multiple sulfatase-deficiency mutations.     

Regulation by SREBP-2 defines a potential link between isoprenoid and adenosylcobalamin metabolism

Mevalonate kinase (MVK) catalyses an early step in cholesterol biosynthesis converting mevalonate to phosphomevalonate. Cob(I)alamin adenosyltransferase (MMAB) converts cob(I)alamin to adenosylcobalamin, functionally required for mitochondrial methylmalonyl-CoA mutase activity and succinyl-CoA formation. These two synthenic genes are found in a head-to-head formation on chromosome 12 in man and chromosome 5 in mouse. The 330bp intergenic region showed several conserved NF-Y sites indicative of potential bidirectional regulatory SREBP synergism. Both MVK and MMAB appear to be regulated in a similar manner, to a large extent by SREBP-2, since their tissue expression pattern was similar and both genes were suppressed by an excess of cholesterol as well as SREBP-2 knockdown. Statin treatment in mice upregulated both Mvk and Mmab mRNA levels indicating that this treatment may be useful in inborn errors of cblB complementation associated with methylmalonic aciduria as well as hyper IgD and periodic fever syndrome (HIDS).     

MeaB is a component of the methylmalonyl-CoA mutase complex required for protection of the enzyme from inactivation

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA. In humans, deficiencies in the mutase lead to methylmalonic aciduria, a rare disease that is fatal in the first year of life. Such inherited deficiencies can result from mutations in the mutase structural gene or from mutations that impair the acquisition of cobalamins. Recently, a human gene of unknown function, MMAA, has been implicated in methylmalonic aciduria (Dobson, C. M., Wai, T., Leclerc, D., Wilson, A., Wu, X., Dore, C., Hudson, T., Rosenblatt, D. S., and Gravel, R. A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15554-15559). MMAA orthologs are widespread in bacteria, archaea, and eukaryotes. In Methylobacterium extorquens AM1, a mutant defective in the MMAA homolog meaB was unable to grow on C(1) and C(2) compounds because of the inability to convert methylmalonyl-CoA to succinyl-CoA (Korotkova N., Chistoserdova, L., Kuksa, V., and Lidstrom, M. E. (2002) J. Bacteriol. 184, 1750-1758). Here we demonstrate that this defect is not due to the absence of adenosylcobalamin but due to an inactive form of methylmalonyl-CoA mutase. The presence of active mutase in double mutants defective in MeaB and in the synthesis of either R-methylmalonyl-CoA or adenosylcobalamin indicates that MeaB is necessary for protection of mutase from inactivation during catalysis. MeaB and methylmalonyl-CoA mutase from M. extorquens were cloned and purified in their active forms. We demonstrated that MeaB forms a complex with methylmalonyl-CoA mutase and stimulates in vitro mutase activity. These results support the hypothesis that MeaB functions to protect methylmalonyl-CoA mutase from irreversible inactivation.     

Oxidative stress and apoptosis in homocystinuria patients with genetic remethylation defects

Oxidative stress has been described as a putative disease mechanism in pathologies associated with an elevation of homocysteine. An increased reactive oxygen species (ROS) production and apoptosis rate have been associated with several disorders of cobalamin metabolism, particularly with methylmalonic aciduria (MMA) combined with homocystinuria cblC type. In this work, we have evaluated several parameters related to oxidative stress and apoptosis in fibroblasts from patients with homocystinuria due to defects in the MTR, MTRR, and MTHFR genes involved in the remethylation pathway of homocysteine. We have also evaluated these processes by knocking down the MTRR gene in cellular models, and complementation by transducing the wild‐type gene in cblE mutant fibroblasts. All cell lines showed a significant increase in ROS content and in MnSOD expression level, and also a higher rate of apoptosis with similar levels to the ones in cblC fibroblasts. The amount of the active phosphorylated forms of p38 and JNK stress‐kinases was also increased. ROS content and apoptosis rate increased in control fibroblasts and in a glioblastoma cell line by shRNA‐mediated silencing of MTRR gene expression. In contrast, wild‐type MTRR gene corrected mutant cell lines showed a decrease in ROS and apoptosis levels. To the best of our knowledge, this study provides the first evidence that an impaired remethylation capacity due to low MTRR and MTR activity might be partially responsible for stress response. J. Cell. Biochem. 114: 183–191, 2012. © 2012 Wiley Periodicals, Inc.     

Identification and quantitation of urinary dicarboxylic acids as their dicyclohexyl esters in disease states by gas chromatography mass spectrometry

Clinical studies were conducted by gas chromatography mass spectrometry selected ion monitoring of urinary dicarboxylic acids as dicyclohexyl esters. The dicyclohexyl esters of the dicarboxylic acids give characteristic electron impact mass spectra suitable for selected ion monitoring. The mass spectra exhibit a prominent acid + 1H ion and an (acid + 1H)-H2O ion for use as quantitating and confirming ions. The cyclohexyl esters are stable for days at room temperature and have excellent chromatographic properties. Dicarboxylic acid quantitation is performed within one hour using only 50 microliter of unpurified urine. A rapid method specifically for methylmalonic acid quantitation is described which has assisted physicians in the diagnosis of pernicious anemia and methylmalonic aciduria. This procedure is applicable for screening urinary organic acids for detection of inborn errors of metabolism. The detection of a child with elevated medium length dicarboxylic acids in the terminal urine specimen is reported. This condition, previously described as an inborn error, is attributed to a terminal event. Finally, an increase in urinary succinic acid paralleling putrescine levels is described during a response to cancer chemotherapy.     

Mouse models for methylmalonic aciduria

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of methylmalonyl-CoA mutase (MCM). MMA is associated with significant morbidity and mortality, thus therapies are necessary to help improve quality of life and prevent renal and neurological complications. Transgenic mice carrying an intact human MCM locus have been produced. Four separate transgenic lines were established and characterised as carrying two, four, five or six copies of the transgene in a single integration site. Transgenic mice from the 2-copy line were crossed with heterozygous knockout MCM mice to generate mice hemizygous for the human transgene on a homozygous knockout background. Partial rescue of the uniform neonatal lethality seen in homozygous knockout mice was observed. These rescued mice were significantly smaller than control littermates (mice with mouse MCM gene). Biochemically, these partial rescue mice exhibited elevated methylmalonic acid levels in urine, plasma, kidney, liver and brain tissue. Acylcarnitine analysis of blood spots revealed elevated propionylcarnitine levels. Analysis of mRNA expression confirms the human transgene is expressed at higher levels than observed for the wild type, with highest expression in the kidney followed closely by brain and liver. Partial rescue mouse fibroblast cultures had only 20% of the wild type MCM enzyme activity. It is anticipated that this humanised partial rescue mouse model of MMA will enable evaluation of long-term pathophysiological effects of elevated methylmalonic acid levels and be a valuable model for the investigation of therapeutic strategies, such as cell transplantation.     

New SUCLG1 patients expanding the phenotypic spectrum of this rare cause of mild methylmalonic aciduria

Deficiencies in two subunits of the succinyl-coenzyme A synthetase (SCS) have been involved in patients with encephalomyopathy and mild methylmalonic aciduria (MMA). In this study, we described three new SUCLG1 patients and performed a meta-analysis of the literature. Our report enlarges the phenotypic spectrum of SUCLG1 mutations and confirms that a characteristic metabolic profile (presence of MMA and C4-DC carnitine in urines) and basal ganglia MRI lesions are the hallmarks of SCS defects. As mitochondrial DNA depletion in muscle is not a constant finding in SUCLG1 patients, this may suggest that diagnosis should not be based on it, but also that alternative physiopathological mechanisms may be considered to explain the combined respiratory chain deficiency observed in SCS patients.