Inborn Error Of Cobalamin Metabolism Research Articles

Streptococcus Pneumonia -Associated Hemolytic Uremic Syndrome: A Case Report

Hemolytic uremic syndrome (HUS), a disease that destroys red blood cells, is the most common cause of sudden, short term acute kidney failure in children. By far, the commonest subgroup (>90% of childhood HUS) is induced by shiga toxin producing bacteria, usually enterohemorrhagic Escherichia coli (EHEC). These patients typically have a prodrome of diarrhea. Another important subgroup that is readily identified on clinical grounds follows invasive Streptococcus pneumonia infection. These infants tend to be younger than those with D + HUS, and the syndrome is very rare after the age of 4 years. They present with pneumonia, empyema, meningitis, and less often, isolated septicemia. Another group of patients may have mutations in complement regulators, and less often, inherited deficiency of von Willebrand protease or an inborn error of cobalamin metabolism. We report a 6-month-old child who developed hemolytic uremic syndrome following S. pneumonia meningitis.Keywords: Hemolytic-Uremic Syndrome; Meningitis; Streptococcus pneumonia; Child

An X-Linked Cobalamin Disorder Caused by Mutations in Transcriptional Coregulator HCFC1

Derivatives of vitamin B12 (cobalamin) are essential cofactors for enzymes required in intermediary metabolism. Defects in cobalamin metabolism lead to disorders characterized by the accumulation of methylmalonic acid and/or homocysteine in blood and urine. The most common inborn error of cobalamin metabolism, combined methylmalonic acidemia and hyperhomocysteinemia, cblC type, is caused by mutations in MMACHC. However, several individuals with presumed cblC based on cellular and biochemical analysis do not have mutations in MMACHC. We used exome sequencing to identify the genetic basis of an X-linked form of combined methylmalonic acidemia and hyperhomocysteinemia, designated cblX. A missense mutation in a global transcriptional coregulator, HCFC1, was identified in the index case. Additional male subjects were ascertained through two international diagnostic laboratories, and 13/17 had one of five distinct missense mutations affecting three highly conserved amino acids within the HCFC1 kelch domain. A common phenotype of severe neurological symptoms including intractable epilepsy and profound neurocognitive impairment, along with variable biochemical manifestations, was observed in all affected subjects compared to individuals with early-onset cblC. The severe reduction in MMACHC mRNA and protein within subject fibroblast lines suggested a role for HCFC1 in transcriptional regulation of MMACHC, which was further supported by the identification of consensus HCFC1 binding sites in MMACHC. Furthermore, siRNA-mediated knockdown of HCFC1 expression resulted in the coordinate downregulation of MMACHC mRNA. This X-linked disorder demonstrates a distinct disease mechanism by which transcriptional dysregulation leads to an inborn error of metabolism with a complex clinical phenotype.

The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning

Mutations in cobalamin or B12 trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B12 processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5'-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ∼116-296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways.

Cobalamin C defect presenting as severe neonatal hyperammonemia

Cobalamin C (Cbl-C) defect is the most common inborn error of cobalamin metabolism which causes a block in the pathway responsible for the synthesis of its two metabolically active forms methyl- and adenosylcobalamin. Cbl-C defect causes the accumulation of methylmalonic acid and homocysteine and decreased methionine synthesis. The clinical presentation of patients with early-onset Cbl-C defect, characterized by a multisystem disease with severe neurological, ocular, hematological, renal, gastrointestinal, cardiac, and pulmonary manifestations, differs considerably from what observed in the "classical" form of methylmalonic aciduria caused by defect of methylmalonyl-CoA mutase. This last condition is in most cases dominated in the neonatal period by a metabolic encephalopathy "intoxication type" with severe hyperammonemia and ketoacidosis. We report a Cbl-C defect patient presenting a neonatal encephalopathy with severe hyperammonemia and ketoacidosis who was successfully treated with peritoneal dialysis. To the best of our knowledge, there are no reported cases of Cbl-C defect showing an acute presentation resembling a classical methylmalonic aciduria. This observation enlarges the spectrum of inherited diseases to be considered in the differential diagnosis of neonatal hyperammonemia.

Hyperpyrexia resulting in encephalopathy in a 14-month-old patient with cblC disease

Cobalamin C (cblC) defect, the most common inborn error of cobalamin metabolism, is a multisystem disorder usually presenting with progressive neurological, haematological and ophthalmological signs. We report on a cblC patient diagnosed in the newborn age who developed nearly normal during the first year of life. During an upper respiratory tract infection with severe hyperpyrexia at the age of 14 months he developed an acute encephalopathic crisis resulting in severe mental retardation and marked internal and external cerebral atrophy. Hyperacute encephalopathic crises have not been observed so far in patients with cblC defect. It remains unclear, if this association is incidental or if the underlying metabolic defect may have predisposed the brain tissue to hyperpyrexia-induced damage.

Vitamin B12 deficiency in a 15-year old boy due to mutations in the intrinsic factor gene, GIF

The discoveries of new genes involved in cobalamin assimilation and metabolism has increased our understanding of the rare inborn errors of cobalamin metabolism (Quadros 2009). The following case documents a compound heterozygote mutation in the gastric intrinsic factor (GIF) gene, with a previously described mutation (c.79+1G>A) and a novel mutation (c.290T>C; M97T) leading to a megaloblastic anaemia in an adolescent.

Clinical and gene mutation analysis in patients with methylmalonic acidemia and homocystelnemia

Objective Methylmalonic acidemia combined with homocysteinemia is an inborn error of cobalamin metabolism with an autosomal recessive mode. The cb1C type is the most common form of this disease,in which the gene was named MMACHC. This study was aimed to analyse the diagnosis, treatment and gene mutations in 14 patients. Methods Measurements of C3 (propionylcarnitine) , C3/C0 (free carnitine)and C3/C2 (acetylcarnitine) in blood were detected by tandem massepectrometry; urine methylmalonic acid were determined by gas-chromatography mass spectrometry; and serum homosysteine were determined with the method of fluorescence polarization immunization. These patients were treated with vitamin β12, L-carnitine, beta;he, folic acid and vitamin β6. The MMACHC gene was screened by PCR combined with DNA direct sequencing in 14 Chinese patients. Results With treatment clinical symptoms of 12 patients were improved obviously; the levels of blood propionylearnitine and homocysteine as well as urine methylmalonic acid were decreased. Two patients were dead without treatments. In this study, five different mutations in 12 patients were detected, only one polymorphism was found in one patient, and in another patient no mutation was detected. We found 609G>A homozygotic mutations in 7 patients, and heterozygotic ones of 609G > A with 658_660delAAG、567_568insT,394C>T and 217C>T in 5 patients, in which 658_660delAAG and 567_568insT were novel mutations.Conclusion The patients with methylmalonic acidemia and homocysteinemia could have an improved outcome after reasonable treatments. The gene mutation detection suggests that 609G>A (W203X) may be the hot spot mutation of MMACHC gene in Chinese patients. Key words: Methylmalonic acidemia; Homocysteinemia; Diagnosis; Treatment; Gene mutation

Epigenetic modification of the gene for the vitamin B12 chaperone MMACHC can result in increased tumorigenicity and methionine dependence

Methionine dependence, the inability of cells to grow when the amino acid methionine is replaced in culture medium by its metabolic precursor homocysteine, is characteristic of many cancer cell lines and some tumors in situ. Most cell lines proliferate normally under these conditions. The methionine dependent tumorigenic human melanoma cell line MeWo-LC1 was derived from the methionine independent non-tumorigenic line, MeWo. MeWo-LC1 has a cellular phenotype identical to that of cells from patients with the cblC inborn error of cobalamin metabolism, with decreased synthesis of cobalamin coenzymes and decreased activity of the cobalamin-dependent enzymes methionine synthase and methylmalonylCoA mutase. Inability of cblC cells to complement the defect in MeWo-LC1 suggested that it was caused by decreased activity of the MMACHC gene. However, no potentially disease causing mutations were detected in the coding sequence of MMACHC in MeWo-LC1. No MMACHC expression was detected in MeWo-LC1 by quantitative or non-quantitative PCR. There was virtually complete methylation of a CpG island at the 5′-end of the MMACHC gene in MeWo-LC1, consistent with inactivation of the gene by methylation. The CpG island was partially methylated (30–45%) in MeWo and only lightly methylated (2–11%) in control fibroblasts. Infection of MeWo-LC1 with wild type MMACHC resulted in correction of the defect in cobalamin metabolism and restoration of the ability of cells to grow in medium containing homocysteine. We conclude that epigenetic inactivation of the MMACHC gene is responsible for methionine dependence in MeWo-LC1.

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

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. 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. 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. 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.

Transcobalamin in cultured fibroblasts from patients with inborn errors of vitamin B 12 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.

Retinal Dysfunction in Combined Methylmalonic Aciduria and Homocystinuria (Cblc) Disease: A Spectrum of Disorders

Cobalamin C methylmalonic aciduria with homocystinuria (cblC disease) is a rare hereditary inborn error of cobalamin metabolism, characterised by neurological, haematological and ophthalmological abnormalities. Three consecutive patients with Cblc disease were examined. Investigations included slit lamp and fundus examination and full-field ERG. A maculopathy associated with both photopic and scotopic abnormal ERG was present in two cases and a salt and pepper retinopathy with abnormal photopic ERG was detected in the third patient. Despite early treatment and regular metabolic controls, all our patients exhibited both retinal and ERG abnormalities. There was no correlation between funduscopic appearance and the type of photoreceptor dysfunction. A literature review disclosed a retinopathy in 29 / 70 cases with cblC disease, with an abnormal ERG in 8 of the 12 tested cases, most with retinopathy. Retinal dysfunction in cblC disease may be more frequent than previously thought, and can involve cones only or both rods and cones. We recommend a formal ocular examination with full-field ERG in patients with Cblc disease.

The adolescent and adult form of cobalamin C disease: clinical and molecular spectrum

Background:Cobalamin C disease is the most common inborn error of cobalamin metabolism with an autosomal recessive mode of inheritance and mutations within the MMACHC gene. Clinical features, including systemic, haematological...

Polymorphic background of methionine synthase reductase modulates the phenotype of a disease-causing mutation

Methionine synthase reductase (MTRR) is the locus of the cblE class of inborn errors of cobalamin metabolism that is characterized by megaloblastic anemia and homocystinuria. Two highly prevalent SNPs, c.66A>G (p.Ile22Met) and c.524C>T (p.Ser175Leu), are found in the MTRR gene. On the basis of the allele frequency of these amino acids and sequence comparison with members of the same family of proteins, the p.Ile22/p.Ser175 sequence is designated as wild type. While characterizing a pathogenic methionine synthase reductase (MSR) mutation, c.166G>A (p.Val56Met), we discovered an interaction between the mutation and one of the polymorphic sites. Thus, when the p.Val56Met mutant was initially expressed in the p.Ile22/p.Ser175 background, we were surprised to find that kinetically, it was virtually indistinguishable from wild-type protein. To determine if the polymorphisms interacted with the p.Val56Met mutation, it was expressed in all four possible genetic backgrounds. We found that in the p.Ile22Met background, the p.Val56Met mutation impacted the kinetics of MSR and an approximately three- to 10-fold higher concentration of the p.Ile22Met/p.Val56Met mutant was required for maximal activation of methionine synthase vs. the range seen with wild-type MSR variants. A comparable (three- to seven-fold) diminution in MSR activity was observed in extracts of fibroblast cells from patients carrying the p.Val56Met mutation on one MSR allele and a null mutation on the other. These results predicted that the patient allele encodes the p.Val56Met mutation and the p.Ile22Met variation, which was confirmed by sequence analysis. This study reveals how a genetic variation can modulate phenotypic expression of a disease-causing mutation.

Mitochondrial vitamin B 12-binding proteins in patients with inborn errors of cobalamin metabolism

Inborn errors of vitamin B 12 (cobalamin, Cbl) metabolism are autosomal recessive disorders and have been classified into nine distinct complementation classes ( cblA- cblH and mut). Disorders affecting methylcobalamin metabolism cause megaloblastic anemia, which may be accompanied by leukopenia and thrombocytopenia, and a variety of neurological problems. Disorders affecting adenosylcobalamin cause methylmalonic acidemia and metabolic acidosis. Previous studies have shown that cobalamin binds to two enzymes in humans: methylmalonyl-CoA mutase in mitochondria and methionine synthase in the cytosol. In this study, cobalamin binding patterns were analyzed in crude mitochondrial fractions obtained from both control and patient fibroblasts that had been incubated with [ 57Co]cyanocobalamin. Crude mitochondrial fractions from control fibroblasts confirmed that the majority of [ 57Co]Cbl eluted with methylmalonyl-CoA mutase. However, in six of the nine disorders, at least one previously unidentified mitochondrial cobalamin binding protein was observed to bind [ 57Co]Cbl. The proportion of [ 57Co]Cbl that binds, is increased compared to controls when a deficiency in either adenosylcobalamin synthesis or utilization prevents binding to methylmalonyl-CoA mutase. Furthermore, unique cobalamin binding profiles emerged demonstrating how known mutations in these patients affect cobalamin binding to as yet unidentified proteins.

Acquired and inherited disorders of cobalamin and folate in children

Cobalamin deficiency in the newborn usually results from cobalamin deficiency in the mother. Megaloblastic anaemia, pancytopenia and failure to thrive can be present, accompanied by neurological deficits if the diagnosis is delayed. Most cases of spina bifida and other neural tube defects result from maternal folate and/or cobalamin insufficiency in the periconceptual period. Polymorphisms in a number of genes involved in folate and cobalamin metabolism exacerbate the risk. Inborn errors of cobalamin metabolism affect its absorption, (intrinsic factor deficiency, Imerslund-Gräsbeck syndrome) and transport (transcobalamin deficiency) as well as its intracellular metabolism affecting adenosylcobalamin synthesis (cblA and cblB), methionine synthase function (cblE and cblG) or both (cblC, cblD and cblF). Inborn errors of folate metabolism include congenital folate malabsorption, severe methylenetetrahydrofolate reductase deficiency and formiminotransferase deficiency. The identification of disease-causing mutations in specific genes has improved our ability to diagnose many of these conditions, both before and after birth.

MRI and1H‐MRS Findings in Early-Onset Cobalamin C/D Defect

Cobalamin C/D defect is an inborn error of cobalamin metabolism causing methylmalonic aciduria and homocystinuria. The early-onset form is characterized by severe neurological impairment. The aim of this study was to evaluate and monitor brain damage in early-onset cbl-C/D defect by conventional MRI and to assess the additional value of 1H-MRS. We retrospectively examined serial MRI studies of 7 patients, performed on a 1.5 T system. Four patients had the first evaluation within the first 4 months of life and three later. The imaging protocol included spin-echo T1-weighted, T2-weighted, IR, and FLAIR. Five patients underwent 1H-MRS, using chemical shift imaging (CSI) in three patients and single voxel spectroscopy (SVS) in two. Three of the patients studied early showed tetraventricular hydrocephalus and diffuse swelling of supratentorial white matter with involvement of the "U" fibres. Two showed patchy cavitating lesions in the basal ganglia. White matter changes became evident at a later stage. In three cases 1H-MRS showed an abnormal peak of lactate in the basal ganglia or in the periventricular white matter. Our study shows severe heterogeneous brain MR abnormalities in cbl-C/D defect. We observed unusual basal ganglia lesions in 30 % of our cases and also found a high incidence of hydrocephalus and supratentorial white matter abnormalities.

Prenatal diagnosis for methylmalonic acidemia and inborn errors of vitamin B 12 metabolism and transport

Vitamin B 12 (cobalamin) is an essential cofactor for two enzymes: methionine synthase (MS), which requires methylcobalamin (MeCbl), and methylmalonyl-CoA mutase (MUT), which requires adenosylcobalamin (AdoCbl). A number of individually rare inborn errors of cobalamin metabolism are known and are distinguished by complementation analysis ( mut, cblA-cblH). From 1984 to 2005, we have performed prenatal diagnosis for 117 high-risk pregnancies. We identified a total of 21 affected pregnancies (18%): cblA, 2/8; cblB, 0/5; cblC, 10/52; cblE, 2/3; cblF, 0/5; cblG, 0/5; transcobalamin deficiency, 0/2; methylmalonyl-CoA mutase ( mut) deficiency, 7/30; and unclassified MMA, 0/7. Studies were performed on amniotic fluid, cultured chorionic villus cells (CCVC), cultured amniocytes (CA), or various combinations of these three types of sample. Analyses done include propionate and methyltetrahydrofolate incorporation into protein and cobalamin cofactor levels (CA: 92%, CCVC: 18%), amniotic fluid metabolite measurement either by gas chromatography/mass spectrometry (GC/MS) or by liquid chromatography–tandem mass spectrometry (LC–MS/MS) (49%), and direct mutation analysis (5%). There was one false negative CCVC result in a pregnancy at risk for cblC and one false positive CCVC in a pregnancy at risk for mutase deficiency. One unaffected pregnancy at risk for an unclassified form of MMA and another unaffected pregnancy at risk for cblC, had higher than control MMA amniotic fluid levels. Our experience suggests that prenatal diagnosis for these disorders should be done by application of two independent methods, and that CA studies appear more reliable than CCVC studies.

Creatine metabolism in combined methylmalonic aciduria and homocystinuria

Methylation is an important aspect of many fundamental biological processes including creatine biosynthesis. We studied five patients with an inborn error of cobalamin metabolism to characterize the relation between homocysteine and creatine metabolism. Plasma guanidinoacetate concentrations were increased, 14.9 +/- 4.8 micromol/L (p < 0.0001), whereas plasma creatine concentrations were in the low reference range, 43.8 +/- 20.7 micromol/L (p = not significant). Individuals with combined methylmalonic aciduria and homocystinuria have a functional impairment of the creatine synthetic pathway probably secondary to a relative depletion of labile methyl groups. The neurotoxic effects of guanidinoacetate may be partly responsible for the observed neurological phenotype.

Complementation studies in the cblAclass of inborn error of cobalamin metabolism: evidence for interallelic complementation and for a new complementation class (cblH)

AIMTo investigate genetic heterogeneity within the cblA class of inborn error of cobalamin metabolism.CONTEXTThecblA disorder is characterised by vitamin B12 (cobalamin) responsive methylmalonic aciduria and deficient synthesis of adenosylcobalamin, required...

Vitamin B 12-responsive methylmalonic aciduria due to a new inborn error of adenosylcobalamin synthesis, cblH

Vitamin B12-responsive methylmalonic aciduria is characterized by deficient synthesis of adenosylcobalamin (AdoCbl) and decreased activity of the AdoCbl-dependent mitochondrial enzyme, methylmalonylCoA mutase. Two distinct disorders of AdoCbl synthesized have been characterized (cblA and cblB) and they can be distinguished on the basis of somatic cell complementation analysis. Such studies have clinical usefulness because in general cblA patients have a better prognosis than do cblB patients. We reported a patient whose fibroblasts had the clinical and biochemical features of the cblA class but complemented cells from all known inborn errors of cobalamin metabolism that cause methylmalonic aciduria, including cblA. The disorder in this patient was designated as cblA variant. Cells from this patient were tested against a panel of 29 cell lines from cblA patients and complemented all members of this panel. These studies strongly suggest that this patient represents a novel complementation class which we have called cblH. Detailed complementation analysis of a panel of 10 cblA lines provided evidence of interallelic complementation. The presence of this interallelic complementation indicates that care must be taken in the use of complementation analysis to identify cblA patients. These studies suggest that additional biochemical steps are required for the reduction and adenosylation of cobalamin. The full clinical spectrum of the new cblH class and its implications remains to be defined.