[show abstract][hide abstract] ABSTRACT: Transcobalamin (TC) transports cobalamin from blood into cells. TC deficiency is a rare autosomal recessive disorder usually presenting in early infancy with failure to thrive, weakness, diarrhoea, pallor, anemia, and pancytopenia or agammaglobulinemia. It can sometimes resemble neonatal leukemia or severe combined immunodeficiency disease. Diagnosis of TC deficiency is suspected based on megaloblastic anemia, elevation of total plasma homocysteine, and blood or urine methylmalonic acid. It is confirmed by studying the synthesis of TC in cultured fibroblasts, or by molecular analysis of the TCN2 gene. TC deficiency is treatable with supplemental cobalamin, but the optimal type, route and frequency of cobalamin administration and long term patient outcomes are unknown. Here we present a series of 30 patients with TC deficiency, including an update on multiple previously published patients, in order to evaluate the different treatment strategies and provide information about long term outcome. Based on the data presented, current practice appears to favour treatment of individuals with TC deficiency by intramuscular injections of hydroxy- or cyanocobalamin. In most cases presented, at least weekly injections (1 mg IM) were necessary to ensure optimal treatment. Most centres adjusted the treatment regimen based on monitoring CBC, total plasma homocysteine, plasma and urine methylmalonic acid, as well as, clinical status. Finally, continuing IM treatment into adulthood appears to be beneficial.
Journal of Inherited Metabolic Disease 12/2013; · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 American Journal of Human Genetics 09/2013; 93(3):506-14. · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of HOCbl to MeCbl, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In 4 'cblG-variant' cases we observed a decreased conversion of CNCbl to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts, and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA respectively, when compared to control cells. In 3-D modelling and docking analysis, both truncated and full size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of cobalamins that is required for Cbl cofactor synthesis.
Human Molecular Genetics 07/2013; · 7.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: A neonate, who was found to have an elevated C3/C2 ratio and minimally elevated propionylcarnitine on newborn screening, was subsequently identified as having the rare cblF inborn error of vitamin B12 (cobalamin) metabolism. This disorder is characterized by the retention of unmetabolized cobalamin in lysosomes such that it is not readily available for cellular metabolism. Although cultured fibroblasts from the patient did not show the expected functional abnormalities of the cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, they did show reduced synthesis of the active cobalamin cofactors adenosylcobalamin and methylcobalamin. Mutation analysis of LMBRD1 established that the patient had the cblF disorder. Treatment was initiated promptly, and the patient showed a robust response to regular injections of cyanocobalamin, and she was later switched to hydroxocobalamin. Currently, at 3 years of age, the child is clinically well, with appropriate development. Adjusted newborn screening cutoffs in Ontario allowed detection of a deficiency that might not have otherwise been identified, allowing early treatment and perhaps preventing the adverse sequelae seen in some untreated patients.
[show abstract][hide abstract] ABSTRACT: Isolated methylmalonic aciduria (MMA) results either from a defect in the mitochondrial enzyme methylmalonylCoA mutase (MCM), or in the intracellular conversion of vitamin B12 (cobalamin) into its active coenzyme adenosylcobalamin (AdoCbl). Mutations in the MMAB gene affect the function of the enzyme ATP:cob(I)alamin adenosyltransferase (ATR) and the production of AdoCbl. Measurement of MCM function in cultured patient fibroblasts, followed by somatic cell complementation analysis in cases where MCM function is decreased, has classically been used to diagnose the cblB cobalamin disorder. A patient with persistent MMA, who could not be diagnosed using traditional somatic cell studies, was subsequently shown by sequencing in a clinical laboratory to contain two variants in the MMAB gene. This observation brings into question whether somatic cell studies have failed to diagnose other cblB patients with mild cellular phenotypes. A high resolution melting analysis (HRMA) assay was developed for the MMAB gene. It was used to scan 96 reference samples and two cohorts of patients: 42 patients diagnosed with cblB by complementation studies; and 181 patients with undiagnosed MMA. MMAB mutations, including one novel nonsense mutation (c.12 C>A [p.C4X]), were identified in all members of the cblB cohort. Four patients with undiagnosed MMA, including the index case described above, were found to contain variants in the MMAB gene: c.185C>T (p.T62M), c.394T>C (p.C132R), c.398C>T (p.S133F), c.521C>T (p.S174L), c.572G>A (p.R191Q). Only the index case was found to have two variants, suggesting that somatic cell studies diagnose almost all cblB patients.
Molecular Genetics and Metabolism 05/2013; · 2.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: The cblE disorder is an inherited disorder of vitamin B12 metabolism that results in elevated levels of homocysteine and decreased methionine in body fluids. Renal complications have been reported in patients with cblC disease, but not in those with cblE disease. The renal complications of cblC disease include thrombotic microangiopathy (TMA), neonatal hemolytic uremic syndrome, chronic renal failure, tubulointerstitial nephritis and proximal renal tubular acidosis. Previously, we reported a patient with cblC disease who had an atypical glomerulopathy that manifested with proteinuria and progressive renal insufficiency. CASE-DIAGNOSIS/TREATMENT: Studies were done on cultured fibroblasts. Renal biopsy tissue was examined by light and electron microscopy. There was decreased incorporation of labeled methyltetrahydrofolate and decreased synthesis of methylcobalamin. Complementation analysis placed the patient into the cblE complementation group. The findings from the histological and ultrastructural studies of renal biopsy were similar, but not identical, to those of idiopathic membranoproliferative glomerulonephritis (MPGN) and overlapped with those of TMA. CONCLUSIONS: We describe a patient with cblE disease who had an atypical glomerulopathy similar to MPGN. Additional findings included migraine headaches, hypothyroidism and livedo reticularis.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Since 1975 cells lines from patients with suspected inborn errors of vitamin B12 metabolism have been referred to our laboratory because of elevations of homocysteine, methylmalonic acid, or both. DESIGN: Cultured fibroblasts from patients were subjected to a battery of tests: incorporation of labelled propionate and methyltetrahydrofolate into cellular macromolecules, to test the functional integrity of methylmalonyl-CoA mutase and methionine synthase, respectively; uptake of labelled cyanocobalamin and synthesis of adenosylcobalamin and methylcobalamin; and, where applicable, complementation analysis. RESULTS: This approach has allowed for the discovery of novel steps in the cellular transport and metabolism of vitamin B12, including those involving cellular uptake, the efflux of vitamin B12 from lysosomes, and the synthesis of adenosylcobalamin and methylcobalamin. For all of these disorders, the responsible genes have been discovered. CONCLUSION: The study of highly selected patients with suspected inborn errors of metabolism has consistently resulted in the discovery of previously unknown metabolic steps and has provided new lessons in biology.
[show abstract][hide abstract] ABSTRACT: Folate and vitamin B(12) metabolism are essential for de novo purine synthesis, and several defects in these pathways have been associated with immunodeficiency. Here we describe the occurrence of severe combined immunodeficiency (SCID) with megaloblastic anemia, leukopenia, atypical hemolytic uremic syndrome, and neurologic abnormalities in which hydroxocobalamin and folate therapy provided partial immune reconstitution. Whole exome sequencing identified compound heterozygous mutations in the MTHFD1 gene, which encodes a trifunctional protein essential for processing of single-carbon folate derivatives. We now report the immunologic details of this novel genetic cause of SCID and the response to targeted metabolic supplementation therapies. This finding expands the known metabolic causes of SCID and presents an important diagnostic consideration given the positive impact of therapy.
[show abstract][hide abstract] ABSTRACT: MMACHC and MMADHC are the genes responsible for cblC and cblD defects of vitamin B(12) metabolism, respectively. Patients with cblC and cblD defects present with various combinations of methylmalonic aciduria (MMA) and homocystinuria (HC). Those with cblC mutations have both MMA and HC whereas cblD patients can present with one of three distinct biochemical phenotypes: isolated MMA, isolated HC, or combined MMA and HC. Based on the subcellular localization of these enzymatic pathways it is thought that MMACHC functions in the cytoplasm while MMADHC functions downstream of MMACHC in both the cytoplasm and the mitochondrion. In this study we determined the subcellular location of MMACHC and MMADHC by immunofluorescence and subcellular fractionation. We show that MMACHC is cytoplasmic while MMADHC is both mitochondrial and cytoplasmic, consistent with the proposal that MMADHC acts as a branch point for vitamin B(12) delivery to the cytoplasm and mitochondria. The factors that determine the distribution of MMADHC between the cytoplasm and mitochondria remain unknown. Functional complementation experiments showed that retroviral expression of the GFP tagged constructs rescued all biochemical defects in cblC and cblD fibroblasts except propionate incorporation in cblD-MMA cells, suggesting that the endogenous mutant protein interferes with the function of the transduced wild type construct.
Molecular Genetics and Metabolism 12/2012; · 2.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Inborn errors of vitamin B(12) (cobalamin) metabolism are characterized by decreased production of active cobalamin cofactors and subsequent deficiencies in the activities of methionine synthase and methylmalonyl-CoA mutase. With the recent discovery of the cblJ defect in two patients with phenotypes mimicking the cblF defect, there are nine genes known to be involved in cobalamin metabolism. The new defect is caused by mutations in the ABCD4 gene, encoding an ABC transporter. At the moment, there is no clear distinction between the cblJ and cblF defects either clinically or biochemically, and both defects result in blocks in the transport of cobalamin from the lysosome to the cytoplasm. A patient was diagnosed with hyperhomocysteinemia and methylmalonic aciduria at the age of 8years. Incorporations of both [(14)C]propionate and [(14)C]methyltetrahydrofolate in cultured fibroblasts were within reference ranges and thus too high to allow for complementation analysis. We observed decreased synthesis of both adenosylcobalamin and methylcobalamin and accumulation of unmetabolized cyanocobalamin. Exome sequencing was performed to identify causative mutation(s) and Sanger re-sequencing was performed to validate segregation of mutation in the family. By this approach, a homozygous mutation, c.423C>G, in the ABCD4 gene was identified. Here, we report the successful application of exome sequencing for diagnosis of a rare inborn error of vitamin B(12) metabolism in a patient whose unusual presentation precluded diagnosis using standard biochemical and genetic approaches. The patient represents only the third known patient with the cblJ disorder.
Molecular Genetics and Metabolism 10/2012; · 2.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: The gene product of MMAA is required for the intracellular metabolism of cobalamin (Cbl). Mutations in this gene lead to the cblA class of disorders, characterized by isolated methylmalonic aciduria. We have been concerned that somatic cell methods of diagnosis may miss patients with mild cellular phenotypes. A high resolution melting analysis (HRMA) assay was developed to rapidly scan the coding exons and flanking intronic regions of the MMAA gene for variants. DNA was scanned by HRMA from 96 unaffected reference individuals, 72 cblA patients confirmed by complementation, and 181 patients with isolated elevated methylmalonic acid, who could not be diagnosed using complementation analysis. Suspected variants were confirmed by Sanger sequencing. In the cblA cohort, HRMA correctly identified all previously known mutations as well as an additional 22 variants, 10 of which had not been previously reported. Novel variants included one duplication (c.551dupG, p.C187LfsX3), one deletion (c.387delC, p.Y129YfsX13), one splice site mutation (c.440-2A>G, splice site), 4 missense mutations (c.748G>A, p.E520K; c.820G>A, p.G274S; c.627G>T, p.R209S; c.826A>G, p.K276E), and 3 nonsense mutations (c.960G>A, p.W320X; c.1075C>T, p.E359X; c.1084C>T, p.Q362X). All novel missense variants affect highly conserved residues and are predicted to be damaging. Scanning of MMAA in the 181 undiagnosed samples revealed a single novel heterozygous missense change (c.821G>A, p.G274D).
Molecular Genetics and Metabolism 09/2012; · 2.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Inherited disorders of vitamin B(12) (cobalamin) have provided important clues to how this vitamin, which is essential for hematological and neurological function, is transported and metabolized. We describe a new disease that results in failure to release vitamin B(12) from lysosomes, which mimics the cblF defect caused by LMBRD1 mutations. Using microcell-mediated chromosome transfer and exome sequencing, we identified causal mutations in ABCD4, a gene that codes for an ABC transporter, which was previously thought to have peroxisomal localization and function. Our results show that ABCD4 colocalizes with the lysosomal proteins LAMP1 and LMBD1, the latter of which is deficient in the cblF defect. Furthermore, we show that mutations altering the putative ATPase domain of ABCD4 affect its function, suggesting that the ATPase activity of ABCD4 may be involved in intracellular processing of vitamin B(12).
[show abstract][hide abstract] ABSTRACT: Derivatives of folic acid are involved in transfer of one-carbon units in cellular metabolism, playing a role in synthesis of purines and thymidylate and in the remethylation of homocysteine to form methionine. Five inborn errors affecting folate transport and metabolism have been well studied: hereditary folate malabsorption, caused by mutations in the gene encoding the proton-coupled folate transporter (SLC46A1); glutamate formiminotransferase deficiency, caused by mutations in the FTCD gene; methylenetetrahydrofolate reductase deficiency, caused by mutations in the MTHFR gene; and functional methionine synthase deficiency, either as the result of mutations affecting methionine synthase itself (cblG, caused by mutations in the MTR gene) or affecting the accessory protein methionine synthase reductase (cblE, caused by mutations in the MTRR gene). Recently additional inborn errors have been identified. Cerebral folate deficiency is a clinically heterogeneous disorder, which in a few families is caused by mutations in the FOLR1 gene. Dihydrofolate reductase deficiency is characterized by megaloblastic anemia and cerebral folate deficiency, with variable neurological findings. It is caused by mutations in the DHFR gene. Deficiency in the trifunctional enzyme containing methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase activities, has been identified in a single patient with megaloblastic anemia, atypical hemolytic uremic syndrome and severe combined immune deficiency. It is caused by mutations in the MTHFD1 gene.
[show abstract][hide abstract] ABSTRACT: The cobalamin F (cblF) defect is caused by disturbed lysosomal release of cobalamin (vitamin B(12)) into the cytoplasm caused by mutations in the LMBRD1 gene. We present the clinical and biochemical characterization of a patient with newly diagnosed cblF disease and a follow-up on a 14-year-old patient. The new patient presented with elevation of propionyl carnitine found on a newborn screen. The patient was small for gestational age, exhibited dysmorphic features and mild developmental delay, and had trigonocephaly and ventricular septal defect. There was biochemical normalization and clinical improvement within 3 weeks of parenteral cobalamin treatment. The other patient presented at 4 weeks of life with failure to thrive and feeding difficulties. She was treated only with monthly cyanocobalamin shots. The patient has never experienced metabolic decompensation. She had short stature and was an average student with no behavioral concerns. Her metabolic derangements normalized after switching to weekly hydroxycobalamin. The available data on 14 patients with confirmed cblF disease suggest variability in age of onset, presenting symptoms, response to treatment, and long-term complications. Common clinical findings include small for gestational age, feeding difficulties, growth failure, and developmental delays. Some patients have congenital heart defects, dysmorphic features, and other congenital anomalies.
[show abstract][hide abstract] ABSTRACT: An infant was investigated because of megaloblastic anaemia, atypical hemolytic uraemic syndrome, severe combined immune deficiency, elevated blood levels of homocysteine and methylmalonic acid, and a selective decreased synthesis of methylcobalamin in cultured fibroblasts.
Exome sequencing was performed on patient genomic DNA.
Two mutations were identified in the MTHFD1 gene, which encodes a protein that catalyses three reactions involved in cellular folate metabolism. This protein is essential for the generation of formyltetrahydrofolate and methylenetetrahydrofolate and important for nucleotide and homocysteine metabolism. One mutation (c.727+1G>A) affects the splice acceptor site of intron 8. The second mutation, c.517C>T (p.R173C), changes a critical arginine residue in the NADP-binding site of the protein. Mutations affecting this arginine have previously been shown to affect enzyme activity. Both parents carry a single mutation and an unaffected sibling carries neither mutation. The combination of two mutations in the MTHFRD1 gene, predicted to have severe consequences, in the patient and their absence in the unaffected sibling, supports causality.
This patient represents the first case of an inborn error of folate metabolism affecting the trifunctional MTHFD1 protein. This report reinforces the power of exome capture and sequencing for the discovery of novel genes, even when only a single proband is available for study.
Journal of Medical Genetics 09/2011; 48(9):590-2. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Derivatives of cobalamin (vitamin B(12)) are required for activity of two enzymes in humans. Adenosylcobalamin is required for activity of mitochondrial methylmalonylCoA mutase and methylcobalamin is required for activity of cytoplasmic methionine synthase. Deficiency in cobalamin, or inability to absorb cobalamin normally, can result in accumulation of methylmalonic acid and homocysteine in blood and urine. Methylmalonic acidemia can result in metabolic acidosis which in severe cases may be fatal. Hyperhomocysteinemia along with hypomethioninemia can result in hematologic (megaloblastic anemia, neutropenia, thrombocytopenia) and neurologic (subacute combined degeneration of the cord, dementia, psychosis) defects. Inborn errors affecting cobalamin absorption (inherited intrinsic factor deficiency, Imerslund–Gra¨ sbeck syndrome) and transport (transcobalamin deficiency) have been described. A series of inborn errors of intracellular cobalamin metabolism, designated cblA-cblG, have been differentiated by complementation analysis. These can give rise to isolated methylmalonic acidemia (cblA, cblB, cblD variant 2), isolated hyperhomocysteinemia (cblD variant 1, cblE, cblG) or combined methylmalonic acidemia and hyperhomocysteinemia (cblC, classic cblD, cblF). All these disorders are inherited as autosomal recessive traits. The genes underlying each of these disorders have been identified. Two other disorders, haptocorrin deficiency and transcobalamin receptor deficiency, have been described, but it is not clear that they have any consistent clinical phenotype.
American Journal of Medical Genetics Part C Seminars in Medical Genetics 02/2011; 157(1):33-44. · 4.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: The cblF disorder, characterized by accumulation of internalized cobalamin in the lysosome, is caused by mutations in the LMBRD1 gene which encodes an integral lysosomal membrane protein. We describe novel mutations in LMBRD1 in three patients: two splice site mutations, c.916-1G>T and c.1339-1G>T, and a 6785 bp deletion encompassing exon 2, c.70-4298_246+2311del6785. The three patients are compound heterozygotes for one novel mutation and the common c.1056delG mutation.
Molecular Genetics and Metabolism 01/2011; 102(4):505-7. · 2.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function.
Human Mutation 08/2010; 31(8):924-9. · 5.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Molecular Genetics and Metabolism 03/2009; 96(4):261-7. · 2.83 Impact Factor