Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment.

In the folate cycle MTHFD1, encoded by MTHFD1, is a trifunctional enzyme containing 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase activity. To date, only one patient with MTHFD1 deficiency, presenting with hyperhomocysteinemia, megaloblastic anaemia, hemolytic uremic syndrome (HUS) and severe combined immunodeficiency, has been identified (Watkins et al J Med Genet 48:590-2, 2011). We now describe four additional patients from two different families. The second patient presented with hyperhomocysteinemia, megaloblastic anaemia, HUS, microangiopathy and retinopathy; all except the retinopathy resolved after treatment with hydroxocobalamin, betaine and folinic acid. The third patient developed megaloblastic anaemia, infection, autoimmune disease and moderate liver fibrosis but not hyperhomocysteinemia, and was successfully treated with a regime that included and was eventually reduced to folic acid. The other two, elder siblings of the third patient, died at 9 weeks of age with megaloblastic anaemia, infection and severe acidosis and had MTFHD1 deficiency diagnosed retrospectively. We identified a missense mutation (c.806C > T, p.Thr296Ile) and a splice site mutation (c.1674G > A) leading to exon skipping in the second patient, while the other three harboured a missense mutation (c.146C > T, p.Ser49Phe) and a premature stop mutation (c.673G > T, p.Glu225*), all of which were novel. Patient fibroblast studies revealed severely reduced methionine formation from [(14)C]-formate, which did not increase in cobalamin supplemented culture medium but was responsive to folic and folinic acid. These additional cases increase the clinical spectrum of this intriguing defect, provide in vitro evidence of disturbed methionine synthesis and substantiate the effectiveness of folic or folinic acid treatment.

Update on transcobalamin deficiency: clinical presentation, treatment and outcome.

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.

Human methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification.

Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, a cofactor for homocysteine methylation to methionine. MTHFR deficiency, an autosomal recessive disorder, results in homocysteinemia. Using degenerate oligonucleotides based on porcine peptide sequence data, we isolated a 90-bp cDNA by PCR from pig liver RNA. This cDNA was used to isolate a human cDNA, the predicted amino acid sequence of which shows strong homology to porcine MTHFR and to bacterial metF genes. The human gene has been localized to chromosome 1p36.3. Two mutations were identified in MTHFR-deficient patients: a missense mutation (Arg to Gln), in a residue conserved in bacterial enzymes, and a nonsense mutation (Arg to Ter).

Defect in vitamin B12 release from lysosomes: newly described inborn error of vitamin B12 metabolism.

Cultured diploid fibroblasts from a patient with a previously undescribed inborn error of cobalamin metabolism accumulate unmetabolized, nonprotein-bound vitamin B12 in lysosomes. These cells are able to endocytose the transcobalamin II-B12 complex and to release B12 from transcobalamin II. The freed vitamin B12 is not released from lysosomes into the cytoplasm of the cell. This suggests that there is a specific lysosomal transport mechanism for vitamin B12 in the human.

Transcobalamin (TC) deficiency--potential cause of bone marrow failure in childhood.

It is unusual for inborn errors of metabolism to be considered in the investigative work-up of pancytopenia. We report a family in which the proband presented with failure to thrive at 2 months of age and subsequent bone marrow failure. A previous sibling had died at 7 months of age with suspected leukaemia. Haematological findings in the proband were significant for pancytopenia, and bone marrow aspiration showed dysplastic changes in all cell lineages. Urinary organic acid analysis revealed elevated methylmalonic acid. The synthesis of transcobalamin II (transcobalamin, TC) by cultured fibroblasts was markedly reduced, confirming the diagnosis of TC deficiency. The proband and his younger asymptomatic sister (also found to have TC deficiency) were homozygous for R399X (c.1195C>T), a novel mutation resulting in the loss of the C- terminal 29 amino acids of TC, a highly conserved region. Response to parenteral vitamin B(12) in the proband was dramatic. At 6 years 3 months of age, physical examination is normal and developmental level is age appropriate. His sister is clinically asymptomatic and is also developing normally. Propionylcarnitine concentrations were not elevated in the newborn screening cards from the proband and sister, but that was for specimens retrieved from storage after 7 years and 5 years, respectively. Inherited and acquired cobalamin disorders should both be considered in the differential diagnosis of bone marrow failure syndromes in young children. Early detection of the metabolic causes of bone marrow failure can ensure prompt recovery in some cases involving the vitamin B(12) pathway.

Genetic heterogeneity among patients with methylcobalamin deficiency. Definition of two complementation groups, cblE and cblG.

A number of patients with megaloblastic anemia and homocystinuria associated with low levels of methylcobalamin synthesis in cultured cells have been recognized. Methionine biosynthesis by intact cells, as determined by incorporation of label from 5-[14C]methyl-tetrahydrofolate into acid-precipitable material, was deficient in cultured skin fibroblasts that were derived from all of these patients. In one group of patients, activity of the methylcobalamin-dependent enzyme, methionine synthase, in cell extracts was within the normal range when the enzyme was assayed under standard conditions. In a second group of patients, methionine synthase activity was decreased under the same assay conditions. Genetic complementation analysis demonstrated the existence of two complementation classes that corresponded to these two groups of patients. The designation cblE has previously been proposed for normal methionine synthase activity. We propose the designation cblG for the mutation in those patients with methylcobalamin deficiency and decreased synthase activity. The results of these studies suggest that the products of at least two loci are required for cobalamin-dependent methionine biosynthesis in mammalian cells.

Altered vitamin B12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis.

Cultured fibroblasts from a recently described patient with homocystinuria and megaloblastic anemia of infancy without methylmalonic aciduria were previously shown to have normal cobalamin uptake and a specific decrease in the proportion of intracellular methylcobalamin. As in control cells but unlike in those from patients with combined homocystinuria and methylmalonic aciduria (cobalamin C and cobalamin D), accumulated 57Co-labeled cobalamin was bound in appropriate amounts and proportion to intracellular binders which are known to be the two vitamin B12-dependent enzymes, methionine synthetase and methylmalonyl-CoA mutase. Despite the association of a normal quantity of intracellular cobalamin with methionine synthetase, the proportion of intracellular cobalamin which was methyl-B12 was below normal and in the range observed in cobalamin C and D cells. This methyl-B12 was decreased by exposure of fibroblasts in culture to nitrous oxide as was observed with control cells. Exposure of control fibroblasts during culture, but not of fibroblasts from this patient, to nitrous oxide significantly reduced the holoenzyme activity of methionine synthetase assayed in cell extracts. In addition, although methionine synthetase activity in cell extracts of control and cells from the patient were similar in the presence of standard assay concentrations of thiols, at low thiol concentrations, methionine synthetase activity in extracts of cells from the patient was much lower than in control extracts. Mixing of control patient extracts corrected this decreased activity in excess of that explained by addition of the individual activities added. The defect of this patient appears to be in a reducing system required for methionine synthesis.

Folate distribution in cultured human cells. Studies on 5,10-CH2-H4PteGlu reductase deficiency.

We have studied the distribution of folate coenzyme forms in cultured human fibroblasts from control lines and from lines derived from nine patients representing all of the published reports of 5,10-CH(2)-H(4)PteGlu reductase deficiency. Based on mobility on DEAE-Sephadex and differential microbiological assay the major folate fractions in extracts of human fibroblasts were 5-CH(3)-H(4)PteGlu, 10-CHO-H(4)PteGlu, and 5-CHO-H(4)PteGlu with smaller fractions, which included 5-CH(3)-H(2)PteGlu, 10-CHO-PteGlu, and H(4)PteGlu. Evidence that the 5-CHO-H(4)PteGlu may have been derived from 5,10-CH=H(4)PteGlu during extraction is presented. In most of the mutant fibroblasts the absolute concentration of 5-CH(3)-H(4)PteGlu was lower than in control cells but the proportion of intracellular folate which was 5-CH(3)-H(4)PteGlu was strikingly lower in mutant cells when determined by chromatography or differential microbiological assay. In both control and mutant cells most of the 5-CH(3)-H(4)-PteGlu was polyglutamate. The proportion of intracellular folate which was polyglutamate was similar in control and mutant cells. A direct relationship was observed between the proportion of cellular folate which was 5-CH(3)-H(4)PteGlu, and both the clinical severity of this disorder and the residual enzyme activity indicating that the distribution of different folates may be an important control of intracellular folate metabolism. These studies indicate that 5,10-CH(2)-H(4)PteGlu reductase is the only significant intracellular pathway for the generation of 5-CH(3)-H(4)PteGlu, that the activity of this enzyme regulates the level of this folate in control and mutant cells under conditions of culture used here, that the majority of intracellular folate is in the polyglutamate form, and that the relative distribution of folates may control folate metabolism by interaction in the various folate reactions.

Genetic characterization of a MUT locus mutation discriminating heterogeneity in mut0 and mut- methylmalonic aciduria by interallelic complementation.

Genetic complementation of fibroblasts from patients with methylmalonic aciduria (MMA) defines a unique class of allelic mutations arising from mutations at the locus encoding the methylmalonyl coenzyme A (CoA) mutase apoenzyme. Various phenotypes of MMA have been delineated including complete absence of enzyme activity (mut0) and abnormal enzyme activity with an elevated Km for adenosylcobalamin (mut-). We describe genetic studies on a cell line (WG1130) from a patient with mut0 MMA which exhibited an unusual complementation phenotype, complementing with three of nine mut0 cell lines and four of five mut- cell lines. This suggests that interallelic complementation occurs between mutant alleles in WG1130 and subsets of alleles associated with both mut0 and mut- phenotypes. The methylmalonyl CoA mutase cDNA was cloned from WG1130 and found to contain a G354----A (Arg93----His) mutation. Gene transfer of this mutant clone into primary fibroblasts from patients with MMA confirms that this mutation expresses a mut0 phenotype when transferred into a mut0 cell line with low levels of mRNA but can contribute to apoenzyme function when transferred into mut cell lines which show correction with WG1130 by somatic cell complementation. These results point to further heterogeneity within both mut0 and mut- and may enable identification of mutations affecting discrete components of apoenzyme function.

Cloning and expression of mutations demonstrating intragenic complementation in mut0 methylmalonic aciduria.

The mut0 mutation resulting in methylmalonyl CoA mutase (MCM) apoenzyme deficiency and methylmalonic aciduria is characterized by undetectable enzyme activity in cell extracts and low incorporation of propionate into cultured cells which is not stimulated by hydroxycobalamin. A mut0 fibroblast cell line (WG1681) from an African-American male infant complemented another mut0 cell line (WG 1130). Cloning and sequencing of cDNA from WG 1681 demonstrated compound heterozygosity for two novel changes at highly conserved sites: G623R and G703R. In addition, two previously described homozygous polymorphisms, H532R and V671I, were found. Hybridization of allele-specific oligonucleotides to PCR amplified MCM exons from the proband and family members identified a clinically normal mother, half-sister, and half-brother as carriers of the G703R change in cis with both polymorphisms. Transfection of each change into a mut0 cell line with very low MCM mRNA (GM1673) demonstrated a lack of stimulation of propionate uptake in the absence and presence of hydroxycobalamin. Cotransfection of each mutation with the previously identified R93H mutation of WG 1130 stimulated propionate uptake, indicating that G623R and G703R are independently capable of complementing the R93H mutation.

A homozygous nonsense mutation in the methylmalonyl-CoA epimerase gene (MCEE) results in mild methylmalonic aciduria.

Methylmalonic aciduria (MMA-uria) is an autosomal recessive inborn error of amino acid metabolism, involving valine, threonine, isoleucine, and methionine. This organic aciduria may present in the neonatal period with life-threatening metabolic acidosis, hyperammonemia, feeding difficulties, pancytopenia, and coma. Most affected patients have mutations in the methylmalonyl-coenzyme A (methylmalonyl-CoA) mutase gene. Mildly affected patients may present in childhood with failure to thrive and recurrent attacks of metabolic acidosis. Both a higher residual activity of methylmalonyl-CoA mutase as well as the vitamin B12-responsive defects (cblA and cblB) may form the basis of the mild disorder. A few patients with moderate MMA-uria are known in whom no defect could be identified. Here we present a 16-year-old female patient with persisting moderate MMA-uria (approximately 50 mmol/mol creatinine). She was born to consanguineous Caucasian parents. Her fibroblast mutase activity was normal and no effect of vitamin B12 supplementation could be established. Reduced incorporation of 14C-propionate into macromolecules suggested a defect in the propionate-to-succinate pathway. We found a homozygous nonsense mutation (c.139C>T) in the methylmalonyl-CoA epimerase gene (MCEE), resulting in an early terminating signal (p.R47X). Both parents were heterozygous for this mutation; they were found to excrete normal amounts of methylmalonic acid (MMA). This is the first report of methylmalonyl-CoA epimerase deficiency, thereby unequivocally demonstrating the biochemical role of this enzyme in human metabolism.

Transport competence of plasma membrane vesicles from cultured human fibroblasts.

We obtained plasma membranes from cultured human skin fibroblasts. The preparation was enriched 10-fold with about 40 percent yield. There was minimal contamination with other cell membranes. Various observations indicated vesicular conformation of a portion of the plasma membranes, notably by electron microscopy and from the effect of osmotic pressure on the distribution of solutes between mass and medium at equilibrium. Other studies indicated that these fibroblast plasma membrane vesicles retained mediated transport processes for a variety of substrates. The evidence included: stereospecific and temperature-dependent uptake of glucose; dependence of L-alanine uptake on sodium ion and an inward-directed transmembrane Na+ gradient; stimulation of L-alanine uptake, with overshoot, by enhancement of the interior-negative transmembrane potential; concentration dependent uptake of methotrexate with apparent competitive inhibition by folinic acid; stimulation of L-lysine uptake by trans-L-arginine. These findings indicate that human fibroblast plasma membrane vesicles could be used to study membrane transport processes and, perhaps, expression of mutant genes that cause inborn errors of transport.

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

AIM: To investigate genetic heterogeneity within the cblA class of inborn error of cobalamin metabolism. CONTEXT: The cblA disorder is characterised by vitamin B12 (cobalamin) responsive methylmalonic aciduria and deficient synthesis of adenosylcobalamin, required for activity of the mitochondrial enzyme methylmalonyl CoA mutase. The cblA gene has not been identified or cloned. We have previously described a patient with the clinical and biochemical phenotype of the cblA disorder whose fibroblasts complemented cells from patients with all known types of inborn error of adenosylcobalamin synthesis, including cblA. METHODS: We have performed somatic cell complementation analysis of the cblA variant fibroblast line with a panel of 28 cblA lines. We have also performed detailed complementation analysis on a panel of 10 cblA fibroblast lines, not including the cblA variant line. RESULTS: The cblA variant line complemented all 28 cell lines of the panel. There was evidence for interallelic complementation among the 10 cblA lines used for detailed complementation analysis; no cell line in this panel complemented all other members. CONCLUSIONS: These results strongly suggest that the cblA variant represents a novel complementation class, which we have designated cblH and which represents a mutation at a distinct gene. They also suggest that the cblA gene encodes a protein that functions as a multimer, allowing for extensive interallelic complementation.

Inherited disorders of vitamin B12 metabolism.

Inherited disorders of vitamin B12 include those which involve the inability of the vitamin to be absorbed from the gut and transported to the appropriate tissues, and those in which the vitamin is not utilised by target cells. The former include intrinsic factor abnormalities, selective malabsorption of vitamin B12 with proteinuria, and deficiencies of transcobalamin I and transcobalamin II. The latter include a defect in the release of free vitamin B12 from lysosomes (cblF), and defects in the formation of both vitamin B12 cofactors (cblC, cblD) or of adenosyl-B12 (cblA, cblB) or methyl-B12 alone (cblE, CblE variant). This article reviews the major clinical manifestations of these diseases, and provides an approach to the diagnosis of transcobalamin II deficiency and the cbl mutations using cultured cells.

Cobalamin and folate deficiency: acquired and hereditary disorders in children.

This review highlights the features of cobalamin and folate deficiency and insufficiency that are particular to children. Maternal deficiency of cobalamin and insufficiency or deficiency of folate are the principal causes of deficiencies of these vitamins in the newborn. Maternal cobalamin deficiency can be caused by pernicious anemia or postgastrectomy, but most often results from a diet lacking in animal protein. The mothers are usually not anemic and failure to thrive and neurological deficits are more common in their infants than is megaloblastic anemia. Inborn errors of cobalamin transport and metabolism present with homocystinuria and methylmalonic aciduria, either alone or in combination. They share many of the clinical features of nutritional cobalamin deficiency. Maternal folate insufficiency results in neural tube defects, fetal loss, prematurity, and fetal growth retardation. Inborn errors of folate metabolism are rare, but polymorphisms affecting the gene for methylenetetrahydrofolate reductase (MTHFR) are common and may have significant health implications. Elevation of plasma methylmalonic acid (MMA) levels reflects a functional lack of cobalamin, whereas elevated total homocysteine levels are associated with a lack of either folate or cobalamin. The determination of these should be part of the investigation of failure to thrive, neurological disorders, and unexplained anemia or cytopenias in children.

Mechanisms of teratogenesis: folic acid and antiepileptic therapy.

Gestational folate deficiency has been associated with abnormal growth and development in both experimental animal and human studies and has been postulated as a putative mechanism for the teratogenic effects of antiepileptic drugs (AEDs). Animal studies have shown that the administration of AEDs results in folate depletion and teratogenic effects. Attempts to prevent the teratogenic effects of AEDs by coadministration of folate have shown variable results, perhaps because of a lack of understanding about the specific effects of AEDs on folate metabolism. Our prospective study of women with epilepsy showed that blood folate levels decreased with increasing plasma AED levels and with the number of AEDs. Low blood folate levels before and/or early in pregnancy were significantly associated with spontaneous abortion and the occurrence of developmental anomalies in the offspring. These findings suggest that folate supplementation might be one means of preventing the occurrence of abnormal pregnancy outcome in women with epilepsy, including neural-tube defects in the offspring.

New disorder of vitamin B12 metabolism (cobalamin F) presenting as methylmalonic aciduria.

An infant with vitamin B12-responsive methylmalonic aciduria and no homocystinuria or megaloblastic anemia presented with stomatitis, glossitis, convulsions, and developmental delay. Cultured fibroblasts showed defective incorporation of both [14C]5-methyltetrahydrofolate and [14C]propionate into protein by whole cells and a decrease of methionine synthase activity in cell extracts. Despite excessive incorporation of [57Co]cyano-B12 by fibroblasts from the patient, free vitamin B12 was unable to efflux from lysosomes, and, therefore, synthesis of both adenosyl-B12 and methyl-B12 was impaired.