Inborn Errors Of Folate Metabolism Research Articles

Methylenetetrahydrofolate reductase deficiency and high-dose FA supplementation disrupt embryonic development of energy balance and metabolic homeostasis in zebrafish.

Folic acid (synthetic folate, FA) is consumed in excess in North America and may interact with common pathogenic variants in methylenetetrahydrofolate reductase (MTHFR); the most prevalent inborn error of folate metabolism with wide-ranging obesity-related comorbidities. While preclinical murine models have been valuable to inform on diet-gene interactions, a recent Folate Expert panel has encouraged validation of new animal models. In this study, we characterized a novel zebrafish model of mthfr deficiency and evaluated the effects of genetic loss of mthfr function and FA supplementation during embryonic development on energy homeostasis and metabolism. mthfr-deficient zebrafish were generated using CRISPR mutagenesis and supplemented with no FA (control, 0FA) or 100μm FA (100FA) throughout embryonic development (0-5days postfertilization). We show that the genetic loss of mthfr function in zebrafish recapitulates key biochemical hallmarks reported in MTHFR deficiency in humans and leads to greater lipid accumulation and aberrant cholesterol metabolism as reported in the Mthfr murine model. In mthfr-deficient zebrafish, energy homeostasis was also impaired as indicated by altered food intake, reduced metabolic rate and lower expression of central energy-regulatory genes. Microglia abundance, involved in healthy neuronal development, was also reduced. FA supplementation to control zebrafish mimicked many of the adverse effects of mthfr deficiency, some of which were also exacerbated in mthfr-deficient zebrafish. Together, these findings support the translatability of the mthfr-deficient zebrafish as a preclinical model in folate research.

Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folate and is involved in the regulation of homocysteine and numerous methylation reactions, including synthesis of neurotransmitters, lipids, DNA, and RNA. Measurement of 5-MTHF in CSF provides diagnostic information regarding disorders affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism and cerebral folate deficiency. Determination of 5-MTHF in CSF (50μL) was performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF-13C5) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400nM) and has an analytical measurement range of 3-1000nM.

Computational algorithmic and molecular dynamics study of functional and structural impacts of non-synonymous single nucleotide polymorphisms in human DHFR gene

Human dihydrofolate reductase (DHFR) is a conserved enzyme that is central to folate metabolism and is widely targeted in pathogenic diseases as well as cancers. Although studies have reported the fact that genetic mutations in DHFR leads to a rare autosomal recessive inborn error of folate metabolism and drug resistance, there is a lack of an extensive study on how the deleterious non-synonymous SNPs (nsSNPs) disrupt its phenotypic effects. In this study, we aim at discovering the structural and functional consequences of nsSNPs in DHFR by employing a combined computational approach consisting of ten recently developed in silico tools for identification of damaging nsSNPs and molecular dynamics (MD) simulation for getting deeper insights into the magnitudes of damaging effects. Our study revealed the presence of 12 most deleterious nsSNPs affecting the native phenotypic effects, with three (R71T, G118D, Y122D) identified in the co-factor and ligand binding active sites. MD simulations also suggested that these three SNPs particularly Y122D, alter the overall structural flexibility and dynamics of the native DHFR protein which can provide more understandings into the crucial roles of these mutants in influencing the loss of DHFR function.

Cerebral folate deficiency: Analytical tests and differential diagnosis.

Cerebral folate deficiency is typically defined as a deficiency of the major folate species 5-methyltetrahydrofolate in the cerebrospinal fluid (CSF) in the presence of normal peripheral total folate levels. However, it should be noted that cerebral folate deficiency is also often used to describe conditions where CSF 5-MTHF is low, in the presence of low or undefined peripheral folate levels. Known defects of folate transport are deficiency of the proton coupled folate transporter, associated with systemic as well as cerebral folate deficiency, and deficiency of the folate receptor alpha, leading to an isolated cerebral folate deficiency associated with intractable seizures, developmental delay and/or regression, progressive ataxia and choreoathetoid movement disorders. Inborn errors of folate metabolism include deficiencies of the enzymes methylenetetrahydrofolate reductase, dihydrofolate reductase and 5,10-methenyltetrahydrofolate synthetase. Cerebral folate deficiency is potentially a treatable condition and so prompt recognition of these inborn errors and initiation of appropriate therapy is of paramount importance. Secondary cerebral folate deficiency may be observed in other inherited metabolic diseases, including disorders of the mitochondrial oxidative phosphorylation system, serine deficiency, and pyridoxine dependent epilepsy. Other secondary causes of cerebral folate deficiency include the effects of drugs, immune response activation, toxic insults and oxidative stress. This review describes the absorption, transport and metabolism of folate within the body; analytical methods to measure folate species in blood, plasma and CSF; inherited and acquired causes of cerebral folate deficiency; and possible treatment options in those patients found to have cerebral folate deficiency.

Functional characterization of missense mutations in severe methylenetetrahydrofolate reductase deficiency using a human expression system.

5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression. Here, we report the in vitro expression of 22 severe MTHFR missense mutations and two known single nucleotide polymorphisms (p.Ala222Val, p.Thr653Met) in human fibroblasts. Significant reduction of MTHFR activity (<20% of wild-type) was observed for five mutant proteins that also had highly reduced protein levels on Western blot analysis. The remaining mutations produced a spectrum of enzyme activity levels ranging from 22-122% of wild-type, while the SNPs retained wild-type-like activity levels. We found increased thermolability for p.Ala222Val and seven disease-causing mutations all located in the catalytic domain, three of which also showed FAD responsiveness in vitro. By contrast, six regulatory domain mutations and two mutations clustering around the linker region showed increased thermostability compared to wild-type protein. Finally, we confirmed decreased affinity for NADPH in individual mutant enzymes, a result previously described in primary patient fibroblasts. Our expression study allows determination of significance of missense mutations in causing deleterious loss of MTHFR protein and activity, and is valuable in detection of aberrant kinetic parameters, but should not replace investigations in native material.

Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folic acid and is involved in regulation of homocysteine and DNA synthesis. Measurement of 5-MTHF in CSF provides diagnostic information regarding diseases affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism. Determination of 5-MTHF in CSF (50 μL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF-(13)C5) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400 nM) and has an analytical measurement range of 3-1000 nM.

A case of severe methylenetetrahydrofolate reductase deficiency presenting as neonatal encephalopathy, seizures, microcephaly and central hypoventilation

Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme in the remethylation of homocysteine to methionine. S-adenosylmethionine, formed from methionine and adenosine triphosphate, is the methyl donor in crucial reactions for brain development and function. MTHFR deficiency is the most prevalent inborn error of folate metabolism and is characterized by hyperhomocysteinemia and hypomethioninemia. A broad clinical spectrum includes psychomotor retardation, microcephaly, seizures, central respiratory failure and death. Premature atherosclerosis, arterial and venous thrombosis or psychiatric manifestations occur in the late onset forms. We report a female neonate with severe MTHFR deficiency presenting with neonatal encephalopathy, congenital microcephaly, seizures, central hypoventilation with gross cerebral atrophy, hypomyelination and pontine hypoplasia on magnetic resonance imaging of brain. She succumbed at 10 wk of age despite treatment with oral S-adenosylmethionine, betaine, folinic acid, riboflavin and hydroxycobalamin. © 2013 - IOS Press and the authors. All rights reserved.

Methylenetetrahydrofolate reductase deficiency: therapy and outcome (with video)

The methylenetetrahydrofolate reductase (MTHFR) deficiency (OMIM 236250) is based on an inborn error of folate metabolism with a broad variability of clinical presentation. Early forms often show a progressive neurological deterioration combined with a severe epileptic encephalopathy. Indicative laboratory findings show homocysteine elevated and methionine reduced. We present the course of the disease in a girl, who was diagnosed with MTHFR-deficient at the age of 6 months (Video).

Congenital MTHFR deficiency causing early-onset cerebral stroke in a case homozygous for MTHFR thermolabile variant

Hyperhomocysteinemia is a risk factor for early-onset venous thrombosis. It can be caused by genetic defects in methionine-homocysteine metabolism. The thermolabile variant of methylene-tetrahydrofolate reductase (MTHFR), c.677C>T, is one of the most common genetic condition, which has been associated with mild to moderate hyperhomocysteinemia, and carriers of this variant are at increased risk of an early-onset stroke-like episode. However, congenital MTHFR deficiency is a rare inborn error of folate metabolism, causing marked hyperhomocysteinemia, and its combination with the thermolabile variant is rarely reported. In this report, we describe a young adult with cerebral infarction. The patient was homozygous for the MTHFR thermolabile variant, but markedly elevated hyperhomocysteinemia led us to investigate the whole MTHFR gene, which revealed two novel MTHFR mutations. This is the first report of MTHFR deficiency in a Korean patient, and one of only a few cases reported in East Asian countries. Despite its rarity, our report underlines the importance of its identification in hyperhomocysteinemia for patient prognosis with appropriate management.

Novel inborn error of folate metabolism: identification by exome capture and sequencing of mutations in the MTHFD1 gene in a single proband

ObjectiveAn 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...

5,10-Methylenetetrahydrofolate reductase deficiency with progressive polyneuropathy in an infant

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most prevalent inborn error of folate metabolism, and has variable clinical manifestations from asymptomatic to severe psychomotor retardation, microcephalus and seizure. In untreated infantile cases, it predominantly affects the central nervous system, which is sometimes fatal. On the other hand, peripheral nerve involvement is uncommon. We present a severe infantile case of MTHFR deficiency that manifested unilateral phrenic nerve palsy with communicating hydrocephalus, developmental delay and died at 11 months of age. An enzymatic study confirmed MTHFR deficiency with residual activity of 0.75% of mean control values in cultured fibroblasts. Mutation analysis of the MTHFR gene revealed homozygous, tandem missense mutations c.[446G>T; 447C>T] in exon 3 of the MTHFR gene converting glycine to valine (Gly149Val). In MTHFR deficiency, betaine may improve the symptoms if started immediately after birth by reducing the level of serum homocysteine and increasing that of methionine. Our results show that we should be aware of possible inborn errors of folate metabolism such as MTHFR deficiency, in infants with unexplained developmental delay manifesting rapidly progressive polyneuropathy.

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.

The molecular basis of glutamate formiminotransferase deficiency.

Glutamate formiminotransferase deficiency, an autosomal recessive disorder and the second most common inborn error of folate metabolism, is presumed to be due to defects in the bifunctional enzyme glutamate formiminotransferase-cyclodeaminase (FTCD). Features of a severe phenotype, first identified in patients of Japanese descent, include elevated levels of formiminoglutamate (FIGLU) in the urine in response to histidine administration, megaloblastic anemia, and mental retardation. Features of a mild phenotype include high urinary excretion of FIGLU in the absence of histidine administration, mild developmental delay, and no hematological abnormalities. We found mutations in the human FTCD gene in three patients with putative glutamate formiminotransferase deficiency. Two siblings were heterozygous for missense mutations, c.457C>T (R135C) and c.940G>C (R299P). Mutagenesis of porcine FTCD and expression in E. coli showed that the R135C mutation reduced formiminotransferase activity to 61% of wild-type, whereas the R299P mutation reduced this activity to 57% of wild-type. The third patient was hemizygous for c.1033insG, with quantitative PCR indicating that the other allele contained a deletion. These mutations are the first identified in glutamate formiminotransferase deficiency and demonstrate that mutations in FTCD represent the molecular basis for the mild phenotype of this disease.

Update on cobalamin, folate, and homocysteine.

Three topics affecting cobalamin, folate, and homocysteine that have generated interest, activity, and advances in recent years are discussed. These are: (I). the application of an expanded variety of tools to the diagnosis of cobalamin deficiency, and how these affect and are affected by our current understanding of deficiency; (II). the nature of the interaction between homocysteine and vascular disease, and how the relationship is affected by vitamins; and (III). the improved understanding of relevant genetic disorders and common genetic polymorphisms, and how these interact with environmental influences. The diagnostic approach to cobalamin deficiency now allows better diagnosis of difficult and atypical cases and more confident rejection of the diagnosis when deficiency does not exist. However, the process has also become a complex and sometimes vexing undertaking. Part of the difficulty derives from the lack of a diagnostic gold standard among the many available tests, part from the overwhelming numerical preponderance of patients with subclinical deficiency (in which isolated biochemical findings exist without clinical signs or symptoms) among the cobalamin deficiency states, and part from the decreased availability of reliable tests to identify the causes of a patient's cobalamin deficiency and thus a growing deemphasis of that important part of the diagnostic process. In Section I, Dr. Carmel discusses the tests, the diagnostic issues, and possible approaches to the clinical evaluation. It is suggested no single algorithm fits all cases, some of which require more biochemical proof than others, and that differentiating between subclinical and clinical deficiency, despite their overlap, may be a helpful and practical point of departure in the evaluation of patients encountered in clinical practice. The arguments for and against a suggested expansion of the cobalamin reference range are also weighed. The epidemiologic data suggest that homocysteine elevation is a risk factor for vascular and thrombotic disease. In Section II, Dr. Green notes that the interactions of metabolism and clinical risk are not well understood and a causative relationship remains unproven despite new reports that lowering homocysteine levels may reduce vascular complications. Genetic and acquired influences may interact in important ways that are still being sorted out. The use of vitamins, especially folate, often reduces homocysteine levels but also carries potential disadvantages and even risks. Folate fortification of the diet and supplement use have also markedly reduced the frequency of folate deficiency, and cobalamin deficiency is now the more common deficiency state, especially among the elderly. Although genetic disorders are rare, they illuminate important metabolic mechanisms and pose diagnostic challenges, especially when clinical presentation occurs later in life. In Section III, Drs. Rosenblatt and Watkins use selected disorders to illustrate the subject. Imerslund-Gräsbeck syndrome, a hereditary disorder of cobalamin absorption at the ileal level, demonstrates genetic heterogeneity. Finnish patients show mutation of the gene for cubilin, the multiligand receptor for intrinsic factor. Surprisingly, Norwegian and other patients have been found recently to have mutations of the AMN (amnionless) gene, mutations that are lethal in mice at the embryonic stage. Two disorders of cobalamin metabolism, cblG and cblE, are now known to arise from mutations of the methionine synthase and methionine synthase reductase genes, respectively. These disorders feature megaloblastic anemia and neurologic manifestations. The folate disorder selected for illustration, methylenetetrahydrofolate reductase (MTHFR) deficiency, paradoxically causes neurological problems but no megaloblastic anemia. This rare deficiency is the most common inborn error of folate metabolism. It is distinct from the very common MTHFR gene polymorphisms, mutations that cause mild to moderate reductions in MTHFR activity but no direct clinical manifestations. The MTHFR polymTHFR polymorphisms, especially the 677C-->T mutation, may contribute to vascular and birth defect risks, while reducing the risk of certain malignancies, such as colon cancer. These polymorphisms and those of genes for other enzymes and proteins related to cobalamin, folate, and homocysteine metabolism may be important role players in frequent interactions between genes and the environment.

The number of dichorionic twin pregnancies is reduced by the common MTHFR 677C→T mutation

In multiple pregnancies, demands for folic acid are considerably increased. The most common inborn error of folate metabolism is mild methylenetetrahydrofolate reductase (MTHFR) deficiency due to the synthesis of a thermolabile variant of the enzyme with impaired catalytic activity which leads to reduced 5-methyltetrahydrofolate (5-methyl-THF) and mildly elevated homocysteine plasma concentrations when folate status is inadequate. To investigate whether the number of offspring is influenced by this mutation, we determined the frequency of the 677C-->T substitution in 156 singleton and 40 twin mothers with dichorionic placentation. The T allele frequency in singleton (0.30) and twin mothers (0.16) was significantly different (P = 0.011). Mothers with the 677C-->T mutation had a 2.28 times lower risk of having a twin pregnancy than those without (95% confidence interval = 1.18-4.66; P = 0.008). Our observation would explain, at least in part, the hereditary trait of multiple gestations and is in agreement with the ethnic distribution pattern of the T allele which has been found to be inversely correlated with the incidence of dichorionic twins. Our findings suggest that the MTHFR 677C-->T mutation interferes with human brood size, probably by influencing the proliferation of rapidly dividing embryonic and maternal cells.

Characterization of six novel mutations in the methylenetetrahydrofolate reductase (MTHFR) gene in patients with homocystinuria.

Severe deficiency of methylenetetrahydrofolate reductase (MTHFR) is the most common inborn error of folate metabolism. Patients are characterized by severe hyperhomocysteinemia, homocystinuria and a variety of neurological and vascular problems. Eighteen rare mutations have been reported in this group of patients. Two polymorphisms which cause mild enzyme deficiencies have been described (677C-->T and 1298A-->C). The first sequence change encodes a thermolabile enzyme and is associated with mild hyperhomocysteinemia. Six novel point mutations are described in patients with severe deficiency of MTHFR, along with their associated polymorphisms and clinical phenotypes. Of the two nonsense mutations (1762A-->T, 1134C-->G) and four missense mutations (1727C-->T, 1172G-->A, 1768G-->A, and 358G-->A), one was identified in the N-terminal catalytic domain, while the others were located in the regulatory C-terminal region. All four residues affected by missense mutations are conserved in one or more MTHFRs of other species. This report brings the total to 24 mutations identified in severe MTHFR deficiency, with two mutations identified in each of 22 patients.

Association of Cerebrospinal Fluid Deficiency of 5-Methyltetrahydrofolate, but Not S-Adenosylmethionine, with Reduced Concentrations of the Acid Metabolites of 5-Hydroxytryptamine and Dopamine

1. Folate deficiency, or inborn errors of folate metabolism, cause reduced turnover of 5-hydroxytryptamine (serotonin), and perhaps dopamine, in the central nervous system. The mechanism by which this occurs are not known. One possibility is that this is mediated by deficiency of the methyl-donor S-adenosylmethionine. 2. To test this in humans, we have measured cerebrospinal fluid concentrations of 5-hydroxyindoleacetic acid and homovanillic acid, metabolites of 5-hydroxytryptamine and dopamine, respectively, in children with inborn errors of the methyl-transfer pathway. These children are naturally deficient in 5-methyltetrahydrofolate, S-adenosylmethionine or both before treatment, and replete with S-adenosylmethionine, but not necessarily with 5-methyltetrahydrofolate, during treatment. 3. Children with subnormal cerebrospinal fluid concentrations of 5-methyltetrahydrofolate had significantly reduced concentrations of 5-hydroxyindoleacetic acid and homovanillic acid. Children with subnormal cerebrospinal fluid concentrations of S-adenosylmethionine did not have significantly reduced concentrations of these metabolites. 4. We conclude that the mechanism by which deficiency of 5-methyltetrahydrofolate causes reduced 5-hydroxytryptamine and dopamine turnover is unlikely to be mediated by S-adenosylmethionine.

Folate and Methotrexate Polyglutamate Tissue Levels in Rhesus Monkeys Following Chronic Low-Dose Methotrexate

Methotrexate (MTX), a mainstay in the treatment of acute lymphoblastic leukemia, is associated with both hepatic and neurologic toxicity. Like a folate, MTX is metabolized to polyglutamated derivatives (MTXGlun) with long intracellular half-lives. These metabolites may contribute to MTX toxicity through a direct effect on cellular metabolism or indirectly through a perturbation of folate homeostasis. To better define the effects of chronic MTX treatment, tissue levels of MTX, MTXGlun, and folate were measured in three monkeys treated with weekly intramuscular MTX for 1 year. Greater than 80% of the total tissue MTX found was in the form of polyglutamated derivatives. Most of these derivatives were MTXGlu3-5 but Glu6-7 were easily detectable. Total tissue folates were measured in liver, kidney, brain and testis with MTX treated animals having a 90% loss of total folate in brain tissue. This is of special interest since inborn errors of folate metabolism are often associated with severe neurologic abnormalities.

Subacute combined degeneration of the cord, dementia and parkinsonism due to an inborn error of folate metabolism.

A 2-year-old girl with 5,10-methylenetetrahydrofolate reductase deficiency developed subacute combined degeneration of the cord and a leuco-encephalopathy which was confirmed at necropsy. Total folate concentrations in serum, red cells and CSF were markedly reduced whereas vitamin B12 concentrations were normal. In addition the patient had Parkinsonism and reduced concentrations of homovanillic acid, 5-hydroxyindoleacetic acid and total biopterins in cerebrospinal fluid. Folic acid administration was accompanied by fits and acute deterioration in the movement disorder. At necropsy the basal ganglia showed no detectable abnormality.

382 DECREASED FOLATE ASSOCIATED WITH METHOTREXATE POLY GLUTAMATE ACCUMULATION IN CHRONICALLY TREATED MONKEYS

Methotrexate (MTX) a mainstay of continuation therapy for acute lymphoblastic leukemia is associated with hepatic and neurotoxicity. The metabolism of MTX to polyglutamates (MTXGLUn), which have long intracellular halflives may be partially responsible for these toxicities. In an effort to model the pharmacodynamics of repetitive, low dose MTX and the folate deficiency previously shown in red cells and livers from patients, the tissue content of MTX and folate from monkeys treated for 1 yr with 50 mgMTX/m2 was analyzed. In addition, the MTXGLUn profiles were also determined by HPLC and radioligand binding assay. The MTX content (pmol/g wet weight) of liver, kidney, brain, testes were 2817, 836 4.9 and 44 respectively. The folate content (pmol/g wet weight) of these same tissues was 11500, 4485, 32 and 474. Control values for folates were 31064, 6500, 286 and 1355 respectively. The predominant MTX derivative(s) found were MTXGLU3-5 but GLU6-7 were easily detectable. Of special interest was the significant folate deficiency found in the brain (90% loss compared to control). Since inborn errors of folate metabolism often present with severe neurological problems not unlike those associated with MTX toxicity (including cerebral calcifications), the biochemical abnormalities associated with repetitive, low dose MTX therapy and its attendant folate deficiency may be responsible for the observed toxicity. Supported by ACS CH228, a scholar award of the Leukemia Society of America, and the NCI.