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Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.

Inherited deficiencies of the L-lysine catabolic pathway cause glutaric aciduria type I and pyridoxine-dependent epilepsy. Dietary modulation of cerebral L-lysine metabolism is thought to be an important therapeutic intervention for these diseases. To better understand cerebral L-lysine degradation, we studied in mice the two known catabolic routes -- pipecolate and saccharopine pathways -- using labeled stable L-lysine and brain peroxisomes purified according to a newly established protocol. Experiments with labeled stable L-lysine show that cerebral L-pipecolate is generated along two pathways: i) a minor proportion retrograde after ε-deamination of L-lysine along the saccharopine pathway, and ii) a major proportion anterograde after α-deamination of L-lysine along the pipecolate pathway. In line with these findings, we observed only little production of saccharopine in the murine brain. L-pipecolate oxidation was only detectable in brain peroxisomes, but L-pipecolate oxidase activity was low (7 ± 2μU/mg protein). In conclusion, L-pipecolate is a major degradation product from L-lysine in murine brain generated by α-deamination of this amino acid.

Background and Hypothesis: Pyridoxine dependent epilepsy (PDE) due to mutations in the ALDH7A1 gene (PDE-ALDH7A1) is caused by α-aminoadipic-semialdehyde-dehydrogenase enzyme deficiency in the lysine pathway resulting in the accumulation of α-aminoadipic acid semialdehyde (α-AASA). Classical presentation is neonatal intractable seizures with a dramatic response to pyridoxine. Pyridoxine therapy does not prevent developmental delays in the majority of the patients. We hypothesized that L-arginine supplementation will decrease accumulation of α-AASA by competitive inhibition of lysine transport into the central nervous system and improve neurodevelopmental and neurocognitive functions in PDE-ALDH7A1., Methods: A 12-year-old male with PDE-ALDH7A1 was treated with l-arginine supplementation as an innovative therapy. Treatment outcome was monitored by cerebral-spinal-fluid (CSF) α-AASA measurements at baseline, 6th and 12th months of therapy. Neuropsychological assessments were performed at baseline and 12th months of therapy., Results: L-arginine therapy was well tolerated without side effects. CSF α-AASA was decreased 57% at 12th months of therapy. Neuropsychological assessments revealed improvements in general abilities index from 108 to 116 and improvements in verbal and motor functioning at 12th months of therapy., Conclusion: The short-term treatment outcome of this novel L-arginine supplementation therapy for PDE-ALDH7A1 was successful for biochemical and neurocognitive improvements., (Copyright © 2014 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Combined D,L-2-hydroxyglutaric aciduria (DL-2HGA; OMIM #615182) is a rare neurometabolic disorder clinically characterized by muscular hypotonia, severe neurodevelopmental dysfunction, and intractable seizures associated with respiratory distress. Biochemically, DL-2HGA patients excrete increased amounts of D- and L-2-hydroxyglutarate (D2HG and L2HG, respectively), with predominance of D2HG, and α-ketoglutarate, and show a decrease in urinary citrate. Impaired function of the mitochondrial citrate carrier (CIC) due to pathogenic mutations within the SLC25A1 gene has been identified as the underlying molecular cause of the disease. CIC mediates efflux of the mitochondrial tricarboxylic acid (TCA) cycle intermediates citrate and isocitrate in exchange for cytosolic malate. Thus, depletion of cytosolic citrate as well as accumulation of citrate inside mitochondria have been considered to play a role in the pathophysiology of DL-2HGA. Here, we report for the first time on a patient with a genetically confirmed diagnosis of DL-2HGA and treatment with either malate or citrate. During malate treatment, urinary malate concentration increased, but beyond that, neither biochemical nor clinical alterations were observed. In contrast, treatment with citrate led to an increased urinary excretion of TCA cycle intermediates malate and succinate, and by trend to an increased concentration of urinary citrate. Furthermore, excretion of D2HG and L2HG was reduced during citrate treatment. Clinically, the patient showed stabilization with regard to frequency and severity of seizures. Treating DL-2HGA with citrate should be considered in other DL-2HGA patients, and its effects should be studied systematically.

Pyridox(am)ine phosphate oxidase (PNPO) deficiency causes severe early infantile epileptic encephalopathy and has been characterized as responding to pyridoxal-5'-phosphate but not to pyridoxine. Two males with PNPO deficiency and novel PNPO mutations are reported and their clinical, metabolic, and video-electroencephalographic (EEG) findings described. The first child showed electro-clinical responses to pyridoxine and deterioration when pyridoxine was withheld. At last review, he has well-controlled epilepsy with pyridoxal-5'-phosphate monotherapy and an autism spectrum disorder. The second child had a perinatal middle cerebral artery infarct and a myoclonic encephalopathy. He failed to respond to pyridoxine but responded well to pyridoxal-5'-phosphate. At the age of 21 months he has global developmental delay and hemiparesis but is seizure-free with pyridoxal-5'-phosphate monotherapy. Plasma and cerebrospinal fluid pyridoxamine levels were increased in both children during treatment with pyridoxine or pyridoxal-5'-phosphate. These observations indicate that differential responses to pyridoxine and pyridoxal-5'-phosphate treatment cannot be relied upon to diagnose PNPO deficiency., (© 2013 Mac Keith Press.)

We have conducted biochemical studies with commercial available pyrroline-5-carboxylate (P5C) reductase (PYCR1) to investigate whether this enzyme plays a role in L-lysine degradation. Our recent studies with antiquitin/ALDH7A1 deficient fibroblasts revealed an alternative genesis of L-pipecolic acid, and we then hypothesized that PYCR1 was responsible for the conversion of Δ(1)-piperideine-6-carboxylate (P6C) into pipecolic acid. We here present evidence that PYCR1 is indeed able to produce L-pipecolic acid from P6C preparations, and the observed K m for this conversion is of the same magnitude as the K m described for the conversion of P5C to L-proline by PYCR1. Urine samples from antiquitin deficient individuals, who accumulate P6C, were also incubated with PYCR1 which resulted in a marked decrease of P6C and a huge increase of L-pipecolic acid as measured by LC-MS/MS, confirming that indeed PYCR1 generates L-pipecolic acid from P6C.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is a disorder caused by recessive mutations in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. Recent observations indicate that the phenotypic range of the disease is much wider than initially thought. Currently, no treatment is available. The aims of our study were (i) to explore a possible genotype-phenotype correlation; and (ii) to identify potential therapeutic agents that modulate the splice site mutations in intron 2 of DARS2, present in almost all patients. A cross-sectional observational study was performed in 78 patients with two DARS2 mutations in the Amsterdam and Helsinki databases up to December 2012. Clinical information was collected via questionnaires. An inventory was made of the DARS2 mutations in these patients and those previously published. An assay was developed to assess mitochondrial aspartyl-tRNA synthetase enzyme activity in cells. Using a fluorescence reporter system we screened for drugs that modulate DARS2 splicing. Clinical information of 66 patients was obtained. The clinical severity varied from infantile onset, rapidly fatal disease to adult onset, slow and mild disease. The most common phenotype was characterized by childhood onset and slow neurological deterioration. Full wheelchair dependency was rare and usually began in adulthood. In total, 60 different DARS2 mutations were identified, 13 of which have not been reported before. Except for 4 of 42 cases published by others, all patients were compound heterozygous. Ninety-four per cent of the patients had a splice site mutation in intron 2. The groups of patients sharing the same two mutations were too small for formal assessment of genotype-phenotype correlation. However, some combinations of mutations were consistently associated with a mild phenotype. The mitochondrial aspartyl-tRNA synthetase activity was strongly reduced in patient cells. Among the compounds screened, cantharidin was identified as the most potent modulator of DARS2 splicing. In conclusion, the phenotypic spectrum of leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is wide, but most often the disease has a relatively slow and mild course. The available evidence suggests that the genotype influences the phenotype, but because of the high number of private mutations, larger numbers of patients are necessary to confirm this. The activity of mitochondrial aspartyl-tRNA synthetase is significantly reduced in patient cells. A compound screen established a 'proof of principle' that the splice site mutation can be influenced. This finding is promising for future therapeutic strategies.

Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, and chondrosarcoma patients. Mutant IDH produces 2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators. We investigated the role of mutant IDH1 using the mouse transplantation assay. Mutant IDH1 alone did not transform hematopoietic cells during 5 months of observation. However, mutant IDH1 greatly accelerated onset of myeloproliferative disease-like myeloid leukemia in mice in cooperation with HoxA9 with a mean latency of 83 days compared with cells expressing HoxA9 and wild-type IDH1 or a control vector (167 and 210 days, respectively, P = .001). Mutant IDH1 accelerated cell-cycle transition through repression of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, and activated mitogen-activated protein kinase signaling. By computational screening, we identified an inhibitor of mutant IDH1, which inhibited mutant IDH1 cells and lowered 2HG levels in vitro, and efficiently blocked colony formation of AML cells from IDH1-mutated patients but not of normal CD34(+) bone marrow cells. These data demonstrate that mutant IDH1 has oncogenic activity in vivo and suggest that it is a promising therapeutic target in human AML cells.

Pyridoxine-dependent epilepsy (PDE) is an inborn error of metabolism resulting from antiquitin deficiency. There is marked elevation of α-amino adipic semi-aldehyde (αAASA), piperidine-6-carboxylate (P6C) and pipecolic acid. The diagnosis can be confirmed by identifying the mutation in the ALDH7A1 gene in chromosome 5q3l. An 8-year-old Indian girl presented with severe developmental delay and seizures and was found to have pyridoxine-dependent epilepsy owing to an antiquitin mutation. Genetic evaluation of the parents allowed antenatal diagnosis to be made during the next pregnancy.

The Krebs cycle is of fundamental importance for the generation of the energetic and molecular needs of both prokaryotic and eukaryotic cells. Both enantiomers of metabolite 2-hydroxyglutarate are directly linked to this pivotal biochemical pathway and are found elevated not only in several cancers, but also in different variants of the neurometabolic disease 2-hydroxyglutaric aciduria. Recently we showed that cancer-associated IDH2 germline mutations cause one variant of 2-hydroxyglutaric aciduria. Complementary to these findings, we now report recessive mutations in SLC25A1, the mitochondrial citrate carrier, in 12 out of 12 individuals with combined D-2- and L-2-hydroxyglutaric aciduria. Impaired mitochondrial citrate efflux, demonstrated by stable isotope labeling experiments and the absence of SLC25A1 in fibroblasts harboring certain mutations, suggest that SLC25A1 deficiency is pathogenic. Our results identify defects in SLC25A1 as a cause of combined D-2- and L-2-hydroxyglutaric aciduria., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

α-Amino adipic semialdehyde (α-AASA) accumulates in body fluids from patients with pyridoxine-dependent epilepsy because of mutations in antiquitin (ALDH7A1) and serves as the biomarker for this condition. We have recently found that the urinary excretion of α-AASA was also increased in molybdenum cofactor and sulfite oxidase deficiencies. The seizures in pyridoxine-dependent epilepsy are caused by lowered cerebral levels of pyridoxal-5-phosphate (PLP), the bioactive form of pyridoxine (vitamin B(6)), which can be corrected by the supplementation of pyridoxine. The nonenzymatic trapping of PLP by the cyclic form of α-AASA is causative for the lowered cerebral PLP levels. We describe 2 siblings with clinically evident pyridoxine-responsive seizures associated with increased urinary excretion of α-AASA. Subsequent metabolic investigations revealed several metabolic abnormities, all indicative for molybdenum cofactor deficiency. Molecular investigations indeed revealed a known homozygous mutation in the MOCS2 gene. Based upon the clinically evident pyridoxine-responsive seizures in these 2 siblings, we recommend considering pyridoxine supplementation to patients affected with molybdenum cofactor or sulfite oxidase deficiencies.

Analysis of α-aminoadipic semialdehyde is an important tool in the diagnosis of antiquitin deficiency (pyridoxine-dependent epilepsy). However continuing use of this test has revealed that elevated urinary excretion of α-aminoadipic semialdehyde is not only found in patients with pyridoxine-dependent epilepsy but is also seen in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency. This should be taken into account when interpreting the laboratory data. Sulphite was shown to inhibit α-aminoadipic semialdehyde dehydrogenase in vitro.

The assessment of urinary α-aminoadipic semialdehyde (α-AASA) has become the diagnostic laboratory test for pyridoxine dependent seizures (PDS). α-AASA is in spontaneous equilibrium with its cyclic form Δ(1)-piperideine-6-carboxylate (P6C); a molecule with a heterocyclic ring structure. Ongoing diagnostic screening and monitoring revealed that in some individuals with milder ALDH7A1 variants, and patients co-treated with a lysine restricted diet, α-AASA was only modestly increased. This prompted us to investigate the diagnostic power and added value of the assessment of urinary P6C compared to α-AASA. Urine samples were diluted to a creatinine content of 0.1 mmol/L, followed by the addition of 0.01 nmol [(2)H(9)]pipecolic acid as internal standard (IS) and 5 μL was injected onto a Waters C(18) T3 HPLC column. Chromatography was performed using water/methanol 97/3 (v/v) including 0.03 % formic acid by volume with a flow rate of 150 μL/min and detection was accomplished in the multiple reaction monitoring mode: P6C m/z 128.1 > 82.1; [(2)H(9)]pipecolic acid m/z 139.1 > 93.1. Due to the dualistic nature of α-AASA/P6C, and the lack of a proper internal standard, the method is semi quantitative. The intra-assay CVs (n = 10) for two urine samples of proven PDS patients with only modest P6C increases were 4.7% and 8.1%, whereas their inter-assay CVs (n = 10) were 16 and 18% respectively. In all 40 urine samples from 35 individuals with proven PDS, we detected increased levels of P6C. Therefore, we conclude that the diagnostic power of the assessments of urinary P6C and α-AASA is comparable.

The organic acidurias D: -2-hydroxyglutaric aciduria (D-2-HGA), L-2-hydroxyglutaric aciduria (L-2-HGA), and combined D,L-2-hydroxyglutaric aciduria (D,L-2-HGA) cause neurological impairment at young age. Accumulation of D-2-hydroxyglutarate (D-2-HG) and/or L-2-hydroxyglutarate (L-2-HG) in body fluids are the biochemical hallmarks of these disorders. The current review describes the knowledge gathered on 2-hydroxyglutaric acidurias (2-HGA), since the description of the first patients in 1980. We report on the clinical, genetic, enzymatic and metabolic characterization of D-2-HGA type I, D-2-HGA type II, L-2-HGA and D,L-2-HGA, whereas for D-2-HGA type I and type II novel clinical information is presented which was derived from questionnaires.

We report two siblings with atypical pyridoxine-dependant epilepsy, modest elevation of biomarkers, in which the open reading frame and the splice sites of ALDH7A1 did not show any mutations. Subsequent genetic analysis revealed a deep homozygous intronic mutation in ALDH7A1 resulting in two types of transcripts: the major transcript containing a pseudoexon, and the minor transcript representing the authentic spliced transcript. In future, this mutation may be targeted with antisense-therapy aiming at exclusion of the pseudoexon., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The recent discovery of heterozygous isocitrate dehydrogenase 2 (IDH2) mutations of residue Arg(140) to Gln(140) or Gly(140) (IDH2(wt/R140Q), IDH2(wt/R140G)) in d-2-hydroxyglutaric aciduria (D-2-HGA) has defined the primary genetic lesion in 50% of D-2-HGA patients, denoted type II. Overexpression studies with IDH1(R132H) and IDH2(R172K) mutations demonstrated that the enzymes acquired a new function, converting 2-ketoglutarate (2-KG) to d-2-hydroxyglutarate (D-2-HG), in lieu of the normal IDH reaction which reversibly converts isocitrate to 2-KG. To confirm the IDH2(wt/R140Q) gain-of-function in D-2-HGA type II, and to evaluate potential therapeutic strategies, we developed a specific and sensitive IDH2(wt/R140Q) enzyme assay in lymphoblasts. This assay determines gain-of-function activity which converts 2-KG to D-2-HG in homogenates of D-2-HGA type II lymphoblasts, and uses stable-isotope-labeled 2-keto[3,3,4,4-(2)H(4)]glutarate. The specificity and sensitivity of the assay are enhanced with chiral separation and detection of stable-isotope-labeled D-2-HG by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Eleven potential inhibitors of IDH2(wt/R140Q) enzyme activity were evaluated with this procedure. The mean reaction rate in D-2-HGA type II lymphoblasts was 8-fold higher than that of controls and D-2-HGA type I cells (14.4nmolh(-1)mgprotein(-1) vs. 1.9), with a corresponding 140-fold increase in intracellular D-2-HG level. Optimal inhibition of IDH2(wt/R140Q) activity was obtained with oxaloacetate, which competitively inhibited IDH2(wt/R140Q) activity. Lymphoblast IDH2(wt/R140Q) showed long-term cell culture stability without loss of the heterozygous IDH2(wt/R140Q) mutation, underscoring the utility of the lymphoblast model for future biochemical and therapeutic studies., (2011 Elsevier B.V. All rights reserved.)

Human induced pluripotent stem cells (iPSCs) have been recently found to harbor genomic alterations. However, the integrity of mitochondrial DNA (mtDNA) within reprogrammed cells has yet to be investigated. mtDNA mutations occur at a high rate and contribute to the pathology of a number of human disorders. Furthermore, the lack of mtDNA integrity may alter cellular bioenergetics and limit efficient differentiation. We demonstrated previously that the derivation of iPSCs is associated with mitochondrial remodeling and a metabolic switch towards glycolysis. Here, we have discovered that alterations of mtDNA can occur upon the induction of pluripotency. Massively parallel pyrosequencing of mtDNA revealed that human iPSCs derived from young healthy donors harbored single base mtDNA mutations (substitutions, insertions, and deletions), both homoplasmic (in all mtDNA molecules) and heteroplasmic (in a fraction of mtDNAs), not present in the parental cells. mtDNA modifications were mostly common variants and not disease related. Moreover, iPSC lines bearing different mtDNA mutational loads maintained a consistent human embryonic stem cell-like reprogramming of energy metabolism. This involved the upregulation of glycolytic enzymes, increased glucose-6-phosphate levels, and the over-expression of pyruvate dehydrogenase kinase 1 protein, which reroutes the bioenergetic flux toward glycolysis. Hence, mtDNA mutations within iPSCs may not necessarily impair the correct establishment of pluripotency and the associated metabolic reprogramming. Nonetheless, the occurrence of pathogenic mtDNA modifications might be an important aspect to monitor when characterizing iPSC lines. Finally, we speculate that this random rearrangement of mtDNA molecules might prove beneficial for the derivation of mutation-free iPSCs from patients with mtDNA disorders., (Copyright © 2011 AlphaMed Press.)

Background: d-3-hydroxybutyrate (3OHB) is an alternative energy substrate for the brain during hypoglycemia, especially during infancy. Supplementation of 3OHB during sustained hypoglycemia in rat pups delays onset of burst suppression coma, but is associated with white matter injury and increased mortality. The biochemical basis for this ambivalent effect is not known. It may be related to an anaplerotic or gluconeogenetic deficit of 3OHB., Methods and Results: We studied clinical alertness, EEG and brain metabolites (acyl-carnitines, amino acids, glycolytic and pentose phosphate intermediates) in 13 day-old rat pups during insulin induced hypoglycemic coma and after treatment with 3OHB alone or in combination with the anaplerotic substrate propionate. Clinically, treatment with 3OHB and propionate resulted in an alert state and EEG improvement, while treatment with 3OHB alone resulted in an improved EEG but animals remained clinically comatose. Biochemically, both treatments resulted in correction of cerebral glutamate and ammonia levels but not of gluconeogenetic substrates and pentose phosphate metabolites., Conclusion: 3OHB treatment restores glutamate metabolism but cannot restore a glycolytic or pentose phosphate pathway deficit. Additional treatment with propionate significantly improved the clinical protective effect of 3OHB in hypoglycemic coma., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Cystinosis is an autosomal recessive lysosomal storage disease caused by mutations in CTNS. The most prevalent CTNS mutation is a homozygous 57-kb deletion that also includes an adjacent gene named SHPK (CARKL), encoding sedoheptulokinase. Patients with this deletion have elevated urinary concentrations of sedoheptulose. Using derivatisation with pentafluorobenzyl hydroxylamine and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we developed a new sensitive method for the quantification of sedoheptulose in dried blood spots. This method can be utilized as a quick screening test to detect cystinosis patients homozygous for the 57-kb deletion in CTNS; which is the most common mutation of cystinosis. Sedoheptulose concentrations in the deleted patients were 6 to 23 times above the upper limit for controls. The assessment of sedoheptulose in a bloodspot from a known cystinosis patient homozygous for the 57-kb deletion retrieved from the Dutch neonatal screening program showed that sedoheptulose was already elevated in the neonatal period. There was no overlap in sedoheptulose levels between cystinosis patients homozygous for the 57-kb deletion and cystinosis patients not homozygous for this deletion. Our presented method can be used prior to mutation analysis to detect cystinosis patients homozygous for the 57-kb deletion. We feel that the presented method enables fast (pre)-symptomatic detection of cystinosis patients homozygous for the 57-kb deletion, allowing early treatment., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: A high percentage of grade II and III gliomas have mutations in the gene encoding isocitrate dehydrogenase (IDH1). This mutation is always a heterozygous point mutation that affects the amino acid arginine at position 132 and results in loss of its native enzymatic activity and gain of alternative enzymatic activity (producing D-2-hydroxyglutarate). The objective of this study was to investigate the cellular effects of R132H mutations in IDH1., Methods: Functional consequences of IDH1(R132H) mutations were examined among others using fluorescence-activated cell sorting, kinome and expression arrays, biochemical assays, and intracranial injections on 3 different (glioma) cell lines with stable overexpression of IDH1(R132H) ., Results: IDH1(R132H) overexpression in established glioma cell lines in vitro resulted in a marked decrease in proliferation, decreased Akt phosphorylation, altered morphology, and a more contact-dependent cell migration. The reduced proliferation is related to accumulation of D-2-hydroxyglutarate that is produced by IDH1(R132H) . Mice injected with IDH1(R132H) U87 cells have prolonged survival compared to mice injected with IDH1(wt) or green fluorescent protein-expressing U87 cells., Interpretation: Our results demonstrate that IDH1(R132H) dominantly reduces aggressiveness of established glioma cell lines in vitro and in vivo. In addition, the IDH1(R132H) -IDH1(wt) heterodimer has higher enzymatic activity than the IDH1(R132H) -IDH1(R132H) homodimer. Our observations in model systems of glioma might lead to a better understanding of the biology of IDH1 mutant gliomas, which are typically low grade and often slow growing., (Copyright © 2011 American Neurological Association.)

Heterozygous somatic mutations in the genes encoding isocitrate dehydrogenase-1 and -2 (IDH1 and IDH2) were recently discovered in human neoplastic disorders. These mutations disable the enzymes' normal ability to convert isocitrate to 2-ketoglutarate (2-KG) and confer on the enzymes a new function: the ability to convert 2-KG to d-2-hydroxyglutarate (D-2-HG). We have detected heterozygous germline mutations in IDH2 that alter enzyme residue Arg(140) in 15 unrelated patients with d-2-hydroxyglutaric aciduria (D-2-HGA), a rare neurometabolic disorder characterized by supraphysiological levels of D-2-HG. These findings provide additional impetus for investigating the role of D-2-HG in the pathophysiology of metabolic disease and cancer.

Thymidine phosphorylase (TP) is often overexpressed in cancer and potentially plays a role in the stimulation of angiogenesis. The exact mechanism of angiogenesis induction is unclear, but is postulated to be related to thymidine-derived sugars. TP catalyzes the conversion of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P), which can be converted to dR-5-P, glyceraldehyde-3-phosphate (G3P) or deoxyribose (dR). However, it is unclear which sugar accumulates in this reaction. Therefore, in the TP overexpressing Colo320 TP1 and RT112/TP cells we determined by LC-MS/MS which sugars accumulated, their subcellular localization (using (3)H-TdR) and whether dR was secreted from the cells. In both TP-overexpressing cell lines, dR-1-P and dR-5-P accumulated intracellularly at high levels and dR was secreted extensively by the cells. A specific inhibitor of TP completely blocked TdR conversion, and thus no sugars were formed. To examine whether these sugars may be used for the production of angiogenic factors or other products, we determined with (3)H-TdR in which subcellular location these sugars accumulated. TdR-derived sugars accumulated in the cytoskeleton and to some extent in the cell membrane, while incorporation into the DNA was responsible for trapping in the nucleus. In conclusion, various metabolic routes were entered, of which the TdR-derived sugars accumulated in the cytoskeleton and membrane. Future studies should focus on which exact metabolic pathway is involved in the induction of angiogenesis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Pyridoxine-dependent epilepsy was recently shown to be due to mutations in the ALDH7A1 gene, which encodes antiquitin, an enzyme that catalyses the nicotinamide adenine dinucleotide-dependent dehydrogenation of l-alpha-aminoadipic semialdehyde/L-Delta1-piperideine 6-carboxylate. However, whilst this is a highly treatable disorder, there is general uncertainty about when to consider this diagnosis and how to test for it. This study aimed to evaluate the use of measurement of urine L-alpha-aminoadipic semialdehyde/creatinine ratio and mutation analysis of ALDH7A1 (antiquitin) in investigation of patients with suspected or clinically proven pyridoxine-dependent epilepsy and to characterize further the phenotypic spectrum of antiquitin deficiency. Urinary L-alpha-aminoadipic semialdehyde concentration was determined by liquid chromatography tandem mass spectrometry. When this was above the normal range, DNA sequencing of the ALDH7A1 gene was performed. Clinicians were asked to complete questionnaires on clinical, biochemical, magnetic resonance imaging and electroencephalography features of patients. The clinical spectrum of antiquitin deficiency extended from ventriculomegaly detected on foetal ultrasound, through abnormal foetal movements and a multisystem neonatal disorder, to the onset of seizures and autistic features after the first year of life. Our relatively large series suggested that clinical diagnosis of pyridoxine dependent epilepsy can be challenging because: (i) there may be some response to antiepileptic drugs; (ii) in infants with multisystem pathology, the response to pyridoxine may not be instant and obvious; and (iii) structural brain abnormalities may co-exist and be considered sufficient cause of epilepsy, whereas the fits may be a consequence of antiquitin deficiency and are then responsive to pyridoxine. These findings support the use of biochemical and DNA tests for antiquitin deficiency and a clinical trial of pyridoxine in infants and children with epilepsy across a broad range of clinical scenarios.

Incidental reports suggest that antenatal treatment of pyridoxine dependent seizures (PDS) may improve neurodevelopmental outcome of affected patients. Two families with PDS are reported, both with two affected siblings. Antenatal treatment with pyridoxine was instituted during the second pregnancy in each family (50 and 60 mg daily from 3 and 10 weeks of gestation, respectively). Perinatal characteristics and neurodevelopmental outcome at 4 (Family A) and 12 (Family B) years of age were compared between the untreated and treated child within each family. Meconium-stained amniotic fluid was present in both first pregnancies and abnormal foetal movements were noticed in one. In the treated infants, pregnancy and birth were uncomplicated. In family A, postnatal pyridoxine supplementation prevented neonatal seizures. Both children in family A were hypotonic and started walking after 2 years of age; both had white matter changes on MRI, and the first child was treated for squint. IQ was 73 and 98 in the antenatally untreated and treated child, respectively. The second child in family B developed seizures on the seventh day, because pyridoxine maintenance therapy had not been instituted after birth. Seizures responded rapidly to pyridoxine supplementation. MRI showed large ventricles and a mega cisterna magna. IQ was 80 and 106 in the antenatally untreated and treated child respectively. Both children had normal motor development. These results suggest that antenatal pyridoxine supplementation may be effective in preventing intrauterine seizures, decreasing the risk of complicated birth and improving neurodevelopmental outcome in PDS.

We performed molecular, enzyme, and metabolic studies in 50 patients with D-2-hydroxyglutaric aciduria (D-2-HGA) who accumulated D-2-hydroxyglutarate (D-2-HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). Enzyme assay of D-2-HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D-2-HGA whose enzyme activity was normal did not have mutations. Significantly lower D-2-HG concentrations in body fluids were observed in mutation-positive D-2-HGA patients than in mutation-negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D-2-HG. Accordingly, we suggest a new classification: D-2-HGA Type I associates with D-2-HGDH deficiency, whereas idiopathic D-2-HGA manifests with normal D-2-HGDH activity and higher D-2-HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D-2-HGA patients with diverse genetic loci will be revealed in future studies., ((c) 2009 Wiley-Liss, Inc.)

The mammalian degradation of lysine is believed to proceed via two distinct routes, the saccharopine and the pipecolic acid routes, that ultimately converge at the level of alpha-aminoadipic semialdehyde (alpha-AASA). alpha-AASA dehydrogenase-deficient fibroblasts were grown in cell culture medium supplemented with either L-[alpha-(15)N]lysine or L-[epsilon-(15)N]lysine to explore the exact route of lysine degradation. L-[alpha-(15)N]lysine was catabolised into [(15)N]saccharopine, [(15)N]alpha-AASA, [(15)N]Delta(1)-piperideine-6-carboxylate, and surprisingly in [(15)N]pipecolic acid, whereas L-[epsilon-(15)N]lysine resulted only in the formation of [(15)N]saccharopine. These results imply that lysine is exclusively degraded in fibroblasts via the saccharopine branch, and pipecolic acid originates from an alternative precursor. We hypothesize that pipecolic acid derives from Delta(1)-piperideine-6-carboxylate by the action of Delta(1)-pyrroline-5-carboxylic acid reductase, an enzyme involved in proline metabolism.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by mutations in the gene encoding L-2-hydroxyglutarate dehydrogenase. An assay to evaluate L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) activity in fibroblast, lymphoblast and/or lymphocyte lysates has hitherto been unavailable. We developed an L-2-HGDH enzyme assay in cell lysates based on the conversion of stable-isotope-labelled L-2-hydroxyglutarate to 2-ketoglutarate, which is converted into L-glutamate in situ. The formation of stable isotope labelled L-glutamate is therefore a direct measure of L-2-HGDH activity, and this product is detected by liquid chromatography-tandem mass spectrometry. A deficiency of L-2-HGDH activity was detected in cell lysates from 15 out of 15 L-2-HGA patients. Therefore, this specific assay confirmed the diagnosis unambiguously affirming the relationship between molecular and biochemical observations. Residual activity was detected in cells derived from one L-2-HGA patient. The L-2-HGDH assay will be valuable for examining in vitro riboflavin/FAD therapy to rescue L-2-HGDH activity.

This study describes the use of cerebral spinal fluid (CSF) and/or urine as source of DNA for mutation analysis combined with multiple displacement amplification. The findings illustrate the opportunities and pitfalls of these methods in the search for identification of the pathogenic mutations in the case that only scarce material is available such as CSF.

Purpose: To describe the pattern of magnetic resonance (MR) imaging abnormalities in l-2-hydroxyglutaric aciduria (L2HGA) and to evaluate the correlation between imaging abnormalities and disease duration., Materials and Methods: MR images in 56 patients (30 male, 26 female; mean age +/- standard deviation, 11.9 years +/- 8.5) with genetically confirmed L2HGA were retrospectively reviewed, with institutional review board approval and waiver of informed consent. At least one complete series of transverse T2-weighted images was available for all patients. The images were evaluated by using a previously established scoring list. The correlation between MR imaging abnormalities and disease duration was assessed (Mann-Whitney or Kruskal-Wallis test)., Results: The cerebral white matter (WM) abnormalities preferentially affected the frontal and subcortical regions. The abnormal subcortical WM often had a mildly swollen appearance (37 patients). Initially, the WM abnormalities were at least partially multifocal (32 patients). In patients with longer disease duration, the WM abnormalities became more confluent and spread centripetally, but the periventricular rim remained relatively spared (41 patients). The mean disease duration in patients with WM atrophy (14.8 years) was significantly longer (P = .001) than that in patients without atrophy (6.7 years). Bilateral involvement of the globus pallidus (55 patients), caudate nucleus (56 patients), and putamen (56 patients) was seen at all stages. The cerebellar WM was never affected. The dentate nucleus was involved bilaterally in 55 of 56 patients., Conclusion: L2HGA has a distinct highly characteristic pattern of MR imaging abnormalities: a combination of predominantly subcortical cerebral WM abnormalities and abnormalities of the dentate nucleus, globus pallidus, putamen, and caudate nucleus. With increasing disease duration, WM abnormalities and basal ganglia signal intensity abnormalities become more diffuse and cerebral WM atrophy ensues.

Objective: Folinic acid-responsive seizures and pyridoxine-dependent epilepsy are two treatable causes of neonatal epileptic encephalopathy. The former is diagnosed by characteristic peaks on cerebrospinal fluid (CSF) monoamine metabolite analysis; its genetic basis has remained elusive. The latter is due to alpha-aminoadipic semialdehyde (alpha-AASA) dehydrogenase deficiency, associated with pathogenic mutations in the ALDH7A1 (antiquitin) gene. We report two patients whose CSF showed the marker of folinic acid-responsive seizures, but who responded clinically to pyridoxine. We performed genetic and biochemical testing of samples from these patients, and seven others, to determine the relation between these two disorders., Methods: CSF samples were analyzed for the presence of alpha-AASA and pipecolic acid. DNA sequencing of the ALDH7A1 gene was performed., Results: Both patients reported here had increased CSF alpha-AASA, CSF pipecolic acid, and known or likely pathogenic mutations in the ALDH7A1 gene, consistent with alpha-AASA dehydrogenase deficiency. Analysis of CSF samples from seven other anonymous individuals diagnosed with folinic acid-responsive seizures showed similar results., Interpretation: These results demonstrate that folinic acid-responsive seizures are due to alpha-AASA dehydrogenase deficiency and mutations in the ALDH7A1 gene. Thus, folinic acid-responsive seizures are identical to the major form of pyridoxine-dependent epilepsy. We recommend consideration of treatment with both pyridoxine and folinic acid for patients with alpha-AASA dehydrogenase deficiency, and consideration of a lysine restricted diet. The evaluation of patients with neonatal epileptic encephalopathy, as well as those with later-onset seizures, should include a measurement of alpha-AASA in urine to identify this likely underdiagnosed and treatable disorder.

Pyridoxine-dependent seizures (PDS) is a rare autosomal recessive disorder causing intractable seizures in neonates and infants. Patients are typically resistant to conventional anticonvulsants but respond well to the administration of pyridoxine. We report two unrelated patients affected with PDS as a result of alpha-aminoadipic semialdehyde (alpha-AASA) dehydrogenase deficiency caused by pathogenic ALDH7A1/antiquitin mutations. Two of the three reported mutations are novel and result in erroneous splicing, as showed by messenger RNA (mRNA) studies. So far, the vast majority of the patients clinically diagnosed as PDS show alpha-AASA dehydrogenase deficiency, caused by mutations in the ALDH7A1 gene. However, despite the availability of reliable biomarkers, early consideration of a pyridoxine trial is still the most important issue in a child with therapy-resistant seizures.

D: -2-Hydroxyglutaric aciduria (D: -2-HGA) is a neurometabolic disorder characterized by elevated levels of D: -2-hydroxyglutarate (D: -2-HG) in physiological fluids. Recent findings revealed that mutations in the D2HGDH gene, encoding D: -2-hydroxyglutarate dehydrogenase, cause D: -2-HGA. So far, a functionalenzyme assay to determine D: -2-hydroxyglutarate dehydrogenase activity, converting D: -2-HG into 2-ketoglutarate (2-KG), has been unavailable. We have now developed a unique enzyme assay for the determination of D: -2-hydroxyglutarate dehydrogenase activity in cells derived from D: -2-HGA patients and controls. The enzyme assay was performed using enantiomerically pure stable-isotope-labelled D: -2-hydroxy[3,3,4,4-(2)H(4)]glutarate. This substrate is convertedby D: -2-hydroxyglutarate dehydrogenase into 2-[3,3,4,4-(2)H(4)]ketoglutarate, which is subsequently converted into L: -[3,3,4,4-(2)H(4)]glutamate by L: -glutamate dehydrogenase, present in saturating amounts in cell homogenates. Enzyme activities were quantified using LC-MS/MS. The mean activities in control fibroblast and lymphoblast homogenates were 298 +/- 207 and 1670 +/- 940 pmol/h per mg protein, respectively. In fibroblast and lymphoblast cell lines derived from patients with pathogenic mutations in the D2HGDH gene, considerably decreased enzyme activities (e.g. <41 pmol/h per mg protein) were found compared with controls. This enzyme assay will have additional utility in further differentiating patients with D: -2-HGA and L: -2-HGA and in assessing the residual activities linked to pathogenic mutations in the D2HGDH gene.

The recent discovery of two defects (ribose-5-phosphate isomerase deficiency and transaldolase deficiency) in the reversible part of the pentose phosphate pathway (PPP) has stimulated interest in this pathway. In this review we describe the functions of the PPP, its relation to other pathways of carbohydrate metabolism and an overview of the metabolic defects in the reversible part of the PPP.

Pyridoxine-dependent seizures are a rare condition recognized when numerous seizures respond to pyridoxine treatment and recur on pyridoxine withdrawal. For decades the diagnosis was confirmed only with pyridoxine treatment withdrawal trial. Recently described biochemical and molecular pathology improved the diagnostic process for those cases in which seizures are caused by alpha amino adipic semialdehyde dehydrogenase deficiency. This article presents a girl with recurrent status epilepticus episodes resistant to phenobarbital and phenytoin and partly responding to midazolam. Eventually the seizures were completely controlled with pyridoxine; however, due to the severe condition of this child when seizing, no trial of withdrawal has been performed. The diagnosis of pyridoxine-dependent seizures was confirmed with biochemical and molecular testing revealing elevated alpha-AASA excretion and the presence of 2 different mutations in the antiquitin ( ALDH7A1) gene. Due to the availability of reliable laboratory testing, confirmation of the diagnosis was made without the life-threatening trial of pyridoxine withdrawal.

The glucose analogue 2-deoxy-D-glucose (2-DG) restrains growth of normal and malignant cells, prolongs the lifespan of C. elegans, and is widely used as a glycolytic inhibitor to study metabolic activity with regard to cancer, neurodegeneration, calorie restriction, and aging. Here, we report that separating glycolysis and the pentose phosphate pathway highly increases cellular tolerance to 2-DG. This finding indicates that 2-DG does not block cell growth solely by preventing glucose catabolism. In addition, 2-DG provoked similar concentration changes of sugar-phosphate intermediates in wild-type and 2-DG-resistant yeast strains and in human primary fibroblasts. Finally, a genome-wide analysis revealed 19 2-DG-resistant yeast knockouts of genes implicated in carbohydrate metabolism and mitochondrial homeostasis, as well as ribosome biogenesis, mRNA decay, transcriptional regulation, and cell cycle. Thus, processes beyond the metabolic block are essential for the biological properties of 2-DG.

Transaldolase (TALDO) deficiency is a rare inborn error of the pentose phosphate pathway. We report the clinical presentation and laboratory findings of a new patient with TALDO deficiency. The two-year-old Arabic boy presented with neonatal onset of anemia and thrombocytopenia, tubulopathy, and rickets and was subsequently found to have cirrhosis and deafness. A comparison with other TALDO deficient patients is given.

The most common mutation in the nephropathic cystinosis (CTNS) gene is a homozygous 57-kb deletion that also includes an adjacent gene carbohydrate kinase-like (CARKL). The latter gene encodes a protein that is predicted to function as a carbohydrate kinase. Cystinosis patients with the common 57-kb deletion had strongly elevated urinary concentrations of sedoheptulose (28-451 mmol/mol creatinine; controls and other cystinosis patients <9) and erythritol (234-1110 mmol/mol creatinine; controls and other cystinosis patients <148). Enzyme studies performed on fibroblast homogenates derived from patients carrying the 57-kb deletion revealed 80% reduction in their sedoheptulose phosphorylating activity compared to cystinosis patients with other mutations and controls. This indicates that the CARKL-encoded protein, sedoheptulokinase (SHK), is responsible for the reaction: sedoheptulose + ATP --> sedoheptulose-7-phosphate + ADP and that deletion of CARKL causes urinary accumulation of sedoheptulose and erythritol., (Copyright 2008 Wiley-Liss, Inc.)

When wounded or attacked by herbivores or pathogens, plants produce a blend of six-carbon alcohols, aldehydes and esters, known as C6-volatiles. Undamaged plants, when exposed to C6-volatiles, respond by inducing defense-related genes and secondary metabolites, suggesting that C6-volatiles can act as signaling molecules regulating plant defense responses. However, to date, the molecular mechanisms by which plants perceive and respond to these volatiles are unknown. To elucidate such mechanisms, we decided to isolate Arabidopsis thaliana mutants in which responses to C6-volatiles were altered. We observed that treatment of Arabidopsis seedlings with the C6-volatile E-2-hexenal inhibits root elongation. Among C6-volatiles this response is specific to E-2-hexenal, and is not dependent on ethylene, jasmonic and salicylic acid. Using this bioassay, we isolated 18 E-2-hexenal-response (her) mutants that showed sustained root growth after E-2-hexenal treatment. Here, we focused on the molecular characterization of one of these mutants, her1. Microarray and map-based cloning revealed that her1 encodes a gamma-amino butyric acid transaminase (GABA-TP), an enzyme that degrades GABA. As a consequence of the mutation, her1 plants accumulate high GABA levels in all their organs. Based on the observation that E-2-hexenal treatment induces GABA accumulation, and that high GABA levels confer resistance to E-2-hexenal, we propose a role for GABA in mediating E-2-hexenal responses.

Background: Eukaryotic cells have evolved various response mechanisms to counteract the deleterious consequences of oxidative stress. Among these processes, metabolic alterations seem to play an important role., Results: We recently discovered that yeast cells with reduced activity of the key glycolytic enzyme triosephosphate isomerase exhibit an increased resistance to the thiol-oxidizing reagent diamide. Here we show that this phenotype is conserved in Caenorhabditis elegans and that the underlying mechanism is based on a redirection of the metabolic flux from glycolysis to the pentose phosphate pathway, altering the redox equilibrium of the cytoplasmic NADP(H) pool. Remarkably, another key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is known to be inactivated in response to various oxidant treatments, and we show that this provokes a similar redirection of the metabolic flux., Conclusion: The naturally occurring inactivation of GAPDH functions as a metabolic switch for rerouting the carbohydrate flux to counteract oxidative stress. As a consequence, altering the homoeostasis of cytoplasmic metabolites is a fundamental mechanism for balancing the redox state of eukaryotic cells under stress conditions.

Employing lymphoblasts derived from two non related patients with L-2-HG aciduria, we examined the origin of L-2-hydroxyglutaric acid (L-2-HG) through incubation with [(13)C6]glucose and [(2)H5]glutamic acid. Formation of labelled 2-ketoglutaric acid (2-KG), citric acid and L-2-HG was determined by GC-MS. The quantitative and qualitative isotopomer pattern following incubation with [(13)C6]glucose was identical for all end-products. Incubations with [(2)H5]glutamic acid as precursor revealed the formation of identical isotopomers for 2-KG and L-2-HG. Our data indicate that 2-KG is the metabolic precursor of L-2-HG, adding to previous studies which revealed that 2-KG is the metabolic precursor of D-2-HG. These data suggest that 2-KG has a pathophysiological role in combined D/L-2-HG aciduria.

Transaldolase deficiency, a recently discovered disorder of carbohydrate metabolism with multisystem involvement, has been diagnosed in 6 patients. Affected patients have abnormal concentrations of polyols in body fluids and in all patients we have previously found increased amounts of a seven-carbon chain carbohydrate which we suspected of being sedoheptulose. We report development of a liquid chromatography-tandem mass spectrometry method for quantitation of the seven-carbon carbohydrates sedoheptulose and mannoheptulose in urine. Additionally, other seven-carbon chain carbohydrates were characterized in urine, including sedoheptitol, perseitol and sedoheptulose 7-phosphate. Transaldolase-deficient patients had significantly increased urinary sedoheptulose and sedoheptulose 7-phosphate, associated with subtle elevations of mannoheptulose, sedoheptitol and perseitol. Our findings reveal novel urinary biomarkers for identification of transaldolase deficiency.

Succinic semialdehyde dehydrogenase (SSADH) deficiency and D-2-hydroxyglutaric aciduria (D-2-HGA) are rare inborn errors of metabolism primarily revealed by urinary organic acid screening. Three patients with proven SSADH deficiency excreted, in addition to GHB considerable amounts of D-2-HG. We examined whether these patients suffered from two inborn errors of metabolism by measuring D-2-HG concentrations in the culture medium of cells from these patients. In addition, mutation analysis of the D-2-hydroxyglutarate dehydrogenase gene was performed. Normal concentrations of D-2-HG were measured in the culture media of fibroblasts or lymphoblasts derived from the three patients. In one patient, we found a heterozygous likely pathogenic mutation in the D-2-hydroxyglutarate dehydrogenase gene. These combined results argue against the hypothesis that the patients are affected with "primary" D-2-HGA in combination with their SSADH deficiency. Moderately increased levels of D-2-HG were also found in urine, plasma, and cerebrospinal fluid samples derived from 12 other patients with SSADH deficiency, revealing that D-2-HG is a common metabolite in this disease. The increase of D-2-HG in SSADH deficiency can be explained by the action of hydroxyacid-oxoacid transhydrogenase, a reversible enzyme that oxidases GHB in the presence of 2-ketoglutarate yielding SSA and D-2-HG.