Mutations in PMM2, a phosphomannomutase gene on chromosome 16p13, in carbohydrate-deficient glycoprotein type I syndrome (Jaeken syndrome).

Carbohydrate-deficient glycoprotein syndrome type 1 (CDG1 or Jaeken syndrome) is the prototype of a class of genetic multisystem disorders characterized by defective glycosylation of glycoconjugates. It is mostly a severe disorder which presents neonatally. There is a severe encephalopathy with axial hypotonia, abnormal eye movements and pronounced psychomotor retardation, as well as a peripheral neuropathy, cerebellar hypoplasia and retinitis pigmentosa. The patients show a peculiar distribution of subcutaneous fat, nipple retraction and hypogonadism. There is a 20% lethality in the first years of life due to severe infections, liver insufficiency or cardiomyopathy. CDG1 shows an autosomal recessive mode of inheritance and has been mapped to chromosome 16p. Most patients show a deficiency of phosphomannomutase (PMM)8, an enzyme necessary for the synthesis of GDP-mannose. We have cloned the PMM1 gene, which is on chromosome 22q13 (ref.9). We now report the identification of a second human PMM gene, PMM2, which is located on 16p13 and which encodes a protein with 66% identity to PMM1. We found eleven different missense mutations in PMM2 in 16 CDG1 patients from different geographical origins and with a documented phosphomannomutase deficiency. Our results give conclusive support to the biochemical finding that the phosphomannomutase deficiency is the basis for CDG1.

A lymphoblast model for IDH2 gain-of-function activity in d-2-hydroxyglutaric aciduria type II: novel avenues for biochemical and therapeutic studies.

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.

Effects of fructosamine-3-kinase deficiency on function and survival of mouse pancreatic islets after prolonged culture in high glucose or ribose concentrations.

Due to their high glucose permeability, insulin-secreting pancreatic beta-cells likely undergo strong intracellular protein glycation at high glucose concentrations. They may, however, be partly protected from the glucotoxic alterations of their survival and function by fructosamine-3-kinase (FN3K), a ubiquitous enzyme that initiates deglycation of intracellular proteins. To test that hypothesis, we cultured pancreatic islets from Fn3k-knockout (Fn3k(-/-)) mice and their wild-type (WT) littermates for 1-3 wk in the presence of 10 or 30 mmol/l glucose (G10 or G30, respectively) and measured protein glycation, apoptosis, preproinsulin gene expression, and Ca(2+) and insulin secretory responses to acute glucose stimulation. The more potent glycating agent d-ribose (25 mmol/l) was used as positive control for protein glycation. In WT islets, a 1-wk culture in G30 significantly increased the amount of soluble intracellular protein-bound fructose-epsilon-lysines and the glucose sensitivity of beta-cells for changes in Ca(2+) and insulin secretion, whereas it decreased the islet insulin content. After 3 wk, culture in G30 also strongly decreased beta-cell glucose responsiveness and preproinsulin mRNA levels, whereas it increased islet cell apoptosis. Although protein-bound fructose-epsilon-lysines were more abundant in Fn3k(-/-) vs. WT islets, islet cell survival and function and their glucotoxic alterations were almost identical in both types of islets, except for a lower level of apoptosis in Fn3k(-/-) islets cultured for 3 wk in G30. In comparison, d-ribose (1 wk) similarly decreased preproinsulin expression and beta-cell glucose responsiveness in both types of islets, whereas it increased apoptosis to a larger extent in Fn3k(-/-) vs. WT islets. We conclude that, despite its ability to reduce the glycation of intracellular islet proteins, FN3K is neither required for the maintenance of beta-cell survival and function under control conditions nor involved in protection against beta-cell glucotoxicity. The latter, therefore, occurs independently from the associated increase in the level of intracellular fructose-epsilon-lysines.

L: -2-Hydroxyglutaric aciduria, a disorder of metabolite repair.

The neurometabolic disorder L: -2-hydroxyglutaric aciduria is caused by mutations in a gene present on chromosome 14q22.1 and encoding L: -2-hydroxyglutarate dehydrogenase. This FAD-linked mitochondrial enzyme catalyses the irreversible conversion of L: -2-hydroxyglutarate to alpha-ketoglutarate. The formation of L: -2-hydroxyglutarate results from a side-activity of mitochondrial L: -malate dehydrogenase, the enzyme that interconverts oxaloacetate and L: -malate, but which also catalyses, very slowly, the NADH-dependent conversion of alpha-ketoglutarate to L: -2-hydroxyglutarate. L: -2-Hydroxyglutarate has no known physiological function in eukaryotes and most prokaryotes. Its accumulation is toxic to the mammalian brain, causing a leukoencephalopathy and increasing the susceptibility to develop tumours. L: -2-Hydroxyglutaric aciduria appears to be the first disease of 'metabolite repair'.

L-2-hydroxyglutaric aciduria, a defect of metabolite repair.

L-2-hydroxyglutaric aciduria is a metabolic disorder in which L-2-hydroxyglutarate accumulates as a result of a deficiency in FAD-linked L-2-hydroxyglutarate dehydrogenase, a mitochondrial enzyme converting L-2-hydroxyglutarate to alpha-ketoglutarate. The origin of the L-2-hydroxyglutarate, which accumulates in this disorder, is presently unknown. The oxidation-reduction potential of the 2-hydroxyglutarate/alpha-ketoglutarate couple is such that L-2-hydroxyglutarate could potentially be produced through the reduction of alpha-ketoglutarate by a NAD- or NADP-linked oxidoreductase. In fractions of rat liver cytosolic extracts that had been chromatographed on an anion exchanger we detected an enzyme reducing alpha-ketoglutarate in the presence of NADH. This enzyme co-purified with cytosolic L-malate dehydrogenase (cMDH) upon further chromatography on Blue Sepharose. Mitochondrial fractions also contained an NADH-linked, 'alpha-ketoglutarate reductase', which similarly co-purified with mitochondrial L-malate dehydrogenase (mMDH). Purified mMDH catalysed the reduction of alpha-ketoglutarate to L-2-hydroxyglutarate with a catalytic efficiency that was about 10(7)-fold lower than that observed with oxaloacetate. For the cytosolic enzyme, this ratio amounted to 10(8), indicating that this enzyme is more specific. Both cMDH and mMDH are highly active in tissues and alpha-ketoglutarate is much more abundant than oxaloacetate and more concentrated in mitochondria than in the cytosol. As a result of this, the weak activity of mMDH on alpha-ketoglutarate is sufficient to account for the amount of L-2-hydroxyglutarate that is excreted by patients deficient in FAD-linked L-2-hydroxyglutarate dehydrogenase. The latter enzyme appears, therefore, to be responsible for a 'metabolite repair' phenomenon and to belong to the expanding class of 'house-cleaning' enzymes.

[L-2-hydroxyglutaric aciduria, an error of metabolism]. / L'acidurie L-2-hydroxyglutarique, une erreur de la réparation métabolique.

The neurometabolic disorder L-2-hydroxyglutaric aciduria was recently shown to be due to a defect in L-2-hydroxyglutarate dehydrogenase. This FAD-linked enzyme catalyses the irreversible conversion of L-2-hydroxyglutarate to alpha-ketoglutarate. The formation of L-2-hydroxyglutarate results from a side-activity of mitochondrial L-malate dehydrogenase, the enzyme which normally catalyses the interconversion of oxaloacetate and L-malate, but which also catalyses the NADH-dependent conversion of alpha-ketoglutarate to L-2-hydroxyglutarate. Though very slow, this activity accounts for the in vivo formation of L-2-hydroxyglutarate. As the latter compound is most likely toxic, L-2-hydroxyglutarate dehydrogenase catalyses a metabolite repair reaction.

The gene mutated in l-2-hydroxyglutaric aciduria encodes l-2-hydroxyglutarate dehydrogenase.

The biochemical defect in L-2-hydroxyglutaric aciduria is still unknown, but the mutated gene has recently been identified on chromosome 14q22. Transfection of human embryonic kidney (HEK) cells with a cDNA encoding the product of the human gene led to a>15-fold increase in L-2-hydroxyglutarate dehydrogenase activity. The overexpressed enzyme had similar biochemical characteristics (including sensitivity to FAD and association with membranes) as the rat liver enzyme. Western blot analysis indicated that it is processed through the removal of a N-terminal approximately 4 kDa fragment, in agreement with a mitochondrial localization. Transfection experiments indicated that the mutations (K81E, E176D, Delta-exon9) found in patients with L-2-hydroxyglutaric aciduria suppressed L-2-hydroxyglutarate dehydrogenase activity. Western blot analysis showed that the three mutated proteins were expressed to various degrees in HEK cells, but were abnormally processed. Taken together, these data indicate that L-2-hydroxyglutaric aciduria is due to a deficiency in L-2-hydroxyglutarate dehydrogenase.

Variability in erythrocyte fructosamine 3-kinase activity in humans correlates with polymorphisms in the FN3K gene and impacts on haemoglobin glycation at specific sites.

BACKGROUND: Part of the fructosamines that are bound to intracellular proteins are repaired by fructosamine 3-kinase (FN3K). Because subject-to-subject variations in erythrocyte FN3K activity could affect the level of glycated haemoglobin independently of differences in blood glucose level, we explored if such variability existed, if it was genetically determined by the FN3K locus on 17q25 and if the FN3K activity correlated inversely with the level of glycated haemoglobin. RESULTS: The mean erythrocyte FN3K activity did not differ between normoglycaemic subjects (n = 26) and type 1 diabetic patients (n = 31), but there was a wide interindividual variability in both groups (from about 1 to 4 mU/g haemoglobin). This variability was stable with time and associated (P < 0.0001) with two single nucleotide polymorphisms in the promoter region and exon 6 of the FN3K gene. There was no significant correlation between FN3K activity and the levels of HbA1c, total glycated haemoglobin (GHb) and haemoglobin fructoselysine residues, either in the normoglycaemic or diabetic group. However, detailed analysis of the glycation level at various sites in haemoglobin indicated that the glycation level of Lys-B-144 was about twice as high in normoglycaemic subjects with the lowest FN3K activities as compared to those with the highest FN3K activities. CONCLUSION: Interindividual variability of FN3K activity is substantial and impacts on the glycation level at specific sites of haemoglobin, but does not detectably affect the level of HbA1c or GHb. As FN3K opposes one of the chemical effects of hyperglycaemia, it would be of interest to test whether hypoactivity of this enzyme favours the development of diabetic complications.

High frequency of cytolytic T lymphocytes directed against a tumor-specific mutated antigen detectable with HLA tetramers in the blood of a lung carcinoma patient with long survival.

We have identified an antigen recognized by autologous CTL on the lung carcinoma cells of a patient who enjoyed a favorable clinical evolution, being alive 10 years after partial resection of the primary tumor. The antigenic peptide is presented by HLA-A2 molecules and encoded by a mutated sequence in the gene coding for malic enzyme, an essential enzyme that converts malate to pyruvate. In the tumor cell line derived from the patient, only the mutated malic enzyme allele is expressed, because of a loss of heterozygosity in the region of chromosome 6 that contains this locus. Tetramers of soluble HLA-A2 molecules loaded with the antigenic peptide stained approximately 0.4% of the patient's blood CD8 T cells. When these cells were stimulated in clonal conditions, 25% of them proliferated, and the resulting clones were lytic and specific for the mutated malic enzyme peptide. T-cell receptor analysis indicated that almost all of these antimalic CTLs shared the same receptor. Antimalic T cells were consistently found in blood samples collected from the patient between 1990 and 1999, at frequencies ranging from 0.1 to 0.4% of the CD8 cells. Their frequency appeared to double within 2 weeks after intradermal inoculation of lethally irradiated autologous tumor cells. These results indicate that nonmelanoma cancer patients may also have a high frequency of blood CTLs directed against a tumor-specific antigen.

Two new cases of serine deficiency disorders treated with l-serine.

OBJECTIVE AND PATIENTS: We report on two new cases of serine deficiency due respectively to 3-phosphoglycerate dehydrogenase (PHGDH) deficiency (Patient 1) and phosphoserine aminotransferase (PSAT1) deficiency (Patient 2), presenting with congenital microcephaly (<3rd centile at birth) and encephalopathy with spasticity. Patient 1 had also intractable seizures. A treatment with oral l-serine was started at age 4.5 years and 3 months respectively. RESULTS: Serine levels were low in plasma and CSF relative to the reference population, for which we confirm recently redefined intervals based on a larger number of samples. l-Serine treatment led in patient 1 to a significant reduction of seizures after one week of treatment and decrease of electroencephalographic abnormalities within one year. In patient 2 treatment with l-serine led to an improvement of spasticity. However for both patients, l-serine failed to improve substantially head circumference (HC) and neurocognitive development. In a couple related to patient's 2 family, dosage of serine was performed on fetal cord blood when the fetus presented severe microcephaly, showing reduced serine levels at 30 weeks of pregnancy. CONCLUSIONS: l-Serine treatment in patients with 2 different serine synthesis defects, led to a significant reduction of seizures and an improvement of spasticity, but failed to improve substantially neurocognitive impairment. Therefore, CSF and plasma serine levels should be measured in all cases of severe microcephaly at birth to screen for serine deficiency, as prompt treatment with l-serine may significantly impact the outcome of the disease. Reduced serine levels in fetal cord blood may also be diagnostic as early as 30 weeks of pregnancy.

Carbohydrate-deficient glycoprotein syndrome type IA (phosphomannomutase-deficiency).

The carbohydrate-deficient glycoprotein or CDG syndromes (OMIM 212065) are a recently delineated group of genetic, multisystem diseases with variable dysmorphic features. The known CDG syndromes are characterized by a partial deficiency of the N-linked glycans of secretory glycoproteins, lysosomal enzymes, and probably also membranous glycoproteins. Due to the deficiency of terminal N-acetylneuraminic acid or sialic acid, the glycan changes can be observed in serum transferrin or other glycoproteins using isoelectrofocusing with immunofixation as the most widely used diagnostic technique. Most patients show a serum sialotransferrin pattern characterized by increased di- and asialotransferrin bands (type I pattern). The majority of patients with type I are phosphomannomutase deficient (type IA), while in a few other patients, deficiencies of phosphomannose isomerase (type IB) or endoplasmic reticulum glucosyltransferase (type IC) have been demonstrated. This review is an update on CDG syndrome type IA.

Novel arguments in favor of the substrate-transport model of glucose-6-phosphatase.

The purpose of this work was to discriminate between two models for glucose-6-phosphatase: one in which the enzyme has its catalytic site oriented toward the lumen of the endoplasmic reticulum, requiring transporters for glucose-6-phosphate, inorganic phosphate (Pi), and glucose (substrate-transport model), and a second one in which the hydrolysis of glucose-6-phosphate occurs inside the membrane (conformational model). We show that microsomes preloaded with yeast phosphoglucose isomerase catalyzed the detritiation of [2-(3)H]glucose-6-phosphate and that this reaction was inhibited by up to 90% by S3483, a compound known to inhibit glucose-6-phosphate hydrolysis in intact but not in detergent-treated microsomes. These results indicate that glucose-6-phosphate is transported to the lumen of the microsomes in an S3483-sensitive manner. Detritiation by intramicrosomal phosphoglucose isomerase was stimulated twofold by 1 mmol/l vanadate, a phosphatase inhibitor, indicating that glucose-6-phosphatase and the isomerase compete for the same intravesicular pool of glucose-6-phosphate. To investigate the site of release of Pi from glucose-6-phosphate, we incubated microsomes with Pb(2+), which forms an insoluble complex with Pi, preventing its rapid exit from the microsomes. Under these conditions, approximately 80% of the Pi that was formed after 5 min was intramicrosomal, compared with <10% in the absence of Pb(2+). We also show that, when incubated with glucose-6-phosphate and mannitol, glucose-6-phosphatase formed mannitol-1-phosphate and that this nonphysiological product was initially present within the microsomes before being released to the medium. These results indicate that the primary site of product release by glucose-6-phosphatase is the lumen of the endoplasmic reticulum.

Study of the regulatory properties of glucokinase by site-directed mutagenesis: conversion of glucokinase to an enzyme with high affinity for glucose.

To identify the amino acids involved in the specific regulatory properties of glucokinase, and particularly its low affinity for glucose, mutants of the human islet enzyme have been prepared, in which glucokinase-specific residues have been replaced. Two mutations increased the affinity for glucose by twofold (K296M) and sixfold (Y214A), the latter also decreasing the Hill coefficient from 1.75 to 1.2 with minimal change in the affinity for ATP. Combining these two mutations with N166R resulted in a 50-fold decrease in the half-saturating substrate concentration (S0.5) value, which became then comparable to the Km of hexokinase II. The location of N166, Y214, and K296 in the three-dimensional structure of glucokinase suggests that these mutations act by favoring closure of the catalytic cleft. As a rule, mutations changed the affinity for glucose and for the competitive inhibitor mannoheptulose (MH) in parallel, whereas they barely affected the affinity for N-acetylglucosamine (NAG). These and other results suggest that NAG and MH bind to the same site but to different conformations of glucokinase. A small reduction in the affinity for the regulatory protein was observed with mutations of residues on the smaller domain and in the hinge region, confirming the bipartite nature of the binding site for the regulatory protein. The K296M mutant was found to have a threefold decreased affinity for palmitoyl CoA; this effect was additive to that previously observed for the E279Q mutant, indicating that the binding site for long-chain acyl CoAs is located on the upper face of the larger domain.

Identification, cloning, and heterologous expression of a mammalian fructosamine-3-kinase.

Fructosamines are thought to play an important role in the development of diabetic complications. Little is known about reactions that could metabolize these compounds in mammalian tissues, except for recent indications that they can be converted to fructosamine 3-phosphates. The purpose of the present work was to identify and characterize the enzyme responsible for this conversion. Erythrocyte extracts were found to catalyze the ATP-dependent phosphorylation of 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine. The enzyme responsible for this conversion was purified approximately 2,500-fold by chromatography on Blue Sepharose, Q Sepharose, and Sephacryl S-200 and shown to copurify with a 35,000-M(r) protein. Partial sequences of tryptic peptides were derived from the protein by nanoelectrospray-ionization mass spectrometry, which allowed for the identification of the corresponding human and mouse cDNAs. Both cDNAs encode proteins of 309 amino acids, showing 89% identity with each other and homologous to proteins of unknown function predicted from the sequences of several bacterial genomes. Both proteins were expressed in Escherichia coli and purified. They were shown to catalyze the phosphorylation of DMF, fructoselysine, fructoseglycine, and fructose in order of decreasing affinity. They also phosphorylated glycated lysozyme, though not unmodified lysozyme. Nuclear magnetic resonance analysis of phosphorylated DMF and phosphorylated fructoseglycine showed that the phosphate was bound to the third carbon of the 1-deoxyfructose moiety. The physiological function of fructosamine-3-kinase may be to initiate a process leading to the deglycation of fructoselysine and of glycated proteins.

A broad spectrum of clinical presentations in congenital disorders of glycosylation I: a series of 26 cases.

INTRODUCTION: Congenital disorders of glycosylation (CDG), or carbohydrate deficient glycoprotein syndromes, form a new group of multisystem disorders characterised by defective glycoprotein biosynthesis, ascribed to various biochemical mechanisms. METHODS: We report the clinical, biological, and molecular analysis of 26 CDG I patients, including 20 CDG Ia, two CDG Ib, one CDG Ic, and three CDG Ix, detected by western blotting and isoelectric focusing of serum transferrin. RESULTS: Based on the clinical features, CDG Ia could be split into two subtypes: a neurological form with psychomotor retardation, strabismus, cerebellar hypoplasia, and retinitis pigmentosa (n=11), and a multivisceral form with neurological and extraneurological manifestations including liver, cardiac, renal, or gastrointestinal involvement (n=9). Interestingly, dysmorphic features, inverted nipples, cerebellar hypoplasia, and abnormal subcutaneous fat distribution were not consistently observed in CDG Ia. By contrast, the two CDG Ib patients had severe liver disease, enteropathy, and hyperinsulinaemic hypoglycaemia but no neurological involvement. Finally, the CDG Ic patient and one of the CDG Ix patients had psychomotor retardation and seizures. The other CDG Ix patients had severe proximal tubulopathy, bilateral cataract, and white matter abnormalities (one patient), or multiorgan failure and multiple birth defects (one patient). CONCLUSIONS: Owing to the remarkable clinical variability of CDG, this novel disease probably remains largely underdiagnosed. The successful treatment of CDG Ib patients with oral mannose emphasises the paramount importance of early diagnosis of PMI deficiency.

Prenatal diagnosis in CDG1 families: beware of heterogeneity.

Carbohydrate-deficient glycoprotein syndrome type 1 (CDG1) is an autosomal recessive, metabolic disorder with severe psychomotor retardation and a high mortality rate in early childhood. Most patients have a deficiency of phosphomannomutase, due to mutations in PMM2, a gene located on chromosome 16p13. Over a period of 18 months we offered prenatal diagnosis to eight families. In six cases and prior to the identification of the gene, the diagnosis was based on linkage analysis and phosphomannomutase measurements. Subsequently direct mutation analysis has been used in two families. It is shown here that phosphomannomutase activities are strongly reduced in cultured amniocytes and trophoblasts of affected foetuses. We refrained from offering prenatal testing in two other families, because either the disease did not link to chromosome 16 and/or normal phosphomannomutase activities were measured in fibroblasts from the proband. This confirms earlier suggestions of heterogeneity for CDG1.

The putative glucose 6-phosphate translocase gene is mutated in essentially all cases of glycogen storage disease type I non-a.

The purpose of this work was to test the hypothesis that mutations in the putative glucose 6-phosphate translocase gene would account for most of the cases of GSD I that are not explained by mutations in the phosphohydrolase gene, ie that are not type Ia. Twenty-three additional families diagnosed as having GSD I non-a (GSDIb, Ic or Id) have now been analysed. The 9exons of the gene were amplified by PCR and mutations searched both by SSCP and heteroduplex analysis. Except for one family in which only one mutation was found, all patients had two allelic mutations in the gene encoding the putative glucose 6-phosphate translocase. Sixteen of the mutations are new and they are all predicted to lead to non-functional proteins. All investigated patients had some degree of neutropenia or neutrophil dysfunction and the clinical phenotype of the four new patients who had been diagnosed as GSD Ic and the one diagnosed as GSD Id was no different from the GSD Ib patients. Since these patients, and the four type Ic patients from two families previously studied, shared several mutations with GSD Ib patients, we conclude that their basic defect is in the putative glucose 6-phosphate translocase and that they should be reclassified as GSD Ib. Isolated defects in microsomal Pi transporter or in microsomal glucose transporter must be very rare or have phenotypes that are not recognised as GSD I, so that in practice there are only two subtypes of GSD I (GSD Ia and GSD Ib).

Structure of the gene mutated in glycogen storage disease type Ib.

We report the structure of the human gene encoding the putative glucose 6-phosphate translocase that is mutated in glycogen storage disease type Ib. Northern blots showed that the encoded 2.4 kb mRNA is mainly expressed in liver and in kidney, but is also present, although in barely detectable amounts, in leucocytes. The gene contains nine exons, one of which (exon 7) is not present in human liver or leucocyte RNA. RT-PCR analysis of mouse RNA indicates that exon 7, which is 63 bp long compared with 66 bp in man, is not expressed in liver and kidney but well in heart and brain. 5'-RACE and RNase protection assays performed on RNAs from human liver, kidney and leucocytes indicated the presence of two main regions of transcription start at approximately -200 and -100 bp with respect to the initiator ATG.

D-glycerate kinase deficiency as a cause of D-glyceric aciduria.

D-Glycerate kinase was measured in human livers thanks to a new, sensitive radiochemical assay. The enzyme was extremely unstable in extracts prepared in water, but was partly stabilized in a homogenization mixture containing inorganic phosphate, D-glycerate and EGTA. When extracted in such a stabilizing mixture, glycerate kinase activity amounted to 0.86 +/- 0.21 U/g in control livers and to 0.03 U/g in the liver of a patient with D-glyceric aciduria. In contrast, D-glycerate dehydrogenase (glyoxylate reductase) and triokinase activities were not deficient in the liver of the same patient. It is concluded that D-glycerate kinase deficiency is a cause of D-glyceric aciduria.

Phosphomannomutase deficiency is a cause of carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein (CDG) syndromes are genetic multisystemic disorders characterized by defective N-glycosylation of serum and cellular proteins. The activity of phosphomannomutase was markedly deficient (< or = 10% of the control activity) in fibroblasts, liver and/or leucocytes of 6 patients with CDG syndrome type I. Other enzymes involved in the conversion of glucose to mannose 1-phosphate, as well as phosphoglucomutase, had normal activities. Phosphomannomutase activity was normal in fibroblasts of 2 patients with CDG syndrome type II. Since this enzyme provides the mannose 1-phosphate required for the initial steps of protein glycosylation, it is concluded that phosphomannomutase deficiency, which is first reported here for higher organisms, is a cause, and most likely the major one, of CDG syndrome type I.