GLUT-1 deficiency syndrome caused by haploinsufficiency of the blood-brain barrier hexose carrier.

The high metabolic requirements of the mammalian central nervous system require specialized structures for the facilitated transport of nutrients across the blood-brain barrier. Stereospecific high-capacity carriers, including those that recognize glucose, are key components of this barrier, which also protects the brain against noxious substances. Facilitated glucose transport in vertebrates is catalyzed by a family of carriers consisting of at least five functional isoforms with distinct tissue distributions, subcellular localizations and transport kinetics. Several of these transporters are expressed in the mammalian brain. GLUT-1, whose sequence was originally deduced from cDNAs cloned from human hepatoma and rat brain, is present at high levels in primate erythrocytes and brain endothelial cells. GLUT1 has been cloned and positionally mapped to the short arm of chromosome 1 (1p35-p31.3; refs 6-8). Despite substantial metabolic requirements of the central nervous system, no genetic disease caused by dysfunctional blood-brain barrier transport has been identified. Several years ago, we described two patients with infantile seizures, delayed development and acquired microcephaly who have normal circulating blood glucose, low-to-normal cerebrospinal fluid (CSF) lactate, but persistent hypoglycorrachia (low CSF glucose) and diminished transport of hexose into isolated red blood cells (RBC). These symptoms suggested the existence of a defect in glucose transport across the blood brain barrier. We now report two distinct classes of mutations as the molecular basis for the functional defect of glucose transport: hemizygosity of GLUT1 and nonsense mutations resulting in truncation of the GLUT-1 protein.

Autosomal recessive inheritance of GLUT1 deficiency syndrome.

GLUT1 deficiency syndrome (GLUT1DS) is understood as a monogenetic disease caused by heterozygous SLC2A1 gene mutations with autosomaldominant and sporadic transmission. We report on a six-year-old girl from an inbred Arab family with moderate global developmental delay, epilepsy, ataxia, hypotonia, and hypoglycorrhachia (CSF glucose 36 mg/dL; CSF lactate 1.09 mmol/L; CSF/blood glucose ratio 0.44). Molecular analysis of the SLC2A1 gene identified a novel homozygous c1402C>T (p. Arg468Trp) mutation in exon 10 in the index patient and her asymptomatic younger sister. The mutation was absent in 120 control alleles of healthy individuals as well as in 400 alleles of other GLUT1DS patients. Arg468 represents a highly conserved, functionally important amino acid residue in the GLUT1 carboxy-terminus essential for substrate recognition and transport. Both unaffected parents were heterozygous for the mutation. A younger brother and two family members were healthy and carried the GLUT1 wild type. A ketogenic diet effectively controlled seizures in the index patient. We conclude that GLUT1DS can be transmitted as an autosomal recessive disease and provide new insights into genetic counselling for this treatable disorder.

Functional consequences of the autosomal dominant G272A mutation in the human GLUT1 gene.

The first autosomal dominant missense mutation (G272A) reported within the human GLUT1 gene and shared by three affected family members was investigated in respect to functional consequences. Substitution of glycine-91 by site-directed mutagenesis with either aspartate or alanine resulted in a significant decrease in transport activity of GLUT1 expressed in Xenopus oocytes. Expression of mutant transporters was confirmed by immunoblot, 2-deoxy-glucose uptake and confocal laser microscopy. The data agree with 3-O-methyl-glucose uptake into patient erythrocytes and indicate that the loss of glycine rather than a hydrophilic side chain (Gly91Asp) defines the functional consequences of this mutation.

Pulse Doppler ultrasound detection, characterization and size estimation of emboli in flowing blood.

A theory describing pulse Doppler ultrasound signals due to backscattering due to emboli in flowing blood is presented. From this theory, the minimum detectable size of a formed-element embolus can be established as a function of carrier frequency and vessel size. Emboli can be sized and characterized, based on the ratio of the amplitude of the Doppler signal during embolus passage through the sample volume to background bloodflow Doppler signal when no embolus is present. This ratio is defined as the "embolus to blood ratio" (EBR). Size estimation of emboli can be done by insonating an embolus with a single frequency and measuring the EBR, only if the embolus does not exceed a certain size, and if the vessel diameter and per cent hematocrit are known. Using two different frequencies, the vessel geometry (diameter and sample volume length) and per cent hematocrit can be eliminated from calculation of embolus size. Sources of uncertainty in the EBR and their effect on embolus size estimation are discussed. Discrimination between gas and formed-element emboli is described, given a detector with sufficient dynamic range, and use of three carrier frequencies. The theory presented here is in agreement with experimental findings of other investigators.

Biochemical assessment and clinical evaluation of a bilirubin adsorbent column (BR-350) in critically ill patients with intractable jaundice.

We investigated the efficacy of an anion-exchange adsorbent column (ASAHI BR-350, DIAMED) for removal of bilirubin and bile acids in five patients with intractable jaundice of various origin. Four litres of plasma were separated by membrane plasma separation (Plasmaflow OP-05) at a rate of 22.5 ml/min. The plasma was then perfused through an anion exchange adsorbent and returned to the venous blood line of the plasma separation. In some of the patients this procedure was combined with regular hemodialysis treatment. The concentration of total bilirubin was cut by 31 to 60%; total bile acids were reduced by 20 to 74%. Three patients recovered and had a favourable outcome. Two patients died despite the bilirubin adsorption treatment. The effects of the adsorbent column on specific blood parameters, including the coagulation system, were measured. Our data suggest that bilirubin adsorption should be examined further as a treatment for critically ill patients with intractable jaundice.

Pyridoxal phosphate-dependent neonatal epileptic encephalopathy.

Pyridox(am)ine-5'-phosphate oxidase converts pyridoxine phosphate and pyridoxamine phosphate to pyridoxal phosphate, a cofactor in many metabolic reactions, including neurotransmitter synthesis. A family with a mutation in the pyridox(am)ine-5'-phosphate oxidase gene presenting with neonatal seizures unresponsive to pyridoxine and anticonvulsant treatment but responsive to pyridoxal phosphate is described. Pyridoxal phosphate should be considered in neonatal epileptic encephalopathy unresponsive to pyridoxine.

Pyridoxal phosphate-dependent neonatal epileptic encephalopathy.

Pyridox(am)ine-5'-phosphate oxidase converts pyridoxine phosphate and pyridoxamine phosphate to pyridoxal phosphate, a cofactor in many metabolic reactions, including neurotransmitter synthesis. A family with a mutation in the pyridox(am)ine-5'-phosphate oxidase gene presenting with neonatal seizures unresponsive to pyridoxine and anticonvulsant treatment but responsive to pyridoxal phosphate is described. Pyridoxal phosphate should be considered in neonatal epileptic encephalopathy unresponsive to pyridoxine.

Seizure control and acceptance of the ketogenic diet in GLUT1 deficiency syndrome: a 2- to 5-year follow-up of 15 children enrolled prospectively.

BACKGROUND: GLUT1 deficiency syndrome is caused by impaired glucose transport into the brain resulting in an epileptic encephalopathy, developmental delay, and a complex motor disorder. A ketogenic diet provides an alternative fuel to the brain and effectively restores brain energy metabolism. METHODS: Fifteen children with GLUT1 deficiency syndrome were enrolled prospectively for a 2.0 - 5.5-year follow-up of the effectiveness of a 3 : 1 LCT ketogenic diet. Eight patients enrolled were described previously, seven patients were novel. RESULTS: Four novel heterozygous GLUT1 mutations were identified. 10/15 patients remained seizure-free on the ketogenic diet in monotherapy. In 2/15 patients seizures recurred after 2(1/2) years despite adequate ketosis, but were controlled by add-on ethosuximide. In one patient seizures were reduced without complete seizure control. No serious adverse effects occurred and parental satisfaction with the diet was good. 2/15 patients discontinued the diet. CONCLUSION: GLUT1 deficiency syndrome represents a complex childhood encephalopathy that can be treated effectively by means of a ketogenic diet. The response to the diet did not correlate to clinical, biochemical, or genetic features of the disease. In contrast to previous reports, our results indicate that epilepsy is not always completely controlled by a ketogenic diet and can recur in a subset of patients.

Technical note: Rationale, development, use and evaluation of an equipment management and image storage system.

Lack of space and organization have become significant problems in the contemporary business world. Modern industries must eliminate mountains of paper and better organize themselves. With the incorporation of a fully automated equipment management system and image storage system, the Jacobi Medical Center has succeeded in correcting these problems. This technical note shows how the use of computer software and hardware components can be integrated to manage electronic patient care equipment. The adoption of this new technology was applied to equipment maintenance, tracking and record-keeping, thereby accomplishing the aims of space conservation and organization. As the components became more advanced, the technology included computer software products that could store more information and electronically effect a more rapid document retrieval. This will eventually allow for a paperless operation. The latest available equipment management software is supported by an image storage system that may contain important signature documents and outside vendor information. The applicability of these systems to the biomedical engineering field is obvious: (a) space efficiency eliminates folders and files; (b) simultaneous technician access to equipment history; and (c) rapid archive retrieval of data as well as vendor information and safety alerts.

[The ketogenic diet in German-speaking countries: update 2003]. / Die ketogene Diät in den deutschsprachigen Ländern im Jahr 2003: eine Standortbestimmung.

BACKGROUND: The ketogenic diet has been used for decades to treat intractable childhood epilepsies. It is also the treatment of choice for GLUT1 deficiency syndrome and pyruvate-dehydrogenase-complex-deficiency. Recent studies have once again confirmed the efficiacy of the diet, but the diet is hardly known in Europe and has never been quite accepted as an effective treatment of childhood epilepsy. PATIENTS: We report retrospective data on 146 children treated with the ketogenic diet in Austria, Switzerland, and Germany. METHOD: In 2000 and 2002, standardized questionaires were sent to 13 neuropediatric departments to evaluate indications, effects and side effects. RESULTS: In children with refractory epilepsy (n = 111), 8 % became seizure-free on the diet. Seizure reduction of > 90 % was achieved in additional 9 % of patients, a seizure reduction of 50-90 % in additional 14 % of patients. There was a great variability between epilepsy departments. All patients with GLUT1 deficiency syndrome (n = 18) and pyruvate-dehydrogenase-complex-deficiency (n = 15) showed clinical improvement. In GLUT1 deficiency syndrome, complete seizure control was achieved in 94 % of patients. Compliance was good in 82 % of all patients regardless of the indication for the diet. CONCLUSION: In contrast to the general restraint towards the ketogenic diet in Europe, our data supports its effectiveness as the treatment of choice for GLUT1-deficiency syndrome und pyruvate-dehydrogenase-complex-deficiency. In children with refractory epilepsy, the ketogenic diet matched the effect of most anticonvulsants and was well tolerated. These data and two workshops resulted in recommendations for the use of the ketogenic diet in children as a basis for a general diagnostic and therapeutic standards to compare and improve the use of the ketogenic diet in Europe.

Deficient transport of dehydroascorbic acid in the glucose transporter protein syndrome.

The glucose transporter protein syndrome (GTPS) is caused by defective transport of glucose across the blood-brain barrier via the glucose transporter GLUT1, resulting in hypoglycorrhachia, infantile seizures, and developmental delay. Recent reports indicated that GLUT1 is a multifunctional transporter. We investigated the transport of vitamin C in its oxidized form (dehydroascorbic acid) via GLUT1 into erythrocytes of 2 patients with GTPS. In both patients, uptake of oxidized vitamin C was 61% of the mothers' values. Our findings are consistent with recent observations that vitamin C is transported in its oxidized form via GLUT1. We speculate that impaired transport of this substrate and perhaps other substrates in GTPS might contribute to the pathophysiology of this condition.

Epilepsy in shunt-treated hydrocephalus.

Although epilepsy is commonly associated with shunt-treated hydrocephalus, its relation to the shunting procedure and the criteria identifying postoperative epilepsy remain controversial. Of 283 patients shunted at Würzburg University Hospital over a 24-year period (1970 to 1994), 182 were followed up for a minimum of 1 year after shunt insertion and entered the study. The data were analyzed retrospectively in 1995 and 1996. Epilepsy was analyzed in relation to the etiology of hydrocephalus, functional status, time and site of shunt insertion, onset of seizures and seizure type, EEG changes, sex, shunt systems, and shunt revisions. Of the 182 patients studied, 37 (20%) developed epilepsy. The incidence of epilepsy varied according to the etiology of hydrocephalus: posthemorrhagic (5%), postinfectious (4%), connatal/miscellaneous/unknown (3%), myelomeningocele (2%), tumor/arachnoidal cyst/aqueduct stenosis (0%). Early shunting and poor functional status was associated with a higher risk for epilepsy. Epilepsy was not influenced by sex, shunt systems, or number of shunt revisions. Twenty-two (12%) of 182 patients developed epilepsy (generalized N=13, focal N=9) after intracranial shunting. Focal EEG abnormalities (N=16) were located mainly at the anatomical site of the shunt (N=14), but only three patients (2%) presented with focal seizures contralateral and focal EEG abnormalities ipsilateral to the site of the shunt. The presence of epilepsy was determined by the etiology of hydrocephalus rather than by surgical intervention. The incidence of postoperative epilepsy (12%) was low. Onset of epilepsy, clinical presentation of seizures, and EEG changes did not appear to be valid criteria for identifying shunt-related epilepsy. Thus, epilepsy as a complication of intracranial shunting might be overestimated in the literature.

Neuroglycopenia in normoglycaemic patients, and the potential benefit of ketosis.

We report a patient with recurrent symptoms of neuroglycopenia due to a defective glucose transport into brain. The potential benefit of ketosis in neuroglycopenia is discussed from the therapeutic concept of a ketogenic diet in GLUT1-deficiency syndrome.

GLUT1-deficiency: barbiturates potentiate haploinsufficiency in vitro.

Barbiturates are known to inhibit glucose transport mediated by the facilitative sugar transporter GLUTI. We have studied such inhibition in children with GLUT1-deficiency. Zero-trans influx of 14C-labeled 3-O-methyl glucose (3OMG) into erythrocytes of patients (n = 3) was 35% of controls (n = 6). Preincubation with 10 mM phenobarbital or pentobarbital reduced patients' 30MG influx to 17%. In patients and controls, preincubation with barbiturates significantly decreased Vmax in a dose-dependent manner (for pentobarbital, IC50 = 0.84 mM, patient 2). The apparent Km in individuals remained largely unchanged. Three-OMG influx without preincubation resulted in a stronger inhibition at lower barbiturate concentrations. The patients' data are discussed in the light of individual missense mutations (patient 1: R126L and K256V; patient 2: T310I; patient 3: S66F) in the GLUTI gene. In conclusion, in controls and patients with GLUT1-deficiency barbiturates interact with GLUT1, lowering its intrinsic activity. The use of barbiturates in this condition for anesthesia or as anticonvulsants could therefore potentially aggravate the existing glucose transport defect and may put these patients at increased risk.

Introduction of a ketogenic diet in young infants.

The ketogenic diet is a rational treatment for pyruvate dehydrogenase complex deficiency (McKusick 312170) and GLUT1 deficiency syndrome (McKusick 138140). An increasing number of patients are diagnosed in early infancy, but few data are available on the introduction of a ketogenic diet in this age group. GLUT1 deficiency syndrome was suspected in four infants presenting with seizures and unexplained hypoglycorrhachia. A ketogenic diet was introduced at 6-28 weeks of age. Ketosis was initiated by fasting, monitored by bedside blood glucose and 3-hydroxybutyrate determinations, and was maintained successfully using supplemented carbohydrate-free infant formula and emulgated triglycerides. All patients developed ketosis within 24 h. 3-Hydroxybutyrate concentrations available at the bedside correlated inversely with the base excess. At glucose levels < or = 40 mg/dl patients remained asymptomatic in the presence of ketones. The ketogenic formula was tolerated well, parental compliance was good, and all patients remained seizure-free on the diet. GLUT1 deficiency was confirmed in two patients; the diet was discontinued in the other two patients. In one infant, failure to thrive on medium-chain triglycerides was effectively reversed using long-chain triglycerides. Urine dipstick analyses failed to detect ketosis in another infant. Adverse effects of the diet were limited to renal stones in one patient. The ketogenic diet can be introduced and maintained successfully in young infants using long-chain fat emulsion. Monitoring 3-hydroxybutyrate at the bedside was useful for metabolic control and superior to urine dipstick analysis. Seizure control was effective and adverse effects were limited, but evaluation of the long-term effects of the ketogenic diet in this age group must await ongoing studies.

Autosomal dominant transmission of GLUT1 deficiency.

GLUT1 deficiency is caused by a defect in the facilitative glucose transporter GLUT1. Impaired glucose transport across brain tissue barriers is reflected by hypoglycorrhachia and results in an epileptic encephalopathy with developmental delay and motor disorders. Recently heterozygous mutations in the GLUT1 gene (1p35-31.3) have been reported in sporadic patients. Parents and siblings carried the GLUT1 wild-type, suggesting a de novo, autosomal dominant condition resulting from GLUT1 haploinsufficiency. We report a father and two children from separate marriages affected by GLUT1 deficiency and carrying a novel heterozygous missense mutation (G272A) in the GLUT1 gene. Mutations were identified by polymerase chain reaction and DNA sequencing and confirmed by restriction fragment digest. The predicted amino acid change (Gly91Asp) affects an Arg-X-Gly-Arg-Arg motif between helices 2 and 3 that represents a cytoplasmic anchor point and is highly conserved among transporters of the major facilitator superfamily down to yeast and bacteria. GLUT1 immunoreactivity was normal, but 3-O-methyl-D-glucose uptake into erythrocytes was significantly reduced, suggesting a quantitatively normal, but functionally impaired, GLUT1 protein at the cell membrane. This is the first report of autosomal dominant transmission of GLUT1 deficiency, confirming that this condition is the result of haploinsufficiency. The Gly-->Asp mutation within a highly conserved sequence highlights its importance for GLUT1 function. GLUT1 deficiency should be considered in patients with epilepsy, mental retardation and motor disorders. Our observations have bearing on the identification of this treatable disorder in pediatric and adult patients, will modify current biochemical protocols which use parental controls and will enable genetic counseling of affected families.

Glucose transporter type 1 deficiency syndrome (Glut1DS): methylxanthines potentiate GLUT1 haploinsufficiency in vitro.

Methylxanthines such as caffeine and theophylline are known to inhibit glucose transport. We have studied such inhibition in the glucose transporter type 1 deficiency syndrome (Glut1DS) by erythrocyte glucose transport assays. Data from four patients with individual mutations in the GLUT1 gene are discussed: patient 1 (hemizygosity), 3 (S66F), 15 (368Ins23), and 17 (R333W). Zero-trans influx of (14)C-labeled 3-O-methyl glucose (3-OMG) into erythrocytes of patients is reduced (patient 1, 51%; 3, 45%; 15, 31%; 17, 52%) compared with maternal controls. Inhibition studies on patients 1, 3, 17, and maternal controls show an IC(50) for caffeine of approximately 1.5 mM both in controls (n = 3) and patients (n = 3) at 5 mM 3-OMG concentration. In the same two groups, kinetic studies show that 3 mM caffeine significantly decreases V(max) (p < 0.005), whereas the decrease in K(m) is significant (p < 0.01) only in the three controls and one patient (patient 3). Kinetic data from individual patients permit us to speculate that the interactions between caffeine and Glut1 are influenced by the mutation. Three mM caffeine also inhibits the transport of dehydroascorbic acid (DHA), another substrate for Glut1. The combined effects of caffeine (3 mM) and phenobarbital (10 mM) on glucose transport, as determined in patient 15 and the maternal control, show no additive or synergistic inhibition. These data indicate that caffeine and phenobarbital have similar Glut1 inhibitory properties in these two subjects. Our study suggests that Glut1DS patients may have a reduced safety margin for methylxanthines. Consumption of methylxanthine-containing products may aggravate the neurologic symptoms associated with the Glut1DS.

Defective glucose transport across brain tissue barriers: a newly recognized neurological syndrome.

Impaired glucose transport across brain tissue barriers causes infantile seizures, developmental delay and acquired microcephaly. Since the first report in 1991 (De Vivo et al, NEJM, 1991) 17 patients have been identified with the glucose transporter protein syndrome (GTPS). The diagnostic feature of the syndrome is an unexplained hypoglycorrhachia in the clinical setting of an infantile epileptic encephalopathy. We review our clinical experience by highlighting one illustrative case: a 6-year old girl who presented at age 2 months with infantile seizures and hypoglycorrhachia. The CSF/blood glucose ratio was 0.33. DNA sequencing identified a missense mutation in exon 7 (C1108T). Erythrocyte GLUT1 immunoreactivity was normal. The time course of 3-O-methyl-glucose (3OMG) uptake by erythrocytes of the patient was 46% that of mother and father. The apparent Km was similar in all cases (2-4 mmol/L), but the apparent Vmax in the patient was only 28% that of the parents (500 versus 1,766 fmol/s/10(6)RBC; p < 0.004). In addition, a 3-month trial of oral thioctic acid also benefited the patient and increased the Vmax to 935 fmol/s/10(6) RBC (p < 3 x 10(-7)). Uptake of dehydroascorbic acid by erythrocytes of the patient was impaired to the same degree as that of 3OMG (Vmax was 38% of that of the mother's), which supports previous observations of GLUT1 being multifunctional. These studies confirm the molecular basis of the GTPS and the multifunctional role of GLUT1. The need for more effective treatment is compelling.

Erythrocyte 3-O-methyl-D-glucose uptake assay for diagnosis of glucose-transporter-protein syndrome.

Glucose transport into the brain is mediated by a facilitative glucose-transporter protein, GLUT-1. A GLUT-1 defect results in the Glucose-Transporter-Protein Syndrome (GTPS), characterized by infantile epilepsy, developmental delay, and acquired microcephaly. The diagnosis is currently based on clinical features, low to normal lactate levels and low glucose levels (hypoglycorrhachia) in the cerebrospinal fluid, and the demonstration of impaired GLUT-1 function in erythrocytes as described here. Blood samples were collected in sodium-heparin or citrate-phosphate-dextrose solution and uptake of 14C-labeled 3-O-Methyl-D-glucose (3OMG into erythrocytes (0.5 mmol/L 3OMG; 1 microCi/mL) was measured at 4C and pH 7.4. Three-OMG influx was terminated at 5-second intervals, washed cells were lysed, and uptake was quantitated by liquid scintillation counting. Patients' uptake (n = 22) was 44 +/- 8% of controls (100 +/- 22%, n = 70). Statistical analyses showed an uptake cut-off point at 60% uptake, a sensitivity of 86% (95%-confidence interval 78 to 94%), and a specificity of 97% (95%-confidence interval 93 to 100%). Gender, age, and ketosis did not influence 3OMG uptake. This assay provides a reproducible and accurate laboratory test for diagnosing the GTPS.