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Reversible infantile hypoglycorrhachia: possible transient disturbance in glucose transport?
Reversible Infantile Hypoglycorrhachia: Possible Transient Disturbance in Glucose Transport? Jo¨rg Klepper, MD*, Darryl C. De Vivo, MD†, David W. Webb, MD‡, Lars Klinge, MD*, and Thomas Voit, MD* Facilitated glucose transporter isoform 1 deficiency syndrome (GLUT1 DS), caused by impaired GLUT1mediated glucose transport into the brain, is characterized by hypoglycorrhachia. The defect in the facilitative glucose transporter isoform 1 (GLUT1) can be confirmed by functional, quantitative, and molecular analyses. Diagnostic difficulties arise when these analyses are normal and hypoglycorrhachia remains unexplained. Three infants presenting with seizures and hypoglycorrhachia at 2, 4, and 6 weeks of age, which suggests GLUT1 deficiency syndrome, are reported. The seizures responded to a ketogenic diet in Patients 1 and 3 and phenobarbitone in Patient 2. Repeated GLUT1 analyses were normal. When treatment was discontinued, all patients remained seizure-free and developed normally. Subsequent lumbar punctures showed the return to normoglycorrhachia. We conclude that these cases might represent a transient disturbance in GLUT1mediated glucose transport. The biomolecular basis for this clinical observation remains unknown. Though no treatment is required, clinical follow-up and repeated lumbar punctures are necessary to distinguish this benign condition from the original GLUT1 deficiency syndrome. © 2003 by Elsevier Inc. All rights reserved. Klepper J, De Vivo DC, Webb DW, Klinge L, Voit T. Reversible infantile hypoglycorrhachia: Possible transient disturbance in glucose transport? Pediatr Neurol 2003;29: 321-325.
From the *Department of Pediatric Neurology, University of Essen, Essen, Germany; †Department of Pediatric Neurology, Columbia University College of Physicians and Surgeons, New York, New York; ‡Department of Neurology, Our Lady’s Hospital for Sick Children, Crumlin, Dublin, Ireland.
© 2003 by Elsevier Inc. All rights reserved. doi:10.1016/S0887-8994(03)00268-6 ● 0887-8994/03/$—see front matter
Introduction Under normal metabolic circumstances, the human brain relies entirely on glucose for energy metabolism. Glucose transport across the blood-brain barrier and into brain cells is mediated by the facilitative glucose transporter GLUT1 [1]. A defect of this transporter results in impaired energy supply to the brain, a condition called GLUT1 deficiency syndrome (GLUT1 DS) [2]. The biochemical hallmark of the disease is hypoglycorrhachia, which is an absolute cerebrospinal fluid glucose value of ⬍40 mg/dL and is also expressed as a reduced ratio of cerebrospinal fluid/blood glucose concentrations (⫾ 0.6 in controls and ⱕ0.33 in patients) [3]. Most patients present with infantile seizures that do not respond to anticonvulsant medication and are often related to fasting. Additional features include developmental delay of various degrees, muscular hypotonia, secondary microcephaly, and a complex motor disorder characterized by ataxia, dystonia, and spasticity. This defect can be confirmed in erythrocytes. Glucose uptake studies and western blot have identified both quantitative and functional GLUT1 defects. Several heterozygous private mutations and autosomal-dominant missense mutations in the GLUT1 gene (1p35-31.3) have been detected [4-7]. An effective treatment is available by means of a ketogenic diet, because ketones enter the brain by a separate facilitative transporter and serve as an alternative fuel for brain metabolism (for review, see [8,9]). Confirmation of GLUT1 deficiency as the cause of hypoglycorrhachia is particularly important in infants, because the clinical features of GLUT1 DS are nonspecific and difficult to evaluate in this age group. In this report, three infants with seizures and hypoglycorrhachia and
Communications should be addressed to: Dr. Klepper; University of Essen, Department of Pediatric Neurology; Hufelandstrae 55; D-45122 Essen; Germany. Received January 9, 2003; accepted April 22, 2003.
Klepper et al: Reversible Hypoglycorrhachia 321
Figure 1. Time course of lumbar punctures, treatment, and follow-up in Patients 1 to 3. Within a period of 3 years (i.e., 156 weeks), the time of follow-up is colored in gray. Each tick label on the x-axis represents 4 weeks. Treatment with a ketogenic diet (KD, black bar) or phenobarbital (PB, white bar) is indicated. Serial lumbar punctures (#) are indicated by arrows (2). Glucose uptake analyses are indicated as vertical, black bars.
without further evidence for a GLUT1 defect are described. Hypoglycorrhachia could not be explained by hypoglycemia or inflammation, and it resolved spontaneously. With the exception of mild hypotonia in Patient 2, all patients continued to develop normally, which suggests a transient disturbance in glucose transport. Patients and Methods Patients 1, 2, and 3 were born to healthy, nonconsanguineous white parents after an uneventful pregnancy and term delivery. Birth weight, length, and head circumference were normal, and no dysmorphic features were present. In each case, the presenting symptoms at 2 to 6 weeks of age were seizures. Patient 1 (female) presented at the age of 4 weeks with a high-pitched scream and sudden stiffening during sleep followed by unresponsiveness, apnea, and cyanosis that lasted 5 to 10 minutes. She recovered spontaneously and was admitted to the local hospital. Two similar episodes occurred in the hospital at 5 and 7 weeks of age. Patient 2 (female) showed three episodes at 6 weeks of age with a clenching of the left fist, opsoclonus, and jerking of the left arm and leg that lasted approximately 5 minutes. A seizure lasting 15 seconds was witnessed with facial grimace and left arm clonic jerking. Postictally, there was some asymmetry of movement with decreased movement of the left arm. This was associated with a brisk left knee jerk and several beats of clonus at the left ankle. Patient 3 (male) developed two generalized, tonic-clonic seizures at 15 days of age. These seizures were associated with unresponsiveness and peculiar eye movements. They lasted approximately 2 minutes and resolved spontaneously. When admitted to the hospital, the general pediatric and neurologic examination was normal except for muscular hypotonia in Patients 1 and 2. No dysmorphic features were observed. Anthropometric measurements and development were appropriate for age. Routine laboratory analyses, as well as infectious and metabolic evaluations, were normal. Repeated electroencephalogram tracings in Patients 1 and 3 showed no epileptic discharges; in Patient 2, spike and slow wave discharges over the right frontal area and an excess of right temporal sharp transients were observed postictally, but they resolved in subsequent tracings. In all patients, electroencephalogram background activity was age appropriate, and ultrasounds and magnetic resonance imaging of the brain were normal. Fasting lumbar punctures to confirm hypoglycorrhachia were performed: 1. 2.
322
after more than 4 hours of fasting to achieve a metabolic “steady state” with blood glucose determination before the lumbar puncture to avoid stress-related hyperglycemia
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with the cerebrospinal fluid analyzed for cells, glucose, protein, and lactate concentrations.
GLUT1 Analyses 3-O-methyl-D-glucose uptake was determined in erythrocytes collected in citrate-dextrose-phosphate anticoagulant solution and shipped on wet ice within 24 hours of drawing blood, as previously described, to avoid the influence of old erythrocytes or lysates. GLUT1 immunoreactivity in erythrocyte membranes and molecular screening of the GLUT1 gene were performed as described previously [10,11].
Results The initial postictal and fasting lumbar punctures showed hypoglycorrhachia in the setting of normoglycemia and normal cerebrospinal fluid lactate suggestive of GLUT1 DS. After treatment with the ketogenic diet (Patient 1 and 3) or phenobarbital (Patient 2), subsequent lumbar punctures documented normoglycorrhachia and normal cerebrospinal fluid parameters (Fig 1; Table 1). Patient 1. The initial lumbar puncture (#1) was bloody and noninformative. In the postictal state (#2), a normal cell count and cerebrospinal fluid protein, as well as hypoglycorrhachia in the setting of normoglycemia and normal cerebrospinal fluid lactate concentrations was determined and later confirmed at 6 weeks after a 6-hour fast (#3). After adhering to the ketogenic diet for 12 weeks, development was normal, and no clinical signs of GLUT1 DS were observed. Normoglycorrhachia was observed on the ketogenic diet (#4), shortly after discontinuing the ketogenic diet (#5), and at the age of 29 weeks (#6). Patient 2. Patient 2 received five lumbar punctures. The initial postictal analysis showed hypoglycorrhachia associated with an elevated cerebrospinal fluid protein and cell count (#1). Cerebrospinal fluid protein concentrations returned to normal, but mild pleocytosis persisted in two subsequent lumbar punctures performed under fasting conditions (#2 and #3). Normal cerebrospinal fluid parameters and hypoglycorrhachia were confirmed 7 weeks later (#4). Phenobarbitone was discontinued at 16 weeks of age. The patient remained seizure-free, age-appropriate
Table 1.
Results and conditions of the lumbar punctures performed on Patients 1 to 3
Lumbar Puncture
#1
Age of patient (weeks) Conditions
#2
Patient 1 #3 #4
#5
4 5 6 16 19 bloody postictal fasting fasting, fasting, off on off diet diet diet CSF cells/L ND 5 10 1 1 CSF protein (mg/dL) ND 45 ND 34.5 39 CSF glucose (mg/dL) ND 33 31 41 47 Blood glucose (mg/dL) ND 86 83 55 84 Ratio CSF/ ND 0.38 0.37 0.75 0.56 blood glucose CSF lactate (mmol/L) ND 0.9 ND 1.1 Blood 3OHB (mmol/L) 2.4 ⬍0.1
#6
#1
#2
Patient 2 #3
#4
#5
#1
Patient 3 #2 #3
#4
29 6 8 8 15 82 2 2 110 122 fasting postictal fasting fasting fasting fasting postictal fasting fasting, fasting, bloody on off diet diet 1 52 14 13 2 0 6 6 0 4 26 62 20 36 29 6 70 60 22.8 19.6 45 21 20 23 36 48 23 28 50 57 90 100 ND 105 86 84 70 83 71 70 0.50 0.21 ND 0.22 0.42 0.57 0.33 0.34 0.70 0.81 1.5 ⬍0.1
development continued, and normoglycorrhachia was confirmed under fasting conditions at 82 weeks of age (#5). Patient 3. On admission, a postictal (#1) and a fasting (#2) lumbar puncture at 2 weeks of age showed hypoglycorrhachia. After adhering to a ketogenic diet for 2 years, normal cerebrospinal fluid parameters and normoglycorrhachia under fasting conditions (#3) were determined and reconfirmed 12 weeks after discontinuing the ketogenic diet (#4). GLUT1 Analyses GLUT1 analyses were performed in Patient 1 at both 6 and 16 weeks of age, in Patient 2 at 67 weeks of age, and in Patient 3 at 4 weeks of age. Functional glucose uptake studies into erythrocytes were normal, as were quantitative assessments of GLUT1-specific immunoreactivity in erythrocyte membranes. Molecular analyses of the GLUT1 gene determined the GLUT1 wild-type in all three patients. Treatment and Follow-up Patient 2 was treated with phenobarbital (weeks 6 to 16 of age). Patients 1 and 3 did not receive anticonvulsant medication. Electroencephalogram-tracings showed ageappropriate activity without epileptic discharges. Patient 1. At 7 weeks of age, Patient 1 began a 3:1 long-chain triglyceride ketogenic diet. The diet was discontinued at 19 weeks, and two subsequent lumbar punctures in the absence of ketosis confirmed normoglycorrhachia. At 21 months of age, she remained seizure-free and normocephalic. In addition, her neurodevelopmental performance was entirely age appropriate. Patient 2. Patient 2 was initially treated with phenobarbitone. She had no further seizures, and she was discharged after 10 days of acyclovir therapy. She was feeding well, fixing, following, and smiling. Her motor asymmetries had resolved. On readmission to the hospital at 14 weeks of age, she continued to feed well and gain weight appropriately. No further seizures occurred, and
1.8
1.7
0.7
1.8 3.3
1.5 ⬍0.1
she was successfully weaned off phenobarbital. At 52 (i.e., 1 year) and 80 weeks (i.e., 1.5 years) of age, she remained well and showed normal development. Patient 3. Initially, Patient 3 was introduced to a 2:1 medium-chain ketogenic diet at 4 weeks of age. Adequate ketosis (3-hydroxy-butyrate [3OHB] in blood ⬎2 mmol/L) was not achieved. She was changed to a longchain triglyceride diet, and the ratio was adjusted to 3:1 as described in detail elsewhere [12]. When ketotic, the patient remained seizure-free, mildly hypotonic, and grew along his individual percentiles. Moderate developmental delay and hypotonia were evident when the ketogenic diet was discontinued at 110 weeks of age (i.e., 2.2 years). He has remained seizure-free, and at 136 weeks of age (i.e., 2.5 years), his muscle tone and development were almost normal. Discussion Infantile seizures are common and unspecific. The combination of infantile seizures with unexplained lowglucose concentrations in the cerebrospinal fluid (hypoglycorrhachia) indicates a defect in glucose transport into brain termed GLUT1 deficiency syndrome (GLUT1 DS). Early diagnosis is important because a ketogenic diet effectively controls seizures and supports the neurologic outcome [9]. Normoglycemia and otherwise normal cerebrospinal fluid parameters distinguish hypoglycorrhachia in GLUT1 DS from secondary causes [13]. A postictal setting and “bloody” lumbar punctures compromise the diagnostic value of the investigation. In Patient 1, lumbar puncture #1 was contaminated with blood and thus uninformative. The glucose-lowering effect of subarachnoidal hemorrhage can be lingering, but lumbar punctures #2 and #3 clearly showed hypoglycorrhachia. In Patient 2, lumbar punctures #1 to #3 showed cerebrospinal fluid pleocytosis and subarachnoidal hemorrhage that possibly lowered the cerebrospinal fluid glucose concentration. No bacterial or viral agents were identified, but meningitis must be considered as an alternative hypothesis to explain this
Klepper et al: Reversible Hypoglycorrhachia 323
patient’s hypoglycorrhachia. In all patients, one of the initial lumbar punctures was performed in the postictal state, but hypoglycorrhachia in the absence of seizures was confirmed by subsequent lumbar punctures within 10 days. The remaining lumbar punctures in Patients 1 and 2 were determined in a controlled fashion, as previously described, to avoid these diagnostic problems, as shown in Patient 3. When a fasting lumbar puncture confirmed hypoglycorrhachia, the preliminary diagnosis of GLUT1 DS was made, and treatment began. Patients 1 and 3 instantly became seizure-free on the ketogenic diet, which is the treatment of choice for GLUT1 DS. Patient 2 initially responded to phenobarbital, which was surprising since this anticonvulsant has been identified as a potential inhibitor of GLUT1 function in vitro and may explain hypoglycorrhachia in lumbar punctures #1 to 4 [14,15]. The diagnosis of GLUT1 DS based on hypoglycorrhachia must be supported by GLUT1 specific investigations; however, in individual cases, the analyses can be normal. In more than half of the patients with GLUT1 DS, a mutation in the GLUT1-gene has been found [7,8]. Normal GLUT1 immunoreactivity in erythrocyte membranes can indicate a functional, rather than quantitative GLUT1 defect [11]; functional analyses of glucose uptake into erythrocytes may not always reflect the GLUT1 defect at the blood-brain barrier (Di Vivo and Klepper, personal observations). The combination of normal development and normal GLUT1 analyses cast doubt on the diagnosis. Because the ketogenic diet affects cerebrospinal fluid and blood glucose levels, lumbar punctures were performed on and off the ketogenic diet. The normoglycorrhachia that was observed suggested a transient and reversible GLUT1 defect. Possible causes remain speculative. In the brains of rats, the expression of GLUT1 and GLUT3 are low during the first postnatal week, which is in keeping with the low rates of cerebral glucose utilization. A rapid increase in GLUT3 to adult levels by 21 to 30 days of age corresponds to the period of neuronal maturation and synaptogenesis, whereas the gradual increase in GLUT1 reflects overall brain growth (neuronal/glial 45-kDa GLUT1) and an increase in blood-brain barrier glucose transport (microvessel 55-kDa GLUT1) [16,17]. No data are available on human postnatal GLUT1 developmental expression, but a delay in GLUT1 upregulation after similar mechanisms could result in reversible clinical symptoms and hypoglycorrhachia. Reversible impairment of GLUT1 function has been described for seizure activity [18], trauma [19,20], infectious agents [21,22], brain insults [23], Genistein [24], GTP analogs [25], antiepileptic drugs, analgesics, and antidepressants [15,26], caffeine [27], and ethyl alcohol [28,29]. With the exception of seizures and phenobarbital, as previously discussed, none of these substances were present in the patients described. Similar observations in other diseases of brain energy metabolism have been reported by De Vivo. In pyruvate carboxylase deficiency, which is a devastating and ulti-
324
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mately fatal disease of brain energy metabolism, a benign variant with normal development was identified [30]. A second case presented with reversible mitochondrial myopathy resulting from cytochrome C oxidase deficiency [31]. In both reports, clinical, metabolic, and biochemical features clearly indicated disease, but no satisfactory explanation for the uniquely benign clinical course could be provided. In summary, maintaining a ketogenic diet in GLUT1 DS throughout childhood should be reconsidered on the basis of our data. In young infants who initially present with seizures and hypoglycorrhachia yet show normal development and normal biochemical and molecular GLUT1 analyses, a discontinuation of the ketogenic diet and subsequent fasting lumbar punctures may be required to distinguish a possible transient disturbance in GLUT1mediated glucose transport into the brain. The authors thank Marcela Garcia-Alvarez, MD, for helpful discussions, Christine Fischer-Lahdo and Danute Bergmann for their dedicated work as laboratory technicians, and Anne Floercken and Elena Gertsen for 3-O-methyl-d-glucose uptake studies and molecular analyses. This work was supported by a grant provided by the Deutsche Forschungsgemeinschaft (DFG KL 1102/2-1).
References [1] Vannucci SJ, Maher F, Simpson IA. Glucose transporter proteins in brain: Delivery of glucose to neurons and glia. Glia 1997;21:2-21. [2] De Vivo DC, Trifiletti RR, Jacobson RI, Ronen GM, Behmand RA, Harik SI. Defective glucose transport across the blood-brain barrier as a cause of persistent hypoglycorrhachia, seizures, and developmental delay. N Engl J Med 1991;325:703-9. [3] De Vivo D, Garcia-Alvarez M, Ronen G, Trifiletti R. Glucose transport protein deficiency: An emerging syndrome with therapeutic implications. International Pediatrics 1995;10:51-6. [4] Brockmann K, Wang D, Korenke CG, et al. Autosomal dominant glut-1 deficiency syndrome and familial epilepsy. Ann Neurol 2001;50:476-85. [5] Klepper J, Willemsen M, Verrips A, et al. Autosomal dominant transmission of GLUT1 deficiency. Hum Mol Genet 2001;10:63-8. [6] Seidner G, Alvarez MG, Yeh JI, et al. GLUT-1 deficiency syndrome caused by haploinsufficiency of the blood-brain barrier hexose carrier. Nat Genet 1998;18:188-91. [7] Wang D, Kranz-Eble P, De Vivo DC. Mutational analysis of GLUT1 (SLC2A1) in Glut-1 deficiency syndrome. Hum Mutat 2000;16: 224-31. [8] Klepper J, Voit T. Facilitated glucose transporter protein type 1 (GLUT1) deficiency syndrome: Impaired glucose transport into brain—a review. Eur J Pediatr 2002;161:295-304. [9] Nordli DR, Jr., De Vivo DC. The ketogenic diet revisited: Back to the future. Epilepsia 1997;38:743-9. [10] Klepper J, Garcia-Alvarez M, O’Driscoll KR, et al. Erythrocyte 3-O-methyl-D-glucose uptake assay for diagnosis of glucose-transporterprotein syndrome. J Clin Lab Anal 1999;13:116-21. [11] Klepper J, Wang D, Fischbarg J, et al. Defective glucose transport across brain tissue barriers: A newly recognized neurological syndrome. Neurochem Res 1999;24:587-94. [12] Klepper J, Leiendecker B, Bredahl R, et al. Introduction of a ketogenic diet in young infants. J Inherit Metab Dis 2002;25:449-60. [13] Fishman RA. Decreased CSF glucose levels. In: Fishman RA, ed. Cerebrospinal fluid in diseases of the nervous system. Philadelphia: WB Saunders, 1992:219-21. [14] Haspel HC, Stephenson KN, Davies-Hill T, et al. Effects of
barbiturates on facilitative glucose transporters are pharmacologically specific and isoform selective. J Membr Biol 1999;169:45-53. [15] Klepper J, Fischbarg J, Vera JC, Wang D, De Vivo DC. GLUT1-deficiency: Barbiturates potentiate haploinsufficiency in vitro. Pediatr Res 1999;46:677-83. [16] Maher F, Vannucci S, Takeda J, Simpson IA. Expression of mouse-GLUT3 and human-GLUT3 glucose transporter proteins in brain. Biochem Biophys Res Commun 1992;182:703-11. [17] Maher F, Vannucci SJ, Simpson IA. Glucose transporter proteins in brain. Faseb J 1994;8:1003-11. [18] Cornford EM, Hyman S, Cornford ME, Landaw EM, DelgadoEscueta AV. Interictal seizure resections show two configurations of endothelial Glut1 glucose transporter in the human blood-brain barrier. J Cereb Blood Flow Metab 1998;18:2642. [19] Cornford EM, Hyman S, Cornford ME, Caron MJ. Glut1 glucose transporter activity in human brain injury. J Neurotrauma 1996;13:523-36. [20] Rosenstein JM, More NS. Immunocytochemical expression of the blood-brain barrier glucose transporter (GLUT-1) in neural transplants and brain wounds. J Comp Neurol 1994;350:229-40. [21] Gamelli RL, Liu H, He LK, Hofmann CA. Alterations of glucose transporter mRNA and protein levels in brain following thermal injury and sepsis in mice. Shock 1994;1:395-400. [22] Kovitz CA, Morgello S. Cerebral glucose transporter expression in HIV infection. Acta Neuropathol (Berl) 1997;94:140-5. [23] Brown GK. Glucose transporters: Structure, function and consequences of deficiency. J Inherit Metab Dis 2000;23:237-46.
[24] Vera JC, Reyes AM, Carcamo JG, et al. Genistein is a natural inhibitor of hexose and dehydroascorbic acid transport through the glucose transporter, GLUT1. J Biol Chem 1996;271:8719-24. [25] Wellner M, Mueckler MM, Keller K. GTP analogs suppress uptake but not transport of D-glucose analogs in Glut1 glucose transporter-expressing Xenopus oocytes. FEBS Lett 1993;327:95-8. [26] Pinkofsky HB, Dwyer DS, Bradley RJ. The inhibition of GLUT1 glucose transport and cytochalasin B binding activity by tricyclic antidepressants. Life Sci 2000;66:271-8. [27] Ho YY, Yang H, Klepper J, Fischbarg J, Wang D, De Vivo DC. Glucose transporter type 1 deficiency syndrome (glut1ds): Methylxanthines potentiate glut1 haploinsufficiency in vitro. Pediatr Res 2001;50: 254-60. [28] Hu IC, Singh SP, Snyder AK. Effects of ethanol on glucose transporter expression in cultured hippocampal neurons. Alcohol Clin Exp Res 1995;19:1398-402. [29] Krauss SW, Diamond I, Gordon AS. Selective inhibition by ethanol of the type 1 facilitative glucose transporter (GLUT1). Mol Pharmacol 1994;45:1281-6. [30] Van Coster RN, Fernhoff PM, De Vivo DC. Pyruvate carboxylase deficiency: A benign variant with normal development. Pediatr Res 1991;30:1-4. [31] Di Mauro S, Nicholson JF, Hays AP, et al. Benign infantile mitochondrial myopathy due to reversible cytochrome c oxidase deficiency. Ann Neurol 1983;14:225-34.
Klepper et al: Reversible Hypoglycorrhachia 325
From the *Department of Pediatric Neurology, University of Essen, Essen, Germany; †Department of Pediatric Neurology, Columbia University College of Physicians and Surgeons, New York, New York; ‡Department of Neurology, Our Lady’s Hospital for Sick Children, Crumlin, Dublin, Ireland.
© 2003 by Elsevier Inc. All rights reserved. doi:10.1016/S0887-8994(03)00268-6 ● 0887-8994/03/$—see front matter
Introduction Under normal metabolic circumstances, the human brain relies entirely on glucose for energy metabolism. Glucose transport across the blood-brain barrier and into brain cells is mediated by the facilitative glucose transporter GLUT1 [1]. A defect of this transporter results in impaired energy supply to the brain, a condition called GLUT1 deficiency syndrome (GLUT1 DS) [2]. The biochemical hallmark of the disease is hypoglycorrhachia, which is an absolute cerebrospinal fluid glucose value of ⬍40 mg/dL and is also expressed as a reduced ratio of cerebrospinal fluid/blood glucose concentrations (⫾ 0.6 in controls and ⱕ0.33 in patients) [3]. Most patients present with infantile seizures that do not respond to anticonvulsant medication and are often related to fasting. Additional features include developmental delay of various degrees, muscular hypotonia, secondary microcephaly, and a complex motor disorder characterized by ataxia, dystonia, and spasticity. This defect can be confirmed in erythrocytes. Glucose uptake studies and western blot have identified both quantitative and functional GLUT1 defects. Several heterozygous private mutations and autosomal-dominant missense mutations in the GLUT1 gene (1p35-31.3) have been detected [4-7]. An effective treatment is available by means of a ketogenic diet, because ketones enter the brain by a separate facilitative transporter and serve as an alternative fuel for brain metabolism (for review, see [8,9]). Confirmation of GLUT1 deficiency as the cause of hypoglycorrhachia is particularly important in infants, because the clinical features of GLUT1 DS are nonspecific and difficult to evaluate in this age group. In this report, three infants with seizures and hypoglycorrhachia and
Communications should be addressed to: Dr. Klepper; University of Essen, Department of Pediatric Neurology; Hufelandstrae 55; D-45122 Essen; Germany. Received January 9, 2003; accepted April 22, 2003.
Klepper et al: Reversible Hypoglycorrhachia 321
Figure 1. Time course of lumbar punctures, treatment, and follow-up in Patients 1 to 3. Within a period of 3 years (i.e., 156 weeks), the time of follow-up is colored in gray. Each tick label on the x-axis represents 4 weeks. Treatment with a ketogenic diet (KD, black bar) or phenobarbital (PB, white bar) is indicated. Serial lumbar punctures (#) are indicated by arrows (2). Glucose uptake analyses are indicated as vertical, black bars.
without further evidence for a GLUT1 defect are described. Hypoglycorrhachia could not be explained by hypoglycemia or inflammation, and it resolved spontaneously. With the exception of mild hypotonia in Patient 2, all patients continued to develop normally, which suggests a transient disturbance in glucose transport. Patients and Methods Patients 1, 2, and 3 were born to healthy, nonconsanguineous white parents after an uneventful pregnancy and term delivery. Birth weight, length, and head circumference were normal, and no dysmorphic features were present. In each case, the presenting symptoms at 2 to 6 weeks of age were seizures. Patient 1 (female) presented at the age of 4 weeks with a high-pitched scream and sudden stiffening during sleep followed by unresponsiveness, apnea, and cyanosis that lasted 5 to 10 minutes. She recovered spontaneously and was admitted to the local hospital. Two similar episodes occurred in the hospital at 5 and 7 weeks of age. Patient 2 (female) showed three episodes at 6 weeks of age with a clenching of the left fist, opsoclonus, and jerking of the left arm and leg that lasted approximately 5 minutes. A seizure lasting 15 seconds was witnessed with facial grimace and left arm clonic jerking. Postictally, there was some asymmetry of movement with decreased movement of the left arm. This was associated with a brisk left knee jerk and several beats of clonus at the left ankle. Patient 3 (male) developed two generalized, tonic-clonic seizures at 15 days of age. These seizures were associated with unresponsiveness and peculiar eye movements. They lasted approximately 2 minutes and resolved spontaneously. When admitted to the hospital, the general pediatric and neurologic examination was normal except for muscular hypotonia in Patients 1 and 2. No dysmorphic features were observed. Anthropometric measurements and development were appropriate for age. Routine laboratory analyses, as well as infectious and metabolic evaluations, were normal. Repeated electroencephalogram tracings in Patients 1 and 3 showed no epileptic discharges; in Patient 2, spike and slow wave discharges over the right frontal area and an excess of right temporal sharp transients were observed postictally, but they resolved in subsequent tracings. In all patients, electroencephalogram background activity was age appropriate, and ultrasounds and magnetic resonance imaging of the brain were normal. Fasting lumbar punctures to confirm hypoglycorrhachia were performed: 1. 2.
322
after more than 4 hours of fasting to achieve a metabolic “steady state” with blood glucose determination before the lumbar puncture to avoid stress-related hyperglycemia
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3.
with the cerebrospinal fluid analyzed for cells, glucose, protein, and lactate concentrations.
GLUT1 Analyses 3-O-methyl-D-glucose uptake was determined in erythrocytes collected in citrate-dextrose-phosphate anticoagulant solution and shipped on wet ice within 24 hours of drawing blood, as previously described, to avoid the influence of old erythrocytes or lysates. GLUT1 immunoreactivity in erythrocyte membranes and molecular screening of the GLUT1 gene were performed as described previously [10,11].
Results The initial postictal and fasting lumbar punctures showed hypoglycorrhachia in the setting of normoglycemia and normal cerebrospinal fluid lactate suggestive of GLUT1 DS. After treatment with the ketogenic diet (Patient 1 and 3) or phenobarbital (Patient 2), subsequent lumbar punctures documented normoglycorrhachia and normal cerebrospinal fluid parameters (Fig 1; Table 1). Patient 1. The initial lumbar puncture (#1) was bloody and noninformative. In the postictal state (#2), a normal cell count and cerebrospinal fluid protein, as well as hypoglycorrhachia in the setting of normoglycemia and normal cerebrospinal fluid lactate concentrations was determined and later confirmed at 6 weeks after a 6-hour fast (#3). After adhering to the ketogenic diet for 12 weeks, development was normal, and no clinical signs of GLUT1 DS were observed. Normoglycorrhachia was observed on the ketogenic diet (#4), shortly after discontinuing the ketogenic diet (#5), and at the age of 29 weeks (#6). Patient 2. Patient 2 received five lumbar punctures. The initial postictal analysis showed hypoglycorrhachia associated with an elevated cerebrospinal fluid protein and cell count (#1). Cerebrospinal fluid protein concentrations returned to normal, but mild pleocytosis persisted in two subsequent lumbar punctures performed under fasting conditions (#2 and #3). Normal cerebrospinal fluid parameters and hypoglycorrhachia were confirmed 7 weeks later (#4). Phenobarbitone was discontinued at 16 weeks of age. The patient remained seizure-free, age-appropriate
Table 1.
Results and conditions of the lumbar punctures performed on Patients 1 to 3
Lumbar Puncture
#1
Age of patient (weeks) Conditions
#2
Patient 1 #3 #4
#5
4 5 6 16 19 bloody postictal fasting fasting, fasting, off on off diet diet diet CSF cells/L ND 5 10 1 1 CSF protein (mg/dL) ND 45 ND 34.5 39 CSF glucose (mg/dL) ND 33 31 41 47 Blood glucose (mg/dL) ND 86 83 55 84 Ratio CSF/ ND 0.38 0.37 0.75 0.56 blood glucose CSF lactate (mmol/L) ND 0.9 ND 1.1 Blood 3OHB (mmol/L) 2.4 ⬍0.1
#6
#1
#2
Patient 2 #3
#4
#5
#1
Patient 3 #2 #3
#4
29 6 8 8 15 82 2 2 110 122 fasting postictal fasting fasting fasting fasting postictal fasting fasting, fasting, bloody on off diet diet 1 52 14 13 2 0 6 6 0 4 26 62 20 36 29 6 70 60 22.8 19.6 45 21 20 23 36 48 23 28 50 57 90 100 ND 105 86 84 70 83 71 70 0.50 0.21 ND 0.22 0.42 0.57 0.33 0.34 0.70 0.81 1.5 ⬍0.1
development continued, and normoglycorrhachia was confirmed under fasting conditions at 82 weeks of age (#5). Patient 3. On admission, a postictal (#1) and a fasting (#2) lumbar puncture at 2 weeks of age showed hypoglycorrhachia. After adhering to a ketogenic diet for 2 years, normal cerebrospinal fluid parameters and normoglycorrhachia under fasting conditions (#3) were determined and reconfirmed 12 weeks after discontinuing the ketogenic diet (#4). GLUT1 Analyses GLUT1 analyses were performed in Patient 1 at both 6 and 16 weeks of age, in Patient 2 at 67 weeks of age, and in Patient 3 at 4 weeks of age. Functional glucose uptake studies into erythrocytes were normal, as were quantitative assessments of GLUT1-specific immunoreactivity in erythrocyte membranes. Molecular analyses of the GLUT1 gene determined the GLUT1 wild-type in all three patients. Treatment and Follow-up Patient 2 was treated with phenobarbital (weeks 6 to 16 of age). Patients 1 and 3 did not receive anticonvulsant medication. Electroencephalogram-tracings showed ageappropriate activity without epileptic discharges. Patient 1. At 7 weeks of age, Patient 1 began a 3:1 long-chain triglyceride ketogenic diet. The diet was discontinued at 19 weeks, and two subsequent lumbar punctures in the absence of ketosis confirmed normoglycorrhachia. At 21 months of age, she remained seizure-free and normocephalic. In addition, her neurodevelopmental performance was entirely age appropriate. Patient 2. Patient 2 was initially treated with phenobarbitone. She had no further seizures, and she was discharged after 10 days of acyclovir therapy. She was feeding well, fixing, following, and smiling. Her motor asymmetries had resolved. On readmission to the hospital at 14 weeks of age, she continued to feed well and gain weight appropriately. No further seizures occurred, and
1.8
1.7
0.7
1.8 3.3
1.5 ⬍0.1
she was successfully weaned off phenobarbital. At 52 (i.e., 1 year) and 80 weeks (i.e., 1.5 years) of age, she remained well and showed normal development. Patient 3. Initially, Patient 3 was introduced to a 2:1 medium-chain ketogenic diet at 4 weeks of age. Adequate ketosis (3-hydroxy-butyrate [3OHB] in blood ⬎2 mmol/L) was not achieved. She was changed to a longchain triglyceride diet, and the ratio was adjusted to 3:1 as described in detail elsewhere [12]. When ketotic, the patient remained seizure-free, mildly hypotonic, and grew along his individual percentiles. Moderate developmental delay and hypotonia were evident when the ketogenic diet was discontinued at 110 weeks of age (i.e., 2.2 years). He has remained seizure-free, and at 136 weeks of age (i.e., 2.5 years), his muscle tone and development were almost normal. Discussion Infantile seizures are common and unspecific. The combination of infantile seizures with unexplained lowglucose concentrations in the cerebrospinal fluid (hypoglycorrhachia) indicates a defect in glucose transport into brain termed GLUT1 deficiency syndrome (GLUT1 DS). Early diagnosis is important because a ketogenic diet effectively controls seizures and supports the neurologic outcome [9]. Normoglycemia and otherwise normal cerebrospinal fluid parameters distinguish hypoglycorrhachia in GLUT1 DS from secondary causes [13]. A postictal setting and “bloody” lumbar punctures compromise the diagnostic value of the investigation. In Patient 1, lumbar puncture #1 was contaminated with blood and thus uninformative. The glucose-lowering effect of subarachnoidal hemorrhage can be lingering, but lumbar punctures #2 and #3 clearly showed hypoglycorrhachia. In Patient 2, lumbar punctures #1 to #3 showed cerebrospinal fluid pleocytosis and subarachnoidal hemorrhage that possibly lowered the cerebrospinal fluid glucose concentration. No bacterial or viral agents were identified, but meningitis must be considered as an alternative hypothesis to explain this
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patient’s hypoglycorrhachia. In all patients, one of the initial lumbar punctures was performed in the postictal state, but hypoglycorrhachia in the absence of seizures was confirmed by subsequent lumbar punctures within 10 days. The remaining lumbar punctures in Patients 1 and 2 were determined in a controlled fashion, as previously described, to avoid these diagnostic problems, as shown in Patient 3. When a fasting lumbar puncture confirmed hypoglycorrhachia, the preliminary diagnosis of GLUT1 DS was made, and treatment began. Patients 1 and 3 instantly became seizure-free on the ketogenic diet, which is the treatment of choice for GLUT1 DS. Patient 2 initially responded to phenobarbital, which was surprising since this anticonvulsant has been identified as a potential inhibitor of GLUT1 function in vitro and may explain hypoglycorrhachia in lumbar punctures #1 to 4 [14,15]. The diagnosis of GLUT1 DS based on hypoglycorrhachia must be supported by GLUT1 specific investigations; however, in individual cases, the analyses can be normal. In more than half of the patients with GLUT1 DS, a mutation in the GLUT1-gene has been found [7,8]. Normal GLUT1 immunoreactivity in erythrocyte membranes can indicate a functional, rather than quantitative GLUT1 defect [11]; functional analyses of glucose uptake into erythrocytes may not always reflect the GLUT1 defect at the blood-brain barrier (Di Vivo and Klepper, personal observations). The combination of normal development and normal GLUT1 analyses cast doubt on the diagnosis. Because the ketogenic diet affects cerebrospinal fluid and blood glucose levels, lumbar punctures were performed on and off the ketogenic diet. The normoglycorrhachia that was observed suggested a transient and reversible GLUT1 defect. Possible causes remain speculative. In the brains of rats, the expression of GLUT1 and GLUT3 are low during the first postnatal week, which is in keeping with the low rates of cerebral glucose utilization. A rapid increase in GLUT3 to adult levels by 21 to 30 days of age corresponds to the period of neuronal maturation and synaptogenesis, whereas the gradual increase in GLUT1 reflects overall brain growth (neuronal/glial 45-kDa GLUT1) and an increase in blood-brain barrier glucose transport (microvessel 55-kDa GLUT1) [16,17]. No data are available on human postnatal GLUT1 developmental expression, but a delay in GLUT1 upregulation after similar mechanisms could result in reversible clinical symptoms and hypoglycorrhachia. Reversible impairment of GLUT1 function has been described for seizure activity [18], trauma [19,20], infectious agents [21,22], brain insults [23], Genistein [24], GTP analogs [25], antiepileptic drugs, analgesics, and antidepressants [15,26], caffeine [27], and ethyl alcohol [28,29]. With the exception of seizures and phenobarbital, as previously discussed, none of these substances were present in the patients described. Similar observations in other diseases of brain energy metabolism have been reported by De Vivo. In pyruvate carboxylase deficiency, which is a devastating and ulti-
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mately fatal disease of brain energy metabolism, a benign variant with normal development was identified [30]. A second case presented with reversible mitochondrial myopathy resulting from cytochrome C oxidase deficiency [31]. In both reports, clinical, metabolic, and biochemical features clearly indicated disease, but no satisfactory explanation for the uniquely benign clinical course could be provided. In summary, maintaining a ketogenic diet in GLUT1 DS throughout childhood should be reconsidered on the basis of our data. In young infants who initially present with seizures and hypoglycorrhachia yet show normal development and normal biochemical and molecular GLUT1 analyses, a discontinuation of the ketogenic diet and subsequent fasting lumbar punctures may be required to distinguish a possible transient disturbance in GLUT1mediated glucose transport into the brain. The authors thank Marcela Garcia-Alvarez, MD, for helpful discussions, Christine Fischer-Lahdo and Danute Bergmann for their dedicated work as laboratory technicians, and Anne Floercken and Elena Gertsen for 3-O-methyl-d-glucose uptake studies and molecular analyses. This work was supported by a grant provided by the Deutsche Forschungsgemeinschaft (DFG KL 1102/2-1).
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