An Infant with Pseudohyperkalemia, Hemolysis, andSeizures: Cation-Leaky GLUT1-Deficiency Syndromedue to a SLC2A1 Mutation
Waleed M. Bawazir, Evelien F. Gevers, Joanna F. Flatt, Ai Leen Ang,Benjamin Jacobs, Caroline Oren, Stephanie Grunewald, Mehul Dattani,Lesley J. Bruce, and Gordon W. Stewart
Bristol Institute for Transfusion Sciences (W.M.B., A.L.A., J.F.F., L.J.B.), National Health Service Blood andTransplant, Bristol BS34 7QH, United Kingdom; Great Ormond Street Hospital for Children (E.F.G., S.G.,M.D.), London WC1N 3RB, United Kingdom; Department of Paediatrics (B.J.), Royal NationalOrthopaedic Hospital, Stanmore, Middlesex HA7 4LP, United Kingdom; Department of Paediatrics(C.O.), Northwick Park Hospital, Harrow HA1 3UJ, United Kingdom; and Division of Medicine (G.W.S.),University College London, London WC1E 6JF, United Kingdom
Context: GLUT1 (glucose transporter 1) deficiency syndrome is a well-known presentation in pe-diatric practice. Very rare mutations not only disable carbohydrate transport but also cause the redcell membrane to be constitutively permeant to monovalent cations, namely sodium andpotassium.
Objective: The aim of this study was to describe the pediatric presentation of a patient with GLUT1deficiency with such a cation-leaky state.
Subject and Methods: The infant presented with erratic hyperkalemia, neonatal hyperbiliru-binemia, anemia, hepatic dysfunction, and microcephaly. Later, seizures occurred and develop-mental milestones were delayed. Magnetic resonance imaging and computerized tomographyscans of the brain showed multiple abnormalities including periventricular calcification. Visualimpairment was present due to the presence of both cataracts and retinal dysfunction.
Results: Measurements of red cell cation content showed extremely leaky red cells (causing thehemolysis) and temperature-dependent loss of potassium from red cells (explaining the hyperka-lemia as pseudohyperkalemia). A trinucleotide deletion in SLC2A1, coding for the deletion ofisoleucine 435 or 436 in GLUT1, was identified in the proband.
Conclusion: This is the fourth pedigree to be described with this most unusual syndrome. Themultisystem pathology probably reflects a combination of glucose transport deficiency at theblood-brain barrier (as in typical GLUT1 deficiency) and the deleterious osmotic effects of a cation-leaky membrane protein in the cells where GLUT1 is expressed, notably the red cell. We hope thatthis detailed description will facilitate rapid diagnosis of this disease entity. (J Clin EndocrinolMetab 97: E987–E993, 2012)
The facilitative, non-sodium-dependent glucose trans-porter known as “glucose transporter 1” (GLUT1) is
the major glucose transporter of the blood-brain barrier.GLUT1 deficiency syndrome (GLUT1DS), caused by de-
fective GLUT1 transport activity at the blood-brain bar-rier, is a well-known but possibly underdiagnosed pedi-atric disorder (1, 2) characterized by microcephaly, mentalretardation, seizures, and hypoglycorrhachia. The pa-
A d v a n c e s i n G e n e t i c s — C l i n i c a l C a s e S e m i n a r
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tients are typically heterozygous for mutations inSLC2A1, coding for GLUT1; the clinical condition is at-tributable to SLC2A1 haploinsufficiency and a gene doseeffect at the blood-brain barrier (3). GLUT1 is also ex-pressed in the membrane of the red cell; GLUT1 activity inthe red cell can be used as a diagnostic test. The protein isalso expressed in lens epithelium (4) and retina (5), but intypical GLUT1DS there is no overt clinical abnormality inthe red cell, lens, or retina.
A very small minority of patients with SLC2A1 muta-tions present with an extended phenotype showing extra-neurological pathology. In all of these cases, it has beenshown by isotopic tracer studies in the accessible red cellthat the membrane is not only defective in glucose trans-
port but also “leaky” to the monovalent cations sodiumand potassium. This cation leakiness causes osmotic in-stability in the erythrocytes and hemolysis (6, 7). Here wegive a detailed pediatric description of a child that has aSLC2A1 mutation resulting in deficient glucose transportand red cell cation leakage, leading to pseudohyperkalemia,neonatal hyperbilirubinemia, periventricular calcificationon computerized tomography (CT) brain scan, nystagmus,cataracts, and liver dysfunction. There is no direct evidence,but it is presumed that all of the pathology that is additionalto the “core” GLUT1DS presentation is attributable the cat-ion-leaky nature of the abnormal protein.
The diagnosis and management of this unusual casecaused much clinical difficulty. The case has two lessons:
TABLE 1. Typical hematology and relevant biochemistry, age 4–12 months
Synacthen testCortisol (zero time) 521 200–600 nmol/liter 18.9 7.2–21.7 �g/dlCortisol, �30 min 808 a nmol/liter 29.3 b �g/dlCortisol, �60 min 822 a nmol/liter 29.8 b �g/dl
DHEAS, Dehydroepiandrosterone sulfate; MCHC, mean cell Hg concentration; MCV, mean red cell volume; WBC, white blood count.a Increment of �170 or stimulated level of �550.b Increment of �6.2 or stimulated level of �19.6.
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first, it illustrates a novel and surprising presentation ofGLUT1DS; and second, the pathology shows how muta-tions in a facilitative glucose transport can cause abnormalsodium and potassium handling.
Case Report
Background and birthThe infant represented the first pregnancy of an Afro-
Caribbean mother and a Caucasian father. She was born at38.7 wk gestation by emergency cesarean section for fetaldistress after a pregnancy complicated by uterine fibroidsand polyhydramnios. The birth weight was 3660 g (75thcentile), and the head circumference was 32.5 cm (9th cen-tile). There was a paternal family history of adrenoleukodys-trophyintwosecondcousins.Thepaternalgrandmotherhadbeen tested and was not a carrier. At 22 h of age, the childpresentedwithsevereconjugatedhyperbilirubinemia[direct,411 �mol/liter (24.0 mg/dl); total, 488 �mol/liter (28.5 mg/dl)], abnormal liver function tests (�-glutamyl transferase,567 IU/liter), and variable hyperkalemia ranging from nor-mal up to around 10 mmol/liter. Sepsis was suspected and
treated for, but cultures were negative.Abdominal ultrasonography showedmild splenomegaly. The toxoplasmosis/rubella/cytomegalovirus/herpes simplex(TORCH) screen was negative; a liver bi-opsy showed nonspecific hepatitis. Thejaundice improved by d 10.
fluctuated from d 1 after birth and wasoften above 9 mmol/liter. Renal func-tion, acid-base status, and the electro-cardiogram were normal. Neither sal-butamol, nor insulin-glucose infusions,nor fludrocortisone were effective. Ad-renal function, plasma ACTH, plasmarenin activity, and aldosterone were allnormal (Table 1). It was noted that atleast some of the variability in mea-sured plasma potassium was related tothe elapsed time between venesectionand analysis. Pseudohyperkalemia wassuspected and was tested for (see Re-sults and Progress).
Neurological pictureIn the immediate postnatal period,
irritability and nystagmus were noted.Milestones were delayed. An electroen-
cephalogram was normal in the first few weeks of life. Abrain CT (Fig. 1, A and B) showed periventricular calci-fication, and an early magnetic resonance imaging scan(Fig. 1, C and D) showed extensive confluent T1w hyper-intensity along the lateral margins of both lateral ventri-cles, extending to the temporal poles bilaterally, whichcorrelated with low T2w signal and some punctate foci ofdiffusion-weighted imaging signal abnormality. Therewas thalamic atrophy. The basal ganglia were normal.These appearances were consistent with periventricularcalcification, consistent with the CT. There was mild gen-eralized sulcal predominance and thinning of the posteriorcorpus callosum, in keeping with global loss of white mat-ter volume. There was a normal sulcal/gyral pattern withno evidence of malformation. The cerebellum was of nor-mal volume. There was a slight delay in myelination, withabsent myelination within the posterior limb of the inter-nal capsule.
Hearing was normal. At 5.5 months, truncal and four-limb hypertonia were noted. Seizures began at approxi-mately 6 months of age; she was admitted to intensive carein status epilepticus and was paralyzed and ventilated.
FIG. 1. Cerebral imaging. A and B, Unenhanced transverse CT showing patchy calcification.C and D, T1-weighted sagittal (C) and transverse (D) magnetic resonance images. Arrowsindicate T1w hyperintensity along the lateral margins of both lateral margins (reflectingcalcification).
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Blood glucose and calcium concentrations were normal.The seizures, which often happened at night or beforefeeds, were controlled with phenytoin.
The intracranial calcification and other neurological fea-tures suggested a diagnosis of the recessive Aicardi-Gout-ieres syndrome. This recessively inherited leukodystrophicneurodegeneration is caused by mutations in one of a se-ries of immune-related genes (TREX1, RNASEH2A,RNASEH2B, RANSEH2C) (8), but no mutation wasfound in any of these.
Ocular and visualNystagmus was a consistent feature. At age 9 months,
nuclear cataracts were noted. The temporal aspects of bothdiscs were considered to be pale, but no other retinal abnor-mality was noted. At 11 months, a right convergent squintand horizontal jerk nystagmus were documented. Electro-retinography and visual-evoked potential studies were con-sistent with both retinal and postretinal dysfunction.
HemolysisAnemia was most severe in the perinatal period [min-
imum hemoglobin (Hb), 6.8 g/dl], coincident with max-imal hyperbilirubinemia. Later, the Hb was typicallynormal or low-normal. The cells were macrocytic, and
reticulocytosis was present. Theblood film showed some echinocytesand stomatocytes (Fig. 2A). Spleno-megaly was present. As will be de-scribed, the hemolysis was later at-tributed to the cation-leaky state ofthe erythrocyte membrane.
Hepatic dysfunctionHyperbilirubinemia was always pres-
ent and was attributable to hemolysis.However, the alanine transaminase and�-glutamyl transferase were always atleast slightly abnormal. At 4 wk, the liverbiopsy showed nonspecific hepatitis.
Method: DNA sequencing analysisGenomicDNAwasisolatedfromblood
samples.Thecodingregionsandsplicesitesof exons 1 to 10 of SLC2A1 from the childand exon 10 of SLC2A1 from the parentswereamplifiedbyPCRusingexon-specificprimers.TheDNAwassequencedaspre-viously described (7).
Results and Progress
In a test for pseudohyperkalemia, whole heparinized bloodof the patient and an adult control was stored at 37, 20, and0 C for up to 6 h (9). Aliquots were taken from the samplesand centrifuged at 0, 1, 2, 4, and 6 h after venipuncture, andthe [K]wasmeasured.Theresultsare showninFig.2B. In thecontrol sample, there was no major change in plasma [K] atany temperature [as isnormal (9)].Bycontrast, in the infant’ssample, theplasma[K]roseatall three temperatures.Therisewas most striking at 0 C, confirming that this blood, whenstored, shows an abnormal rise in plasma [K] due to leakageof potassium from red cells. The very rapid rise at 0 C isconsistent with the cryohydrocytosis phenotype of stomato-cytosis (9). Measurements of intracellular sodium and po-tassium revealed a gross abnormality in fresh red cells: theintracellular sodium was very high, and the potassium waslow (Table 1). These data showed that the cells were veryleaky to these cations. Intracellular sodium concentration([Na]) and [K] in the parents’ red cells were normal (data notshown). These data showed that, first, the hyperkalemia wasduetoatemperature-dependentartifact,andsecond, that theinfant had markedly abnormal red cells with a very dramaticabnormality in intracellular [Na] and [K], even in fresh cells,consistent both with the hemolysis and with a diagnosis ofcryohydrocytosis, a member of the cation-leaky hereditarystomatocytosis group (10).
FIG. 2. Blood film and pseudohyperkalemia test. A, Wright-Giemsa-stained blood film,showing largely normal red cells with a few echinocytes (e) and occasional stomatocytes (s). B,Pseudohyperkalemia: whole heparinized blood was stored at the temperatures shown.Aliquots were taken at the times indicated and spun, and the supernatant plasma wascollected. Plasma [K] was estimated by flame photometry. In the patient’s case (upper panel),the [K] was normal at zero time, then rose quickly after venesection, especially at 0 C. In thecontrol case (lower panel), there was only minimal change at any temperature, consistentwith clinical experience.
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Because we had previously shown that two adult pa-tients with neurological retardation and abnormal cation-leaky red cells (11) had mutations in SLC2A1 encoding forGLUT1 (7), we sequenced SLC2A1. The infant washeterozygous for an ATC trinucleotide deletion, codingfor isoleucine 435 or 436. The parents were normal. Thisis the same mutation that was found in a previous adultpatient labeled sdCHC(B) (7).
The corollary of this finding was that at least part of theneurological presentation should be due to deficiency ofGLUT1-mediated transport of glucose across the blood-brainbarrier.Afasting lumbarpuncturewasperformed.Thecerebrospinal fluidglucoseconcentrationwas2.0mmol/liter(36.0 mg/dl) compared with a simultaneous plasma glucoseconcentration of 3.8 mmol/liter (68.5 mg/dl), confirming thepresence of hypoglycorrhachia, consistent with deficienttransport of glucose across the blood-brain barrier.
Western blots showed that GLUT1 was expressed at nor-mal levels in the red cell membrane of the affected child,suggesting that the mutant protein is expressed and stablyinserted into the membrane (Fig. 3B). The cells showed re-duced expression of the 32-kDa protein stomatin (Fig. 3C),a “raft” protein of poorly understood function that is mis-trafficked in cation-leaky red cells (12). “Lutheran” is anIg-likeproteinof theredcellmembranethatactsasareceptorfor laminin (13). As in the previous cases (7), the expressionof Lutheran was increased in the abnormal cells (Fig. 3D).There was no abnormality in the membranes of the hema-tologically normal parents (data not shown).
Thus, an unusual form of GLUT1 deficiency was diag-nosed.The infantwasstartedonaketogenicdiet.Theseizure
frequency decreased and, in the opinionof both parents and clinicians, the infantbecame less irritable. The hyperkalemiawas understood to be red cell-based,temperature-dependent pseudohyper-kalemia. It was ensured that the elapsedtime between venipuncture and analysiswasminimized,andmeasuredpotassiumlevels were thereafter always normal.
Discussion
This case confirms the association be-tween a hematoneurological syndromeand mutations in SLC2A1 and, for thefirst time, illustrates the pediatric presen-tation in detail. As described for the vastmajority of SLC2A1 mutations, the pa-tient is heterozygous, and the mutationis de novo (1–3). As shown in the pre-vious adult cation-leaky case (7), the al-
tered conformation of the GLUT1 protein has two qual-ities: first, there is loss of glucose transport function; andsecond, there is occurrence of a nonspecific “leak” to themonovalent cations sodium and potassium, which can beseen and measured in red cells.
The dual nature of the mutant GLUT1 protein can ac-count for the complex clinical picture in this child. First,there is the picture of GLUT1DS, caused by the haploin-sufficiency of glucose transport at the blood-brain barrier(1, 3), which could account for microcephaly, develop-mental delay, and seizures and is confirmed by hypogly-corrhachia and the positive therapeutic response to theketogenic diet. Second, and not seen in the simple loss offunction GLUT1DS, there is hemolytic anemia due to acation leak, with associated pseudohyperkalemia attrib-utable to the bizarre temperature dependence of the cationleak. In addition to the hemolysis, this child demonstratesmarkedly abnormal brain imaging with periventricularcalcification, leading to a suspected diagnosis of Aicardi-Goutieres syndrome (8). There was also visual impair-ment, with probable retinal dysfunction and cataracts,and hepatic dysfunction. In our two adult cases, cataractsand hepatosplenomegaly were noted, but no brain imag-ing was available (11).
Although the hemolysis was certainly attributable tothe cation leak, cation movements are difficult to assess inthe other tissues and cell types. GLUT1 is expressed in lensepithelium (4) and retina (5). It is possible that the periven-tricular calcification, which is probably a form of dystro-phic calcification (14), is due to cellular injury at the
FIG. 3. Western blots. Red cell membranes were prepared by hypotonic lysis (18), separatedon 10% Laemmli gels, and immunoblotted using the indicated antibodies as described (7). Clanes, Control; P lanes, proband. A, Coomassie stain. The absence of a Coomassie stainingband at 32 kDa represents the deficiency of stomatin, confirmed in blot C. B, Anti-GLUT1antibody. The GLUT1 protein is heavily glycosylated and is seen as a broad 50- to 100-kDaband. In the sdCHC patient, the amount of GLUT1 was not reduced, suggesting that themutant glut1 is expressed normally. C, Anti-stomatin antibody. The protein is reduced inexpression but not entirely absent. D, Anti-Lutheran antibody. The Lutheran protein wasincreased in the sdCHC sample. An actin (or protein 4.2) loading control is shown beneaththe blots.
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blood-brain barrier; the hepatic dysfunction could be dueto a cation leak in cells within the liver. This is speculationbecause measurements of cation content and flux are notpossible in these human tissues.
The hematological findings in this child are similar tothose observed in our previous cases (11), with the excep-tion of the red cell morphology. The cells are very leaky tosodium and potassium at 37 C, as evidenced by the veryabnormal intracellular [Na] and [K] in the fresh cells. Onstorage at 0 C, the cells very quickly lose potassium, con-sistent with a cryohydrocytosis phenotype, in which thecells are leakiest at 0 C (9, 11). The cells also lose potas-sium when stored in heparin at 37 C; this is probably dueto energy deprivation, the very active cells quickly using upglucose in the surrounding plasma. There is minimal glu-cose utilization at 0 C, so this point cannot explain the lossof potassium at low temperatures.
Pseudohyperkalemia (an artifactual rise in apparentplasma potassium due to in vitro loss of potassium fromblood cells) is a well-recognized aspect of leaky cell con-ditions of the hereditary stomatocytosis group (10). It wasvery marked in this case and caused much clinical con-sternation, especially because the infant had seizures.Pseudohyperkalemia can be recognized by the random na-ture of the readings, by the lack of background pathology(renal or adrenal failure, drug treatment, diabetes, acido-sis), and by the recognition that those samples that showthe highest readings have been stored the longest beforeanalysis.
Phenotypic variation is a feature of GLUT1DS (1), andthis quality applies to these cation-leaky cases. Two indi-vidual patients and one pedigree have been described withcation-leaky GLUT1DS. In the pedigree, which showedthe deletion Q282-S285 in GLUT1, the neurology was lesssevere, manifesting as paroxysmal exercise-induced dys-kinesia (6). As in the present case, the red cell morphologywas echinocytic. In the male adult case previously de-scribed by us, labeled sdCHC(A) (7), who showed thesubstitution gly286asp, the neurology was similarly severeto the present case, and the red cell morphology was sto-matocytic. In case sdCHC(B) (7), where the mutation wasthe same as in the present case, the neurology was likewisesevere, but the morphology was not described. The expla-nation for this difference in red cell morphology is unclear.Variations in modifying genes are possible, but it is alreadyknown that there is marked phenotypic variability in theGLUT1DS phenotype dependent on the exact SLC2A1variation.
Note that this condition is different from neuroacan-thocytosis, which also shows spiculated red cells with aneurological phenotype. In neuroacanthocytosis, the on-set of neurological symptoms occurs later in life, and the
conditions are caused by mutations in VPS13A, JPH3,XK, or PANK2 (15).
As in our previous cases (11), the stomatin protein wasdeficient from the membrane. Mistrafficking of stomatinduring erythropoiesis is seen in these very leaky GLUT1mutants, in the rhesus-associated glycoprotein mutants(16), and in dog red cells with a genetic polymorphism thatdown-regulates the NaK pump (17). The mechanism andpurpose behind this mistrafficking is not understood; forthe present case, stomatin can be considered to be a bio-marker for cells that are high in intracellular sodium andlow in potassium.
We hope that this report will facilitate diagnosis in sim-ilar cases.
Acknowledgments
We thank the parents for permission to publish this report. Manyclinicians contributed to the care of the infant, including Dr.C. D. Notaney (Wembley), Prof. Anil Dhawan (Kings CollegeHospital), Dr. Courtney Wusthoff (Hammersmith Hospital),Dr. Sepali Wijesinghe (Brent), and Dr. Alki Liasis (Great Or-mond Street Hospital). We thank Dr. Bari Levinson (San Fran-cisco) for invaluable advice. We thank Advocacy for Neuroac-anthocytosis for generous support.
Address all correspondence and requests for reprints to: Gor-don Stewart, Division of Medicine, University College London,Rayne Building, University Street, London WC1E 6JF, UnitedKingsom. E-mail: [email protected].
This work was supported by Advocacy for Neuroacantho-cytosis (to G.W.S.) and by the United Kingdom National HealthService R&D Directorate (to W.M.B., A.L.A., J.F.F., and L.J.B.).
Author Contributions: A.L.A., W.M.B., and J.F.F., proteingel analysis and gene sequencing; B.J., C.O., and S.G., key clin-ical contributions; E.F.G., M.D., G.W.S., and L.J.B., authorship.
Disclosure Summary: No author has any conflict of interest toreport.
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