MI Medicine International 2754-3242 2754-1304 D.A. Spandidos MI-4-6-00181 10.3892/mi.2024.181 Articles Clinical and genetic analysis of children with glucose transporter type 1 deficiency syndrome QianHao * YingGuohuan * XuHaifeng * WangShangyu WuBing WangXin QiHongdan HeMingying Ud DinM. Jalal HuangTingting WuYimei ZhangGang Department of Neurology, Children's Hospital of Nanjing Medical University, Nanjing, Jiangsu 210006, P.R. China Correspondence to: Dr Gang Zhang or Ms. Yimei Wu, Department of Neurology, Children's Hospital of Nanjing Medical University, 72 Guangzhou Road, Nanjing, Jiangsu 210006, P.R. China wym891203@163.com zhanggangnjmu@126.com

*Contributed equally

 Nov-Dec 2024 19 07 2024 4 6 57 26 03 2024 01 07 2024 Copyright: © 2024 Qian et al. 2024 This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.

Glucose transporter type 1 deficiency syndrome (GLUT1-DS) is a rare metabolic encephalopathy with a wide variety of clinical phenotypes. In the present study, 15 patients diagnosed with GLUT1-DS were selected, all of whom had obvious clinical manifestations and complete genetic testing. Their clinical data and genetic reports were collated. All patients were provided with a ketogenic diet (KD) and an improvement in their symptoms was observed during a follow-up period of up to 1 year. The results revealed that the 15 cases had clinical symptoms, such as convulsions or dyskinesia. Although none had a cerebrospinal fluid/glucose ratio <0.4, the genetic report revealed that all had the solute carrier family 2 member 1 gene variant, and their clinical symptoms basically improved following the use of the KD. GLUT1-DS is a genetic metabolic disease that causes a series of neurological symptoms due to glucose metabolism disorders in the brain. Low glucose levels in cerebrospinal fluid and genetic testing are key diagnostic criteria, and the KD is a highly effective treatment option. By summarizing and analyzing patients with GLUT1-DS, summarizing clinical characteristics and expanding their gene profile, the findings of the present study may be of clinical significance for the early recognition and diagnosis of the disease, so as to conduct early treatment and shorten the duration of brain energy deficiency. This is of utmost importance for improving the prognosis and quality of life of affected children.

 glucose transporter type 1 deficiency syndrome children solute carrier family 2 member 1 ketogenic diet epilepsy Funding: The present study was partly supported by the Epilepsy Research Fund of CAAE (grant no. CU-A-2021-17), and a key project of Nanjing Municipal Health Bureau (grant no. ZKX21047). Introduction

The human brain comprises a mere 2% of the weight of an adult individual; however, it requires a full 25% of the energy consumed by the individual, and the brain of a child requires 3-fold the amount of energy as does the brain of an adult (1). Glucose, being the main energy source in biological material, is also the major energy source in the central nervous system; yet, it cannot pass through the lipid bilayer structure of the cell membrane into cells freely, and must traverse the blood-brain barrier (BBB) via primarily, the glucose transporter 1 (GLUT1) to deliver glucose to the brain (2). The level of GLUT1 can consequently affect brain function and the general health of an individual (3).

GLUT1 deficiency syndrome (GLUT1-DS) is a rare metabolic disease of the nervous system, first reported by De Vivo et al in 1991(4). The current incidence is ~1:83,000(5) and the majority of cases are sporadic. It mainly occurs due to mutations in the solute carrier family 2 member 1 (SLC2A1) gene, resulting in the reduced or partial loss of GLUT1 expression; thus, glucose cannot effectively pass through the BBB, leading to neuronal energy deficiency, impaired brain function, and finally, to a series of neurological symptoms (6). Low glucose levels have been found in the cerebrospinal fluid (CSF) of these patients. A low CSF/blood glucose ratio (<0.4) is one of the most critical parameters in the diagnosis of GLUT1-DS (7,8). Furthermore, the diagnosis can be genetically confirmed if a pathogenic variant is detected in the SLC2A1 gene (9). Therefore, the diagnosis can be made following the detection of a pathogenic variant in SLC2A1 or the CSF criteria either alone, or in combination (10). The disease is classically characterized by low intelligence, microcephaly, seizures and movement disorders. According to the clinical phenotype of children, the disease can be divided into classic and non-classic GLUT1-DS. The onset of GLUT1-DS is early and the symptoms are severe. The main manifestations of chronic encephalopathy are infantile drug-resistant epilepsy, developmental delay, acquired microcephaly, mixed dyskinesia with ataxia and dystonia. The onset of atypical GLUTl-DS occurs at a relatively late stage and mainly presents with mild to moderate clinical symptoms, such as episodic dystonia, migraines and atypical childhood absence episodes. The ketogenic diet (KD), which has been shown to provide more ketone bodies as an alternate source of energy for brain metabolism, is currently the most effective treatment (11). The KD refers to a high-fat, low-carbohydrate diet, in which more fat is metabolized to produce ketone bodies that cross the BBB via monocarboxylate transporters to replace glucose as energy for the brain.

The present study describes the clinical manifestations, genetic testing, treatment and prognosis of 15 patients with GLUT1-DS diagnosed at the authors' hospital.

 Patients and methods <sec> <title>Study participants

A total of 15 patients diagnosed with GLUT1-DS at the Department of Neurology of Children's Hospital of Nanjing Medical University (Nanjing, China) were included in the present retrospective cohort study. The inclusion criteria were as follows: i) Children diagnosed with GLUT1-DS in the Neurology Department of the hospital, meeting the latest 2020 diagnostic criteria as outlined by the ‘Glut1 Deficiency Syndrome (Glut1DS): State of the art in 2020 and recommendations of the international Glut1DS study group’ (7); ii) children who underwent advanced genetic testing revealing an SLC2A1 mutation; iii) children with comprehensive clinical data; iv) Children whose parents/guardians volunteered for them to participate in the study, demonstrated good compliance and were cooperative during follow-up. The exclusion criteria were as follows: i) Poor family compliance, uncooperative patients and children with missing clinical data; ii) clearly an acquired brain disease accompanied by corresponding structural abnormalities or the existence of immune, infectious and organic causes.

The present study included 15 patients and they were monitored for up to 1 year. Extensive clinical data, including sex, age at onset, age at diagnosis, clinical manifestations, blood glucose levels, CSF glucose levels, imaging examination results, electroencephalography (EEG) results, genetic test results and disease remission were collected (Table I, Table II and Table III). As regards the treatment response, the anti-seizure drugs and treatment regimens used in the overall treatment of all children are summarized in Table IV. Moreover, the mutation sites were predicted by PyMol Viewer (https://pymol.org/) and SWISS-MODEL (https://swissmodel.expasy.org/). The residues of the missense mutation site and the nearby functional sites were illustrated using PyMol Viewer (the SLC2A1 gene homologous protein model (id: 6THA) was established in the SWISS-MODEL, and the homologous protein was then imported into PyMol Viewer for visual molecular docking).

The present study involving human participants was reviewed and approved by the Ethics Committee of the Children's Hospital of Nanjing Medical University. Written informed consent to participate in the study was provided by the legal guardian/next of kin of each participant.

 Patient data

Clinical data, neuroimaging studies and the results of EEG were obtained from medical records and the examination of the patients during clinical visits. The patients underwent a lumbar puncture in a fasting state (following 4-6 h of fasting) as part of the initial enrollment in the study. Blood samples for glucose measurements were obtained prior to the lumbar procedure to avoid stress-related hyperglycemia. The genomic DNA of the patients and their parents was extracted from EDTA blood samples according to standard procedures and was carried out by Chigene Genetics. The parents of the patients provided written informed consent.

 Results <sec> <title>Clinical characteristics

Of the 15 included patients, 7 were males and 8 were females. Their average age was 4 years and 11 months (range, 1 year and 8 months to 12 years and 6 months). The average age at seizure onset was 27.71 months (range, 12 to 57 months).

The clinical features, seizure types and neuroimaging features of the patients are summarized in Table I. Of these 15 children, 10 had seizures. The seizures were of mixed types, including 8 cases of generalized seizure, the most common seizure type, and 2 cases of focal seizure. Of the 15 cases, 9 were classified as classical and 6 as non-classical GLUT1-DS. In total, 11 children were treated with anti-epileptic medications prior to KD therapy; however, the medications had a minimal effect. Movement disorders were reported in 6 patients. Developmental delay was observed in 14 patients, and none of the children had microcephaly. Case nos. 6 and case 13 presented with confusion at the beginning of the disease. Of note, case nos. 6 and 13 (shown in Table I) exhibited frequent convulsions accompanied by changes in consciousness in a short period of time and were admitted to the pediatric intensive care unit of the hospital. Following active treatment, their condition improved.

 Biochemical characteristics and neuroimaging results

A fasting lumber puncture was performed in 9 patients, CSF was analyzed and a low CSF glucose level at 1.34-2.36 mmol/l (average, 1.85 mmol/l), combined with a low CSF/blood glucose ratio of 0.33-0.51 were observed. All patients underwent brain magnetic resonance imaging (MRI) and 13 patients underwent EEG. Only 9 cases exhibited abnormalities on the brain MRI, such as arachnoid cyst, ventricle plump, and hypomyelination and abnormal signals in the right frontal lobe. The EEG revealed generalized epileptiform discharges or background slowing in 8 cases. No abnormalities were found in the remaining 5 cases. These data are summarized in Table II. Biochemical and ultrasound results were also obtained. Each child underwent a cardiac examination and no obvious murmurs were heard. No obvious abnormalities were found on the cardiac ultrasound, electrocardiography and myocardial enzyme spectrum. No notable liver and kidney abnormalities were found in combination with the biochemical and ultrasound results.

 Molecular genetic analysis

All patients underwent genetic testing and were found to carry pathogenic mutations in the SLC2A1 gene, including 10 patients with missense, 2 patients with frameshift mutations and 3 patients with splice-site mutations (Table III).

A missense variant [chr1:43395436, NM_006516.2: c.695(exon6)G>A, p.Arg232His] in the SLC2A1 gene was found in case no. 4, which originated from his father, although his father did not have the phenotype. This mutation was previously reported in a patient with absent epilepsy (12), but not in the GLUT1-DS patient population, at least to the best of our knowledge, and it was defined as likely pathogenic. However, according to the Human Gene Mutation Database (HGMD; http://www.hgmd.cf.ac.uk/ac/index.php), it shared the same missense mutation or non-frame-shifting mutation as the identified pathogenic mutation, or shared the same amino acid changes. Combined with the clinical manifestations in this child, it was considered that this gene variant is related to the phenotype of patients, and is a new mutation that has not been previously reported.

A total of 15 site mutations were detected in these children, of which nine mutations have been previously reported (13-17). In addition to case no. 4, the mutation sites in case nos. 6, 7, 11, 12 and 15 have not been previously reported, at least to the best of our knowledge. In patient no. 6, we found a missense variant [chr1:43396415, NM_006516.2: c.398(exon4)G>A, p.Cys133Tyr] in the SLC2A1 gene, which was described as likely pathogenic, while this missense variation was located in the pathogenic hot spot (there are more than three harmful mutations within the range of ~10 bp), and case no. 6 had clinical manifestations, such as convulsions, developmental lag and a markedly decreased glucose content in CSF. Therefore, it was concluded that the mutation is consistent with the phenotype and is a new, previously unidentified mutation in exon 4.

In case no. 7, two site mutations were found in the SLC2A1 gene, namely chr1:43424514, NM_006516.2: c.275+2(IVS3)T>G and chr1:43424514, NM_006516.2: c.192(exon4)G>C; in fact, the former was defined as a pathogenic mutation as the loss of function (LOF) mutation led to a possible loss of gene function, while the base change in the latter did not affect the transcription of amino acids and generally did not lead to this disease.

Gene sequencing maps were representatively selected for case nos. 4, 5, 6 and 7. The residues of the missense mutation site and the nearby functional sites were illustrated using PyMol Viewer (https://pymol.org/); the SLC2A1 gene homologous protein model (id: 6THA) was established in the SWISS-MODEL (https://swissmodel.expasy.org/), and the homologous protein was then imported into PyMol Viewer for visual molecular docking. The mutations may affect the protein structure by altering hydrogen bonding and spatial conformation (Fig. 1). The protein prediction software SWISS-MODEL (6THA: X-ray Diffraction, 2.40 Å. ‘Crystal structure of human sugar transporter GLUT1 (SLC2A1) in the inward conformation’; Released 2020-11-25) was used to predict the three-dimensional structure of proteins caused by missense/frameshift mutations in addition to the splice site mutation in case no. 7. It was predicted that c.997C>T in case 1, c.695G>A missense mutations in case no. 4, and c.715dup frameshift mutation in case no. 3 affected the protein structure (Fig. 2). However, missense mutations in case nos. 2, 5 and 6 did not cause changes in protein structure.

 Response to treatment

In the early stages of the disease, the children with convulsions were treated with anti-epileptic therapy, and almost all received various drugs, as shown in Table IV, had a minimal effect in controlling the seizures. Therefore, when they were diagnosed with GLUT1-DS, they received the KD and were followed-up for 1 year. The results revealed that case nos. 1, 2, 3, 6, 8, 9, 11 and 14 had no recurrent convulsions, while case nos. 7 and 15, who had convulsions as the first symptom, still had occasional convulsions due to a short period on the KD, but had fewer attacks than previously. In addition, the 6 children with mobility disorders as their first symptom exhibited an improvement in motor instability after the KD. The main symptom in case no. 4 was delayed speech and motor development; however, when he started KD therapy, his motor skills markedly improved and he can now walk, say single words and call out to individuals. Dyspraxia in case no. 5 also markedly improved. The KD emphasizes the proportion of fat in the diet, and the proportion commonly used in clinical practice ranges from 2:1 to 4:1.

Blood glucose and blood ketones were monitored throughout the period of KD treatment, and there were no severe side-effects at any stage of the KD, and none of the patients dropped out of the study.

 Discussion

GLUT1 is the earliest identified GLUT protein, and contains 492 amino acids and 12 alpha-helical stranded transmembrane proteins. The sequence is highly conserved, and the known human GLUT1 protein has 98% homology with the amino acid sequence of rat, mouse, rabbit and pig (18).

SLC2A1, is the only gene related to GLUT1-DS, and Mueckler et al (19) successfully cloned it from the cDNA library of the human hepatoma cell line for the first time in 1985, which contains 10 exons and 9 introns. The SLC2A1 gene mutation is the main cause of GLUT1-DS, and this has been found in >90% of reported cases (20), which is basically a heterozygous mutation, including missense mutation, transcoding mutation, nonsense mutation, splicing site mutation, large fragment deletion and interposition (21). All 15 patients in the present study had the SLC2A1 gene mutation, 66.7% of which were missense mutations.

The clinical manifestations of GLUT1-DS vary and can be divided into three types. Type I is typical, accounting for ~85% of all cases, the main manifestations of which are mental retardation and epilepsy, with or without motor disorders. Type II is mainly manifested by low intelligence, dyskinesia, but without epilepsy; Type III, is characterized by paroxysmal movement-induced dyskinesia, with or without epilepsy, and the latter two types are also known as atypia, accounting for ~15% of cases.

Epilepsy is one of the most common clinical symptoms of GLUT1-DS, and ~90% of patients have different forms of seizures. Generalized epileptiform spike-wave patterns are the most common. Hewson et al (22) reviewed the seizure patterns of 99 patients with GLUT-DS, of whom 89% had extensive spikes or spike-wave electrical activity on EEG. Of all seizure types, generalized tonic-clonic seizures are the most common, and other heterogeneous seizure types, including absence, myoclonic and tonic-clonic seizures have also been reported (23). In addition, some studies have found that the form of EEG was age-dependent. Children <2 years of age are more likely to exhibited focal characteristics on the EEG, and a trend in focal slowing down or epileptic discharge can be observed (24). Nonetheless, in the present study, 10 of the 15 children had seizures, and most were generalized, which is consistent with other studies (25,26). Due to mutation of the SLC2A1 gene, the function of GLUTl is impaired, the glucose level in the central nervous system is reduced, and the energy supply is insufficient, resulting in abnormal changes in nerve excitability. Therefore, LOF mutation caused by SLC2A1 gene mutation is associated with the occurrence of epilepsy and other symptoms in patients with GLUT1-DS.

GLUT1-DS has also been shown to be associated with a variety of movement disorders, including, but not limited to choreoathetosis, dystonia, ataxia and alternating hemiplegia. Ataxia is one of the most common movement disorders, but is usually chronic and occurs with other classic symptoms (27). The retrospective study by Pons et al (28) also confirmed this point. They observed the clinical manifestations of 57 patients with GLUT1-DS and found that 89% were accompanied by gait disorders, with ataxic-spasm and ataxia being the most common, 86% of them had limb dystonia, 75% had mild chorea, 16% had myoclonus, 21% had dyskinesia and 28% had a non-epileptic emergency, including ataxia, weakness, Parkinson's disease and non-motor dyskinesia (28). Ramm-Pettersen et al (29) demonstrated that these movement disorders reflect chronic nutrient deficiencies during brain development, which may be alleviated by a long-term use of the KD. In the present study, there were 6 children with motor disorders, mainly manifested as paroxysmal lower limb weakness and were prone to falls, which were improved by the KD. In the 15 patients in the present study, the authors attempted to analyze the association between the treatment response and mutation pattern. Due to the small sample size, no differences were found. When reviewing previous studies (6,29-34), no significant differences were found between the treatment response and mutation pattern. This may be related to the degree of damage to the gene itself.

The present study also found that all 15 children had varying degrees of language motor development lag, which is consistent with known research results, as the developing brain in patients with GLUT1-DS is unable to obtain sufficient energy for its growth from infancy, leading to cerebral dysfunction (35). As shown in a previous study involving 181 patients with GLUT1-DS, ~69.7% of neurodevelopmental abnormalities were closely related to the incidence of epilepsy, and in epileptic patients, 75.6% had varying degrees of mental motor lag (35). However, due to the small number of cases included herein, the authors were unable to confirm the association between developmental status and the incidence of epilepsy.

For the diagnosis of GLUT1-DS, CSF/blood glucose ratio and gene testing are critical. Among the 15 children in the present study, the CSF/glucose ratio fluctuated between 0.33 and 0.51, and 4 of the 9 children had ratio results >0.4, which lacked high sensitivity and specificity. Combined with the diagnostic criteria for children (07), the absolute decrease in CSF glucose level is more reliable than the decrease in CSF glucose/glucose ratio in the clinic. All the children in the present study had completed genetic testing, and SLC2A1 gene mutations were found. However, in recent years, the 3-O-methyl-D-Glucose (3-OMG) uptake assay has also emerged as a new test (14). GLUT1 protein is not only widely expressed in brain capillary endothelial cells, astrocytes, neurons and microglia, but is also highly expressed on erythrocyte membranes, and is a key carrier protein of glucose 3-OMG into erythrocytes; therefore, the 3-OMG uptake assay can be used to evaluate the function of GLUT1. When the quantity of GLUT1 protein is reduced or its function is lost, the ability of red blood cells to take up glucose decreases. Yang et al (36) studied 109 patients with suspected GLUT1-DS and found that gene mutations were detected in 70 of 74 patients with a reduced glucose intake, compared with only 1 in 35 patients with a normal glucose intake. By inference (36), the majority of patients with GLUT1-DS had a decreased erythrocyte glucose uptake, and when they increased the erythrocyte glucose uptake rate from 60 to 70% of the control group, the sensitivity and specificity reached 99 and 100%, which has resulted in the 3-OMG uptake assay being a critical complementary means of genetic testing and has great significance in confirming GLUT1-DS. However, the 3-OMG uptake assay requires fresh red blood cells with metabolic function; therefore, transporting samples over a long distance will be a key issue in promoting this assay.

The findings of the present study demonstrated that regardless of the initial symptoms, there was an improvement after the KD, which is a high-fat, low-carbohydrate diet. The KD was effective in all 15 children with epilepsy, movement disorders and cognitive improvement, and these symptoms improved within 3 months following the initiation of the KD. The KD can replace glucose with ketone bodies as fuel for the brain, preventing seizures, movement disorders and cognitive impairment, and the earlier it is commenced, the greater its effectiveness (37,38).

Leen et al (39) conducted a study on 57 patients with GLUT1-DS, and found that the KD was effective in 86% of the patients with epilepsy, and significantly reduced movement disorder in 48% of the patients with classical phenotype and 71% of the patients with non-classical phenotype. Ramm-Pettersen (29) demonstrated following the long-term observation of patients with GLUT1-DS, that those who were diagnosed and started the KD within a few months after birth had better psychomotor development, while those who commenced the KD later significantly lagged behind their peers. This conclusion has also been verified by other studies (11,22).

In a previous study, researchers from several hospitals in China have retrospectively collected clinical data from 19 children with GLUT1-DS; all patients were treated with the KD, which was effective in 18 cases in terms of seizure control, 11 cases had dyskinesia improvement and 18 cases had cognitive function improvement (40). In the present study, it was found that the KD was effective in improving children's symptoms. This is consistent with the findings of previous studies both nationally and internationally (29,40).

The traditional KD has a strict energy ratio, maintaining the blood ketone body level in the range of 3-4 mg/dl. While maintaining that level of ketone bodies, the body is prone to fatigue, diarrhea and other side-effects, particularly in adult patients, and can reduce the compliance of patients. The Modified Atkins Diet (MAD) can maintain blood ketone levels, the therapeutic effect is similar to KD, and it is better tastewise (41). In previous studies, among patients with GLUT1-DS who were treated with the MAD, the frequency of seizures was reduced by 70-90, and >50% of patients exhibited an improvement in persistent motor disorder symptoms, such as paroxysmal dyskinesia or ataxia (41-43). The MAD was effective for GLUT1-DS, and could further reduce the fat content in the dietary ratio, which has long-term significance for improving food taste and reducing the adverse reactions associated with the high ester diet, and can help improve the compliance of patients with GLUT1-DS.

On the other hand, GLUT1-DS is mainly due to the deficiency of GLUT1 protein expression, which causes metabolic disorders in the brain; thus, gene replacement will become a new therapeutic direction. Nakamura et al (44) generated an adenovirus vector aAV9/3 in which human SLC2A1-myc-DDK was expressed under the human GLUT1 promoter (AAV-GLUT1), that was intracerebroventricularly injected into GLUT1-deficient mice, and the distribution of exogenous GLUT1 protein in the brain and other organs was analyzed. Their results revealed that exogenous GLUT1 protein was strongly expressed in the cerebral cortex, hippocampus and thalamus, while motor function and CSF glucose levels were significantly improved. Their study provides a basis for gene-level therapy of GLUT1-DS in the future (44).

The present study had certain limitations, which should be mentioned. These include the small sample size and only a 1-year follow-up time. In the future, the authors' research group aims to further expand the collection of cases.

In conclusion, GLUT1-DS is a genetic metabolic disease that causes a series of neurological symptoms due to glucose metabolism disorders in the brain. The decrease in the CSF/blood glucose ratio in CSF is a key indicator in the diagnosis of this disease. The diagnosis in the majority of cases can be confirmed by SLC2A1 gene testing. GLUT1-DS is a genetic disorder and the most effective treatment is currently the KD. However, the disease is difficult to diagnose given its clinical features, with the mean time to diagnosis in the present study cohort being 4 years and 8 months. Therefore, by reporting relevant cases, this will publicize the disease more and enrich the gene pool, and will provide the relevant basis for earlier diagnosis. Early diagnosis can result in early treatment, which will reduce the duration of nervous system energy deficiency in order to improve the prognosis of affected children.

 Acknowledgements

Not applicable.

 Availability of data and materials

The datasets generated and/or analyzed during the current study are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the datasets should be directed to the corresponding author.

 Authors' contributions

HaQ analyzed and interpreted the patient data and was a major contributor to the article. GY and HX participated in the data analysis and in the writing of the manuscript. SW participated in the writing of the manuscript, conducted data analysis and interpretation, and conducted a rigorous review of the relevant content of the manuscript. BW and XW conducted the data management and participated in the writing of the manuscript. HoQ and MH participated in data collection and analysis. MJUD and TH were involved in project management and contributed to the analysis and interpretation of the data. YW and GZ contributed significantly to the conceptualization and design of the work, assisted in the acquisition of data, provided resources and guidance for this paper, and was involved in its final approval. All authors have read and approved the final manuscript. HaQ and GY confirm the authenticity of all the raw data.

 Ethics approval and consent to participate

The present involving human participants was reviewed and approved by the Children's Hospital of Nanjing Medical University (Nanjing, China). Written informed consent to participate in this study was provided by the legal guardians/next of kin of the participants.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 References RaichleMEGusnardDAAppraising the brain's energy budgetProc Natl Acad Sci USA99102371023920021214948510.1073/pnas.172399499 TangMMonaniURGlut1 deficiency syndrome: New and emerging insights into a prototypical brain energy failure disorderNeurosci Insights162633105521101150720213458970810.1177/26331055211011507 SeidnerGAlvarezMGYehJIO'DriscollKRKlepperJStumpTSWangDSpinnerNBBirnbaumMJDe VivoDCGLUT-1 deficiency syndrome caused by haploinsufficiency of the blood-brain barrier hexose carrierNat Genet181881911998946275410.1038/ng0298-188 De VivoDCTrifilettiRRJacobsonRIRonenGMBehmandRAHarikSIDefective glucose transport across the blood-brain barrier as a cause of persistent hypoglycorrhachia, seizures, and developmental delayN Engl J Med3257037091991171454410.1056/NEJM199109053251006 Soto-InsugaVLópezRGLosada-Del PozoRRodrigo-MorenoMCayuelasEMGiráldezBGDíaz-GómezESánchez-MartínGGarcíaLOSerratosaJMGrupo Español de Genética de las Epilepsias de la Infancia (GEGEI)Glut1 deficiency is a rare but treatable cause of childhood absence epilepsy with atypical featuresEpilepsy Res154394120193103524310.1016/j.eplepsyres.2019.04.003 PawlikWOkulewiczPPawlikJKrzywińska-ZdebEDiagnostic and clinical manifestation differences of glucose transporter type 1 deficiency syndrome in a family with SLC2A1 Gene MutationInt J Environ Res Public Health19327920223532896510.3390/ijerph19063279 KlepperJAkmanCArmenoMAuvinSCervenkaMCrossHJDe GiorgisVDella MarinaAEngelstadKHeussingerNGlut1 Deficiency Syndrome (Glut1DS): State of the art in 2020 and recommendations of the international Glut1DS study groupEpilepsia Open535436520203291394410.1002/epi4.12414 KochHWeberYGThe glucose transporter type 1 (Glut1) syndromesEpilepsy Behav91909320193007604710.1016/j.yebeh.2018.06.010 De GiorgisVVeggiottiPGLUT1 deficiency syndrome 2013: Current state of the artSeizure2280381120132389083810.1016/j.seizure.2013.07.003 Gburek-AugustatJHeinzeAAbou JamraRMerkenschlagerAHemiplegic Migraine in Glut1 deficiency syndrome and paroxysmal dyskinesia at ketogenic diet induction: Case report and literature reviewMov Disord Clin Pract796597020203316356910.1002/mdc3.13087 SchwantjeMVerhagenLMvan HasseltPMFuchsSAGlucose transporter type 1 deficiency syndrome and the ketogenic dietJ Inherit Metab Dis4321622220203160554310.1002/jimd.12175 LarsenJJohannesenKMEkJTangSMariniCBlichfeldtSKibaekMvon SpiczakSWeckhuysenSFranguMThe role of SLC2A1 mutations in myoclonic astatic epilepsy and absence epilepsy, and the estimated frequency of GLUT1 deficiency syndromeEpilepsia56e203e20820152653743410.1111/epi.13222 HullyMVuillaumier-BarrotSLe BizecCBoddaertNKaminskaALascellesKde LonlayPCancesCdes PortesVRoubertieAFrom splitting GLUT1 deficiency syndromes to overlapping phenotypesEur J Med Genet5844345420152619338210.1016/j.ejmg.2015.06.007 GrasDCousinCKappelerCFungCWAuvinSEssidNChungBHDa CostaLHainqueELutonMPA simple blood test expedites the diagnosis of glucose transporter type 1 deficiency syndromeAnn Neurol8213313820172855618310.1002/ana.24970 MauriADuseAPalmGPrevitaliRBovaSMOlivottoSBenedettiSCosciaFVeggiottiPCeredaCMolecular Genetics of GLUT1DS Italian pediatric cohort: 10 novel disease-related variants and structural analysisInt J Mol Sci231356020223636234710.3390/ijms232113560 ItoYTakahashiSKagitani-ShimonoKNatsumeJYanagiharaKFujiiTOguniHNationwide survey of glucose transporter-1 deficiency syndrome (GLUT-1DS) in JapanBrain Dev3778078920152548768410.1016/j.braindev.2014.11.006 JiXNXuCJGaoZJChenSHXuKMChenQGlucose transporter 1 deficiency syndrome: Features of movement disorders, diagnosis and treatmentZhongguo Dang Dai Er Ke Za Zhi2020921320182953012110.7499/j.issn.1008-8830.2018.03.009(In Chinese) KumagaiAKDwyerKJPardridgeWMDifferential glycosylation of the GLUT1 glucose transporter in brain capillaries and choroid plexusBiochim Biophys Acta119324301994803819110.1016/0005-2736(94)90328-x MuecklerMCarusoCBaldwinSAPanicoMBlenchIMorrisHRAllardWJLienhardGELodishHFSequence and structure of a human glucose transporterScience2299419451985383959810.1126/science.3839598 WillemsenMAVissersLEVerbeekMMvan BonBWGeuerSGilissenCKlepperJKwintMPLeenWGPenningsMUpstream SLC2A1 translation initiation causes GLUT1 deficiency syndromeEur J Hum Genet2577177420172837881910.1038/ejhg.2017.45 RajaMKinneRKPathogenic mutations causing glucose transport defects in GLUT1 transporter: The role of intermolecular forces in protein structure-functionBiophys Chem200-20191720152586319410.1016/j.bpc.2015.03.005 HewsonSBrungaLOjedaMFImhofEPatelJZakMDonnerEJKobayashiJSalomonsGSMercimek-AndrewsSPrevalence of genetic disorders and GLUT1 deficiency in a ketogenic diet clinicCan J Neurol Sci45939620182914422510.1017/cjn.2017.246 SulsAMullenSAWeberYGVerhaertKCeulemansBGuerriniRWuttkeTVSalvo-VargasADeprezLClaesLREarly-onset absence epilepsy caused by mutations in the glucose transporter GLUT1Ann Neurol6641541920091979863610.1002/ana.21724 LearyLDWangDNordliDR JrEngelstadKDe VivoDCSeizure characterization and electroencephalographic features in Glut-1 deficiency syndromeEpilepsia4470170720031275247010.1046/j.1528-1157.2003.05302.x WolkingSBeckerFBastTWiemer-KruelAMayerTLercheHWeberYGFocal epilepsy in glucose transporter type 1 (Glut1) defects: Case reports and a review of literatureJ Neurol2611881188620142502294210.1007/s00415-014-7433-5 PeeraerADamianoJABellowsSTSchefferIEBerkovicSFMullenSAHildebrandMSEvaluation of GLUT1 variation in non-acquired focal epilepsyEpilepsy Res133545720172841998010.1016/j.eplepsyres.2017.04.007 TchapyjnikovDMikatiMAAcetazolamide-responsive episodic ataxia without baseline deficits or seizures secondary to GLUT1 Deficiency: A case report and review of the literatureNeurologist23171820182926603910.1097/NRL.0000000000000168 PonsRCollinsARotsteinMEngelstadKDe VivoDCThe spectrum of movement disorders in Glut-1 deficiencyMov Disord2527528120102006342810.1002/mds.22808 Ramm-PettersenAStabellKENakkenKOSelmerKKDoes ketogenic diet improve cognitive function in patients with GLUT1-DS? A 6- to 17-month follow-up studyEpilepsy Behav3911111520142524012210.1016/j.yebeh.2014.08.015 GramerGWolfNIVaterDBastTSanterRKamsteegEJWeversRAEbingerFGlucose transporter-1 (GLUT1) deficiency syndrome: Diagnosis and treatment in late childhoodNeuropediatrics4316817120122262295610.1055/s-0032-1315433 Ramm-PettersenANakkenKOHaavardsholmKCSelmerKKGLUT1-deficiency syndrome: Report of a four-generation Norwegian family with a mild phenotypeEpilepsy Behav70(Pt A)1420172840752310.1016/j.yebeh.2017.02.016 CastellottiBRagonaFFreriESolazziRCiardulloSTricomiGVenerandoASalisBCanafogliaLVillaniFScreening of SLC2A1 in a large cohort of patients suspected for Glut1 deficiency syndrome: Identification of novel variants and associated phenotypesJ Neurol2661439144820193089538610.1007/s00415-019-09280-6 HashimotoNKagitani-ShimonoKSakaiNOtomoTTominagaKNabatameSMogamiYTakahashiYImaiKYanagiharaKSLC2A1 gene analysis of Japanese patients with glucose transporter 1 deficiency syndromeJ Hum Genet5684685120112201181710.1038/jhg.2011.115 HuQShenYSuTLiuYXuSClinical and genetic characteristics of chinese children with GLUT1 deficiency syndrome: Case report and literature reviewFront Genet1273448120213488089910.3389/fgene.2021.734481 HaoJKellyDISuJPascualJMClinical aspects of glucose transporter type 1 deficiency: Information from a global registryJAMA Neurol7472773220172843753510.1001/jamaneurol.2017.0298 YangHWangDEngelstadKBagayLWeiYRotsteinMAggarwalVLevyBMaLChungWKDe VivoDCGlut1 deficiency syndrome and erythrocyte glucose uptake assayAnn Neurol70996100520112219037110.1002/ana.22640 De GiorgisVMasnadaSVaresioCChiappediMAZanaboniMPascaLFilippiniMMacasaetJAValenteMFerrarisCOverall cognitive profiles in patients with GLUT1 Deficiency SyndromeBrain Behav9e0122420193071435110.1002/brb3.1224 YuMMiaoJLvYWangXZhangWShaoNMengHA challenging diagnosis of atypical glut1-DS: A case report and literature reviewFront Neurol1154933120213358448910.3389/fneur.2020.549331 LeenWGKlepperJVerbeekMMLeferinkMHofsteTvan EngelenBGWeversRAArthurTBahi-BuissonNBallhausenDGlucose transporter-1 deficiency syndrome: The expanding clinical and genetic spectrum of a treatable disorderBrain133(Pt 3)65567020102012993510.1093/brain/awp336 YuLFZhangYQDuanJNiYGongXYLuZYLiaoJXLuXPShiZNLeiMFClinical characteristics and ketogenic diet therapy of glucose transporter type 1 deficiency syndrome in children: A multicenter clinical studyZhonghua Er Ke Za Zhi5888188620203312045810.3760/cma.j.cn112140-20200822-00819(In Chinese) AmalouSGrasDIleaAGrenecheMOFrancoisLBellavoineVDelanoeCAuvinSUse of modified Atkins diet in glucose transporter type 1 deficiency syndromeDev Med Child Neurol581193119920162727352610.1111/dmcn.13167 Ohshiro-SasakiAShimboHTakanoKWadaTOsakaHA three-year-old boy with glucose transporter type 1 deficiency syndrome presenting with episodic ataxiaPediatr Neurol509910020142420004010.1016/j.pediatrneurol.2013.09.002 HaberlandtEKarallDJudVBaumgartnerSSZotterSRostasyKBaumannMScholl-BuergiSGlucose transporter type 1 deficiency syndrome effectively treated with modified Atkins dietNeuropediatrics4511711920142388846810.1055/s-0033-1349225 NakamuraSMuramatsuSITakinoNItoMJimboEFShimazakiKOnakaTOhtsukiSTerasakiTYamagataTOsakaHGene therapy for Glut1-deficient mouse using an adeno-associated virus vector with the human intrinsic GLUT1 promoterJ Gene Med20e301320182962479010.1002/jgm.3013

Structural analysis of WT and the variant SLC2A1 with mutations. The residues of missense mutant sites together with the nearby functional site are illustrated in WT and variant SLC2A1 using PyMol Viewer. The computed hydrogen bonds are shown as yellow dashed lines. Residues of the mutant sites are highlighted in orange solid lines. WT, wild-type.

Structural analysis of wild-type and the variant SLC2A1 with missense mutations and frameshift mutation. Protein prediction software (SWISS-MODEL) was used to predict the three-dimensional structure of proteins. (A) Wild-type, (B) case no. 1, (C) case no. 3, (D) case no. 4. (B-D) Case nos. 1, 3 and 4 were mutant.

Clinical data of the patients with GLUT1-DS in the present study.

  Clinical features
Patient no. Sex Current age Age at onset Microcephaly MD DD Seizure type Phenotype Anti-epileptic agent
1 F 2 years, 3 months 1 year - - + GTCS on VPA Classical VPA,
2 F 3 years, 7 months 2 years - - + GTCS, myoclonic; sporadic absences Classical VPA, LTG, CZP
3 M 4 years, 6 months 3 years - Paroxysmal hypokinesia + - Non-classical -
4 M 2 years, 4 months 1 year - - + - Non-classical -
5 F 6 years, 9 months 4 years - Paroxysmal hypokinesia +, IQ 55 - Non-classical -
6 M 2 years, 8 months 2 years, 8 months - - +, DQ 56, MI 77 Frequent absences Classical VPA, LEV
7 M 5 years 2 years - - +, IQ 40 GTCS Classical VPA, OCBZ, Lacosamide
8 M 2 years, 7 months 3 months - + + Focal seizure Classical OCBZ
9 M 12 years, 6 months 1 year - - + Focal seizure Classical VPA, LTG
10 F 6 years 3 years - + + - Non-classical -
11 M 11 years, 9 months 4 years - + + Generalized seizure Non-classical VPA
12 F 3 years, 6 months - + + - Non-classical VPA
13 F 1 years, 8 months 1 year, 1 month - - - Generalized seizure Classical LEV, OCBZ
14 F 5 years, 1 month 2 years, 7 months - - + Generalized seizure Classical LEV, VPA, TPM
15 F 3 years, 9 months 3 years, 1 month - - + Generalized seizure Classical VPA, LTG

F, female; M, male; DD, developmental delay; MD, movement disorder; +, positive; -, negative; GTCS, generalized tonic–clonic seizure; VPA, valproic acid; LTG, lamotrigine; CZP, clonazepam; LEV, levetiracetam; OCBZ, oxcarbazepine; TPM, topiramate.

Laboratory findings and neuroimaging results of patients with GLUT1-DS.

Patients no. Blood glucose (mmol/l) CSF glucose (mmol/l) CSF/blood glucose ratio Brain MRI EEG
1 3.8 N/A N/A Arachnoid cyst; Bilateral ventricle plump Epileptiform discharge
2 4.8 1.62 0.33 Normal Epileptiform discharge
3 5.0 N/A N/A Normal Normal
4 4.6 2.36 0.51 Hypomyelination; Bilateral ventricle plump Normal
5 3.4 1.34 0.39 Abnormal signals in right frontal lobe Normal
6 4.6 1.98 0.43 Normal Background Slowing
7 4.5 1.56 0.35 Left ventricle plump Background Slowing, δ activity
8 4.37 N/A N/A Bilateral ventricle plump Normal
9 5.4 1.95 0.36 Normal Epileptiform discharge
10 4.37 N/A N/A Left ventricle plump N/A
11 5.12 2.17 0.42 Bilateral ventricle plump Epileptiform discharge
12 3.96 1.54 0.39 Normal Boundary electroencep halogram
13 4.57 2.17 0.47 Several small punctate FLAIR signals in the frontal parietal cortex on both sides N/A
14 3.9 N/A N/A Elevated T2W1 signals in bilateral paraventricular terminal zone Normal
15 3.96 N/A N/A Normal Background Slowing

N/A, no information available.

Mutation details of patients with GLUT1-DS.

Patients no. Location in gene Nucleotide Amino acid Source Type of mutation Phenotype
1 - c.997C>T p.R333W De novo Missense mutation Classical
2 - c.457C>T p.A153C De novo Missense mutation Classical
3 - c.715dup p.H239P*2 De novo Frameshift mutation Non-classical
4 Exon 6 c.695G>A p.R232H Father Missense mutation Non-classical
5 Exon 4 c.376C>T p.P126C De novo Missense mutation Non-classical
6 Exon 4 c.398G>A p.C133Y De novo Missense mutation Classical
7 IVS 3 c.275+2T>G - De novo Splice-site mutation Classical
8 IVS 5 c.680-11G>A - De novo Splice-site mutation Classical
9 Exon 7 c.884C>T p.T295M Father Missense mutation Classical
10 Exon 9 c.1199G>A p.R400H De novo Missense mutation Non-classical
11 IVS 5 c.680-1G>T - De novo Splice-site mutation Non-classical
12 Exon 9 c.1077_1090del p.E359fs*17 De novo Frameshift mutation Non-classical
13 Exon 8 c.997C>T p.R333W Mother Missense mutation Classical
14 Exon 4 c.377G>A p.R126H De novo Missense mutation Classical
15 Exon 4 c.464C>T p.A155V De novo Missense mutation Classical

IVS, intervening sequence; DNA mutations were numbered using the reference sequence NM_006516.1.

Treatment and response of the patients with GLUT1-DS.

  Effect of ketogenic diet on
Patients no. Anti-epileptic agent Clinical features Movement disorder Developmental delay Seizure type
1 VPA DD, seizure N/A + +
2 VPA, LTG, CZP DD, seizure N/A + +
3 - MD, DD +/- + +
4 - DD N/A + N/A
5 - MD, DD + + N/A
6 VPA, LEV DD, seizure N/A + +
7 VPA, OCBZ, Lacosamide DD, seizure N/A N/A +/-
8 OCBZ MD, DD, seizure N/A N/A +
9 VPA, LTG DD, seizure N/A N/A +
10 - MD, DD +/- N/A N/A
11 VPA MD, DD, seizure +/- + +
12 VPA MD, DD N/A N/A N/A
13 LEV, OCBZ Seizure N/A N/A N/A
14 LEV, VPA, TPM DD, seizure N/A N/A +
15 VPA, LTG DD, seizure N/A N/A +/-

The seizure type is shown in Table I. VPA, valproic acid; LTG, lamotrigine; CZP, clonazepam; LEV, levetiracetam; OCBZ, oxcarbazepine; TPM, topiramate; DD, developmental delay; MD, movement disorder; N/A, no information available; Movement disorder: +, total disappearance of movement disorder; +/-, reduction of frequency and/or severity of movement disorder; Developmental delay: +, positive; Seizure type: +, seizure-free; +/-, reduction of seizures.