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. 2023 Jan 2;34:100954. doi: 10.1016/j.ymgmr.2022.100954

Establishment of a flow cytometry screening method for patients with glucose transporter 1 deficiency syndrome

Sachie Nakamura a, Yasushi Ito b,c, Hiroko Hayakawa d, Shiho Aoki a, Takanori Yamagata a, Hitoshi Osaka a,⁎
PMCID: PMC9817163  PMID: 36618999

Abstract

Objective

We assessed the usefulness of flow cytometry as a functional assay to measure glucose transporter 1 (GLUT1) levels on the surface of red blood cells (RBCs) from Japanese patients with glucose transporter 1 deficiency syndrome (Glut1DS).

Methods

We recruited 13 genetically confirmed Glut1DS patients with a solute carrier family 2 member 1 (SLC2A1) mutation (eight missense, one frameshift, two nonsense, and two deletion) and one clinically suspected Glut1DS-like patient without an SLC2A1 mutation, and collected whole blood with informed consent. We stained pelleted RBCs (1 μL) from the patients with a Glut1.RBD ligand and anti-glycophorin A antibody, which recognizes a human RBC membrane protein, and analyzed the cells using flow cytometry.

Results

Relative GLUT1 levels quantified by flow cytometry in 11 of 13 patients with definite Glut1DS were 90% below those of healthy controls. Relative GLUT1 levels were not reduced in two of 13 Glut1DS patients who had a missense mutation and no intellectual disability and one Glut1DS-like patient without an SLC2A1 mutation. Relative GLUT1 levels were significantly reduced in Glut1DS patients with an SLC2A1 mutation, more severe intellectual disability, and spasticity.

Conclusions

This method to detect GLUT1 levels on RBCs is simple and appears to be an appropriate screening assay to identify severe Glut1DS patients in the early stage before the development of irreversible neurologic damage caused by chronic hypoglycorrhachia.

Keywords: Glucose transporter 1 deficiency syndrome (Glut1DS), GLUT1, SLC2A1, Flow cytometry, Screening method

Highlights

  • •

    Assessment of the flow cytometry to measure GLUT1 levels on RBCs from Japanese Glut1DS patients

  • •

    Simplicity of the flow cytometry using Glut1.RBD ligand

  • •

    Correlation with the type of SLC2A1 mutation and ID severity in relative GLUT1 levels on RBCs measured by flow cytometry.

1. Introduction

Glucose transporter 1 deficiency syndrome (Glut1DS; OMIM #606777) is caused by haploinsufficiency of solute carrier family 2 member 1 (SLC2A1; cytogenic location: 1p34.2), which encodes GLUT1. GLUT1 is expressed mainly on the endothelial cells of the blood-brain barrier, but also on other cell types including astrocytes, oligodendrocytes, and neurons of the central nervous system [[1], [2], [3], [4], [5]]. Impaired hexose transport by heterozygous mutation of SLC2A1 causes energy failure and results in irreversible neurologic dysfunction [[6], [7], [8]]. Glut1DS patients with the “classic” severe phenotype present with intractable infantile-onset seizures, subsequent delayed neurologic development, and complex movement disorders with a combination of ataxia, spasticity, and dystonia. Some Glut1DS patients develop paroxysmal exercise-induced dyskinesia [9]. Currently, Glut1DS is treated with ketogenic diet (KD) therapy, which consists of high-fat and carbohydrate-restricted meals [[10], [11], [12]]. KD therapy is effective for seizures and ataxia [11,12]; however, it does not prevent intellectual disability (ID) and leads to hyperlipidemia, which is a potential risk factor for the development of cardiovascular disease and acute pancreatitis [7,9,11,13,14].

Previously, we established a functional test for Glut1DS using an enzymatic photometric 2-deoxyglucose uptake assay [15]. This assay can predict the effect of SLC2A1 mutations on GLUT1 transport, but is complicated because it uses cultured cells transfected with an expression vector with the corresponding SLC2A1 mutation. In other functional tests of Glut1DS, the erythrocyte 3-O-methyl-d-glucose (3-OMG) uptake assay using radioisotopes is useful for patients with a pathogenic SLC2A1 mutation that shows an approximately 50% reduction in uptake, but some patients demonstrate normal 3-OMG uptake [6,7]. We consider that the ideal screening method should be simple, less invasive, and require a small sample.

In a previous report, GLUT1 levels on the surface of red blood cells (RBCs) from Glut1DS patients measured by flow cytometry using a Glut1.RBD ligand was shown to be significantly reduced in comparison with healthy controls [16,17]. In the present study, we assessed the usefulness of flow cytometry as a screening approach to measure GLUT1 levels on gated CD235a and Glut1.RBD double-positive RBCs from Japanese patients with Glut1DS.

2. Materials and methods

2.1. Patients and controls

We recruited 14 patients (age at the time of the survey: 4–30 years old; 11 males) (Table 1) confirmed by clinical symptoms, cerebrospinal fluid (CSF)/blood glucose ratio, and genetic analysis. and compared them with eight healthy, neurologically non-impaired controls. The secondary structure of GLUT1 and the amino acid positions of the missense, frameshift, and nonsense mutations of SLC2A1 of the participants (Patients #1–11) are shown in Fig. 1. All participants provided informed consent and we collected whole blood with medical information. We obtained clinical and laboratory data including sequence analysis of SLC2A1 from all participants from the relevant medical institutions. We measured CSF glucose and blood glucose levels after an overnight fasting period in all patients except for Patient #12. We collected clinical information from attending doctors including age at onset, seizure type, ID before KD therapy, chronic movement disorders, and paroxysmal movement disorders. We evaluated intelligence quotient (IQ) by the Wechsler Intelligence Scale for Children – Third Edition in children aged ≥5 years (Patient #6), the Tanaka–Binet Intelligence Scale (Japanese version of the Stanford–Binet test) in children aged ≥2 years (Patients #2, 4, 7, 10, and 14), and the New Kyoto Scale of Psychological Development in children aged ≥100 days (Patients #1, 3, 5, 8, and 13). We classified ID with reference to the current Wechsler IQ classification (IQ range, very superior: >130; superior: 120–129; high average: 109–119; average: 90–109; low average: 80–89; borderline: 70–79; mild ID: 50–69; moderate ID: 35–49; severe ID: 20–34; profound severe ID: <20). This study was approved by the Clinical Research Ethics Committee of Jichi Medical University.

Table 1.

Clinical summary of the 14 patients.

Patient no. Age / sex Mutation Type of SLC2A1 mutation CSF/blood glucose Onset (months) Seizure ID before treatment ID at time of survey Chronic movement disorder Paroxysmal movement disorder Ketogenic diet Relative GLUT1 (%)
1 6Y2M/F c.971C > T (p.Ser324Leu) [8] missense 0.41 15 FS(I), GTCS average low average – – poor compliance 95.8
2 11Y7M/ M c.196 T > A (p.Ser66Thr) [18] missense 0.36 12 GTCS low average borderline ataxia – effective 99.2
3 7Y6M/F c.377G > A (p.Arg126His) [6] missense 0.45 7 GTS mild borderline ataxia ataxia effective 83.1
4 17Y4M/ M c.971C > T (p.Ser324Leu) [8] missense 0.37 12 FS(I) mild mild ataxia hyperkinesia, ataxia, dystonia not done 89.8
5 4Y2M /F c.971C > T (p.Ser324Leu) [8] missense 0.42 12 – mild mild – – poor compliance 87.6
6 23Y11M/ M c.1199G > A (p.Arg400His) [8] missense 0.45 8 MS, aAb moderate mild ataxia, Spa, dystonia hemifacial spasm not done 68.7
7 10Y11M/ M c.997C > T (p.Arg333Trp) [6,8] missense 0.36 3 GTCS, AS, aAb severe severe ataxia ataxia poor compliance 88.8
8 9Y8M/ M C.458G > A (p.Arg153His) [19] missense 0.42 2 MS profound severe severe ataxia, Spa myoclonus, chorea, Uni weakness effective 63.4
9 23Y11M/M c.745_746insC (p.Arg249fs) [6,8,19] frameshift 0.3 4 MS, AS, aAb severe severe ataxia, Spa, dystonia Spa effective 78.6
10 19Y10M/ M c.988C > T (p.Arg330X) [8,19] nonsense 0.36 4 AS, aAb severe severe ataxia, Spa, dystonia Spa effective 68
11 30Y5M/ M c.84C > G (p.Tyr28X) [8] nonsense 0.3 2 MS, FS(I), GTCS profound severe profound severe ataxia, Spa, dystonia myoclonus, choreoathetosis ineffective 87.6
12 23Y3M/ M del(1)(p34.2p34.1)[20] deletion not examined 4 GTS, FS(I) profound severe profound severe ataxia, Spa hyperkinesia, Spa, weakness not done 74.5
13 4Y8M/M del (1)(p34.3p34.1)[21] deletion 0.33 4 MS profound severe profound severe ataxia - effective 81.6
14 18Y8M/M
- (Glut1DS like)		 - 0.69 31 GTCs, aAb, MS severe severe ataxia, Spa, dystonia hyperkinesia not done 99.1
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aAb: atypical absence seizures, AS: atonic seizures, FS(I): focal seizures (impaired awareness), GTCS: generalized tonic-clonic seizures, GTS: generalized tonic seizures,

ID: intellectual disability, MS: myoclonic seizures. Spa; spasticity, Uni; unilateral.

Fig. 1.

Fig. 1

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eGFP-fused Glut1.RBD ligand and the binding site of GLUT1. eGFP-fused Glut1. RBD is a flow cytometry reagent that binds to ECL6 of GLUT1 (amino acids: 423–429, purple). This reagent integrates the receptor-binding domains of HTLV-1 and -2 to bind to ECL6 of GLUT1 at virus entry. The secondary structure of GLUT1 and the amino acid positions of missense, frameshift, and nonsense mutations of SLC2A1 of the participants (Patients #1–11) are indicated using colored dots and on the right side of the figure. Pt, patient.

2.2. Flow cytometry analysis of GLUT1 on the surface of RBCs

We collected 0.5 mL nonfasted venous blood at room temperature in an EDTA-2Na tube and centrifuged it at 3000 rpm for 10 min within 24 h. We suspended 1 μL isolated RBCs in 2 mL of 0.1% bovine serum albumin (BSA), and then recentrifuged them at 500 ×g for 3 min. After removing the supernatant, we diluted the RBCs in 0.1% BSA at 2.5 × 106 cells/mL. For each sample, we confirmed the cell count and cell morphology using an automated cell counter (Thermo Fisher Scientific, Waltham, MA). We selected the same size of cells without hemolysis and anomalies. Then we centrifuged the tube at 500 ×g for 3 min and removed the supernatant. Unlike the conventional method, we resuspended the pelleted RBCs in 100 μL of 0.1% BSA and stained them with 5 μL enhanced green fluorescent protein (eGFP)-fused Glut1.RBD (METAFORA Biosystems, Evry, France) (Fig. 1) and 20 μL phycoerythrin (PE)-fused anti-CD235a (glycophorin A) (Beckman Coulter, Inc., Brea, CA) at 37 °C for 20 min. eGFP-fused Glut1.RBD is a ligand that binds to extracellular loop 6 (ECL6; amino acids 423–429) of GLUT1. ECL6 is a binding site for the human T cell leukemia virus (HTLV)-1 and HTLV-2 receptor binding domains at virus entry [17]. This reagent integrates the HTLV receptor binding domains to bind to ECL6 of GLUT1. The PE-fused anti-glycophorin A antibody recognizes a human RBC membrane protein. Then, we washed the pelleted RBCs with phosphate-buffered saline, fixed them in 2% paraformaldehyde, and measured them on a BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences, Franklin Lakes, NJ). During measurement on the flow cytometer, the total cell count was 10,000 cells for each sample, and we gated CD235a and Glut1.RBD double-positive RBCs and excluded debris. We performed data analysis using FlowJo (Ver. 10. 8. 0) software (BD Biosciences, Franklin Lakes, NJ).

2.3. Statistical analysis

Relative GLUT1 levels quantified by flow cytometry are indicated as a percentage of the mean of healthy controls. Relative GLUT1 levels are expressed as the mean ± standard error of the mean. We used Student's t-test for comparisons between two groups, and Dunnett's test for multiple comparisons. The significance level for statistical comparison was P < 0.05.

3. Results

3.1. Characteristics of the patients

We recruited 14 patients (13 patients with an SLC2A1 mutation and one patient with Glut1DS-like syndrome who had ataxia on overnight fasting and no SLC2A1 mutation) for this study (Table 1). We identified eight missense, one frameshift, two nonsense, and two deletion SLC2A1 mutations in the 13 patients [6,8,[19], [20], [21], [22]]. We identified the same missense mutation, c.971C > T, p. Ser324Leu, in three siblings (Patients #1, 4, and 5). One patient with a microdeletion and profound severe ID (Patient #12) refused a CSF assay and received only molecular analysis. The CSF/blood glucose ratio in 12 patients with an SLC2A1 mutation indicated a low range of 0.3–0.45, but the CSF/blood glucose ratio of the Glut1DS-like patient was within the normal range (0.69; normal ratio, 0.6) [7]. The age at onset of the initial clinical symptoms of the patients with an SLC2A1 mutation ranged from 2 to 15 months. Details of the clinical symptoms (seizure type, ID severity before KD therapy and at the time of the survey, and type of chronic or paroxysmal movement disorders) are shown in Table 1. Ten of the 13 patients were treated with KD therapy after the diagnosis of Glut1DS and all patients with epilepsy were treated with antiepileptic drugs.

3.2. GLUT1 levels quantified by flow cytometry

Relative GLUT1 levels on RBCs quantified by flow cytometry are indicated as a percentage compared with healthy controls (Table 1). Relative GLUT1 levels on RBCs from the healthy controls (2 males, 11 females) were 97.2, 95.9, 97.1, 99.6, 95.8, 97.5, 97.2, 96.8, 94.8, 90.3, 96.8, 98.7, and 96.9%. The mean value of the healthy controls (n = 13) was 96.5 ± 0.61% (range: 90.3–99.6%). The relative GLUT1 level was not reduced in the Glut1DS-like patient without an SLC2A1 mutation, and this patient was excluded from statistical analysis. The mean relative GLUT1 levels in the Glut1DS patients with an SLC2A1 mutation (n = 13) was 82.0 ± 3.0% (range: 63.4–99.2%), which was significantly reduced in comparison with the healthy controls (P < 0.05). Typical histograms of a case with mild ID and a missense mutation and a case with profound severe ID and a missense mutation in comparison with healthy controls are shown in Fig. 2. The histograms of Glut1DS patients indicated a left shift related to ID severity. A scatter diagram of relative GLUT1 levels on RBCs is shown in Fig. 3. GLUT1 levels in 11 of 13 patients with an SLC2A1 mutation were <90% of those of healthy controls, but two patients with a missense mutation and average-low average IQ did not have reduced GLUT1 levels. Our results indicated a sensitivity of 84.6% and specificity of 100% (cut-off value 90%).

Fig. 2.

Fig. 2

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Typical histograms of GLUT1 levels on RBCs of Glut1DS patients. Green line: Glut1DS case with a missense mutation and mild ID (c.971C > T, p.Ser324Leu); blue line: Glut1DS case with a missense mutation and profound severe ID (c.458G > A, p.Arg153His); red line: healthy control without an SLC2A1 mutation. The horizontal axis is the fluorescence intensity of GFP (GFP-fused GLUT1, GLUT1-GFP) and the vertical axis is the RBC count. All samples were analyzed for 10,000 RBCs and gated CD235a-PE- and GLUT1-GFP-positive cell populations. The histograms of Glut1DS patients indicated a left-side shift related to ID severity.

Fig. 3.

Fig. 3

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Scatter diagram of relative GLUT1 levels on RBCs. Relative GLUT1 levels on RBCs of all patients are indicated as a percentage in the scatter diagram. Relative GLUT1 levels in healthy controls and a Glut1DS-like patient were not reduced. Relative GLUT1 levels in 11 of 13 patients with Glut1DS were < 90% of those of healthy controls. Pink: healthy controls (n = 13); green: Glut1DS-like patient; yellow-brown: Glut1DS patients with a missense mutation (n = 8); purple: Glut1DS patient with a frameshift mutation; light blue: Glut1DS patients with a nonsense mutation (n = 2); blue: Glut1DS patients with a deletion (n = 2). In the missense mutation group, we divided it into three groups according to ID severity before KD therapy; pale yellow: average and low average IQ patients (n = 2); yellow: mild-moderate ID patients (n = 4); brown: severe-profound severe ID patients (n = 2).

We assessed relative GLUT1 levels on RBCs classified according to genotype and ID severity (Fig. 4, Fig. 5). Relative GLUT1 levels were 96.5 ± 0.61% in the normal SLC2A1 group (n = 13), 84.5 ± 4.4% in the missense group (n = 8), and 78.0 ± 3.3% in the frameshift, nonsense, and deletion group (n = 5). Relative GLUT1 levels were significantly reduced for every type of SLC2A1 mutation compared with the normal SLC2A1 group (P < 0.05), and the relative GLUT1 levels in the frameshift, nonsense, and deletion group were lower than those in the missense group (Fig. 4). Relative GLUT1 levels were 96.5 ± 0.61% in the healthy control normal IQ group (n = 13), 82.2 ± 4.7% in the mild-moderate ID group (n = 4), and 77.5 ± 3.6% in the severe-profound severe ID group (n = 7) (Fig. 5). Two patients with average or low average IQ before KD therapy had median GLUT1 levels of 97.5% (99.2 and 95.8%). In the mild-moderate and severe-profound severe ID groups, relative GLUT1 levels were significantly reduced compared with healthy controls (P < 0.05) (Fig. 5). We also compared relative GLUT1 levels between different levels of ID at the time of the survey because the ID of some of the Glut1DS patients who received KD therapy had worsened because of poor compliance. However, the relative GLUT1 levels of the Glut1DS patients with ID at the time of the survey had not changed compared with before KD therapy (Supplemental Fig. 1). The relative GLUT1 levels of the Glut1DS patients with spasticity were significantly reduced compared with those without spasticity (P < 0.05) (Supplemental Fig. 2). There was no significant difference in relative GLUT1 levels according to seizure type.

Fig. 4.

Fig. 4

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Relative GLUT1 levels on RBCs classified according to genotype. Relative GLUT1 levels were significantly reduced in the missense group and frameshift/nonsense/deletion group compared with the healthy controls.

Fig. 5.

Fig. 5

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Relative GLUT1 levels on RBCs classified according to ID severity before KD therapy. In the mild-moderate and severe-profound severe groups, relative GLUT1 levels were significantly reduced. Two of the median GLUT1 levels in the average and low average ID groups were approximately equivalent to those in healthy controls.

4. Discussion

We confirmed that relative GLUT1 levels measured by flow cytometry were well correlated with the type of SLC2A1 mutation, ID severity before KD therapy, and presence of spasticity at the time of the survey. The erythrocyte 3-OMG uptake assay is useful for the diagnosis of Glut1DS, but it requires specific facilities capable of dealing with radioisotopes. The flow cytometry assay used in the present study can be performed at many facilities and requires less time than the erythrocyte 3-OMG uptake assay [23,24]. In 2017, flow cytometry analysis of GLUT1 expression on RBCs was reported to indicate a significant reduction in expression and no overlap between their patient groups and healthy controls in 78% of Glut1DS patients [16]. Another study analyzed a case with two inherited missense mutations of SLC2A1 and her parents, and found a reduction of GLUT1 expression on RBCs measured by flow cytometry, despite showing normal values in the erythrocyte 3-OMG assay [25]. Moreover, in a recent prospective population-based national cohort, flow cytometry analysis revealed a high incidence of SLC2A1 mutations in patients with childhood-onset genetic epilepsy [26]. By analyzing the surface GLUT1 levels of Japanese Glut1DS patients, we also provide support for the usefulness of the flow cytometric method and demonstrated a correlation between GLUT1 levels and the mutational and clinical severity of Glut1DS.

A CSF assay has been developed for the diagnosis of Glut1DS [20,27]. The CSF/blood glucose ratio is reportedly below the 10th percentile in 91% of Glut1DS patients [28]. In other reviews, the cut-off values for CSF glucose levels and the CSF/glucose ratio for the diagnosis of Glut1DS were defined as <40 mg/dL and < 0.45, respectively [[6], [7], [8]]. We reported that CSF/blood glucose levels were significantly lower in Glut1DS patients with a missense mutation than in Glut1DS patients with a truncating mutation, but there was no significant difference in ID severity [8]. This is in contrast with the flow cytometric method presented here, which demonstrated an association with ID severity in severe Glut1DS patients. Lumbar puncture for CSF collection is an invasive procedure and requires a fasting time of at least 4–6 h, which is a risk factor for hypoglycemia, especially in children. The flow cytometric method is less burdensome and risky than CSF collection and does not require fasting.

As a limitation of the present study, we failed to detect a reduction of surface GLUT1 expression for two variants. Especially for the p. Ser324Leu variant, three siblings (Patients #1, 4, and 5) with different ID (one average ID and two mild ID) suggested a correlation between relative GLUT1 levels on RBCs (95.8, 89.8, and 87.6%, respectively) and ID severity. Furthermore, we did not analyze the expression of GLUT1 mRNA according to type and mutated allele in this analysis, which may reveal that genetic heterogeneity is related to the skewed expression of the wild-type and mutant allele. The reduction of GLUT1 levels measured by this assay was influenced by the expression levels of GLUT1 and structural changes of the RBD-binding site (extracellular loop 6 of GLUT1) [16,29,30]. Therefore, this method may fail to detect SLC2A1 mutations that decrease glucose transport ability without a structural change in the RBD-binding site, including mutations altering the “transition state change” of GLUT1 [29].

In our mutations, Arg153His and Arg333Trp were suggested to destabilize salt bridges between the GLUT1 side chains and impair sugar translocation [29]. The relative GLUT1 levels in severe cases with Arg153His and Arg333Trp were able to capture the influence of these mutations as a reduction of 63.4% and 88.8%, respectively. Moreover, Arg126 is located close to the outer face of GLUT1, and Arg126His was predicted to alter the conformational stability and cause a dysfunction of sugar binding in the exofacial site [30]. The relative GLUT1 level for Arg126His was 83.1%, indicating a moderate decrease and reflecting a structural change of the exofacial site. These reductions suggest that function changes of GLUT1 are well correlated with structural changes of the RBD-binding site.

Some of the patients with a missense mutation and normal or mild ID were not detected in our analysis. Therefore, a final diagnosis combined with an SLC2A1 mutation assay and analysis of CSF glucose levels is necessary for patients with normal or mild ID.

5. Conclusions

Relative GLUT1 levels on RBCs measured by flow cytometry were well correlated with the SLC2A1 mutation associated with the surface expression of GLUT1, ID severity before KD therapy, and the presence of spasticity associated with upper motor neuron disorders under chronic hypoglycorrhachia. This method is simple and less invasive compared with the CSF assay as a screening method for Glut1DS. In the future, this method may become an appropriate approach for identifying severe Glut1DS patients in the early stage before the development of neurological damage.

Funding

This work was supported by grants from the Japan Society for the Promotion of Science, JSPS KAKENHI [grant number 19K17374], grants for the Project for Health Research on Infants, Children, Adolescents, and Young Adults from the Agency of Medical Research and Development, and grants from the Japan Agency for Medical Research and Development (AMED) [grant numbers 19lm0203049h0002, JP20ae0201007].

CRediT authorship contribution statement

Sachie Nakamura: Conceptualization, Methodology, Formal analysis, Funding acquisition, Visualization, Writing – original draft. Yasushi Ito: Resources, Data curation, Investigation, Validation. Hiroko Hayakawa: Formal analysis, Software. Shiho Aoki: Formal analysis, Software. Takanori Yamagata: Supervision. Hitoshi Osaka: Project administration, Funding acquisition, Writing – review & editing.

Acknowledgements

We are grateful to all of the patients who sent valuable information and cooperate collecting the blood in the study.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymgmr.2022.100954.

Appendix A. Supplementary data

Supplementary material Supplementary Fig. 1 Relative GLUT1 levels on RBCs classified according to ID severity at the time of the survey. After starting KD therapy, antiepileptic drugs, and thyrotropin-releasing hormone-tartrate, ID severity improved in four patients. We assessed relative GLUT1 levels on RBCs classified according to ID severity at the time of the survey. The relative GLUT1 levels of the low average-borderline ID group, mild ID group, and severe-profound severe ID groups were 92.7 ± 4.9%, 82.0 ± 6.7%, and 77.5 ± 3.6%, respectively.

Supplementary Fig. 2 Relative GLUT1 levels on RBCs classified according to spasticity. Spasticity was caused by upper neuron disorders under chronic hypoglycorrhachia. Relative GLUT1 levels were significantly reduced in the patients with spasticity (73.4 ± 3.5%) compared to those without spasticity (89.4 ± 2.3%).

mmc1.pptx (245KB, pptx)

Data availability

No data was used for the research described in the article.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material Supplementary Fig. 1 Relative GLUT1 levels on RBCs classified according to ID severity at the time of the survey. After starting KD therapy, antiepileptic drugs, and thyrotropin-releasing hormone-tartrate, ID severity improved in four patients. We assessed relative GLUT1 levels on RBCs classified according to ID severity at the time of the survey. The relative GLUT1 levels of the low average-borderline ID group, mild ID group, and severe-profound severe ID groups were 92.7 ± 4.9%, 82.0 ± 6.7%, and 77.5 ± 3.6%, respectively.

Supplementary Fig. 2 Relative GLUT1 levels on RBCs classified according to spasticity. Spasticity was caused by upper neuron disorders under chronic hypoglycorrhachia. Relative GLUT1 levels were significantly reduced in the patients with spasticity (73.4 ± 3.5%) compared to those without spasticity (89.4 ± 2.3%).

mmc1.pptx (245KB, pptx)

Data Availability Statement

No data was used for the research described in the article.

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