Zaman SM et al. (2018), Development of a rapid functional assay that pr...

XB-ART-55576

Neurol Genet 2018 Dec 06;46:e297. doi: 10.1212/NXG.0000000000000297.

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Development of a rapid functional assay that predicts GLUT1 disease severity.

Zaman SM , Mullen SA , Petrovski S , Maljevic S , Gazina EV , Phillips AM , Jones GD , Hildebrand MS , Damiano J , Auvin S , Lerche H , Weber YG , Berkovic SF , Scheffer IE , Reid CA , Petrou S .

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Objective: To examine the genotype to phenotype connection in glucose transporter type 1 (GLUT1) deficiency and whether a simple functional assay can predict disease outcome from genetic sequence alone. Methods: GLUT1 deficiency, due to mutations in SLC2A1, causes a wide range of epilepsies. One possible mechanism for this is variable impact of mutations on GLUT1 function. To test this, we measured glucose transport by GLUT1 variants identified in population controls and patients with mild to severe epilepsies. Controls were reference sequence from the NCBI and 4 population missense variants chosen from public reference control databases. Nine variants associated with epilepsies or movement disorders, with normal intellect in all individuals, formed the mild group. The severe group included 5 missense variants associated with classical GLUT1 encephalopathy. GLUT1 variants were expressed in Xenopus laevis oocytes, and glucose uptake was measured to determine kinetics (Vmax) and affinity (Km). Results: Disease severity inversely correlated with rate of glucose transport between control (Vmax = 28 ± 5), mild (Vmax = 16 ± 3), and severe (Vmax = 3 ± 1) groups, respectively. Affinities of glucose binding in control (Km = 55 ± 18) and mild (Km = 43 ± 10) groups were not significantly different, whereas affinity was indeterminate in the severe group because of low transport rates. Simplified analysis of glucose transport at high concentration (100 mM) was equally effective at separating the groups. Conclusions: Disease severity can be partly explained by the extent of GLUT1 dysfunction. This simple Xenopus oocyte assay complements genetic and clinical assessments. In prenatal diagnosis, this simple oocyte glucose uptake assay could be useful because standard clinical assessments are not available.

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Species referenced: Xenopus laevis
Genes referenced: slc2a1

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	Figure 1. Distribution of disease-causing and population variants along the SLC2A1 geneWe used lollipops-v1.3.1 (github.com/pbnjay/lollipops/releases) to plot the distribution of gnomAD's 159 filter-passed missense variants (blue circles) based on the SLC2A1 canonical transcript (NM_006516.2; uniprot P11166). These represent normal variation in the gene. We also plotted the distribution of our 14 studied missense variants (black diamonds), with unfilled diamonds representing those studied in this article and that were reported in the ClinVar and HGMD screen, as described above. First, a search for “pathogenic,” “likely pathogenic,” or “likely pathogenic; Pathogenic” missense variants in ClinVar (accessed in December 2016) was performed, subsequently a search for “DM” classified variants was performed based on HGMD (hgmd2016.3). A review of the relevant entries and their associated literature found that 32 SLC2A1 case-ascertained missense variants (red circles) were accompanied with written commentary that the variants either arose de novo in the patient (n = 29 variants) or there was evidence of the variant segregating among all (and >3) affected carriers and without >1 unaffected carriers in the pedigree (n = 3 variants).
	Figure 2. Effects on glucose transport of variants leading to mild GLUT1 diseaseVariants in the mild cohort (orange) compared with the average reference curve (green). Curves (A–I) demonstrate a broad range of residual GLUT1 protein function, with (A–H) probands indicating a significant decrease in glucose uptake (p < 0.0001) when compared with the unaffected curve. Variant I, although presented with a mild phenotype, showed elevated glucose transport (p <0.0001). (J) Highlights the separation between the average of all mild variants (excluding the gain-of-function variant, I) at each concentration compared with the average unaffected curve.
	Figure 3. Effects on glucose transport of variants leading to severe GLUT1 disease(A–E) (red curves) variants associated with severe GLUT1 encephalopathy phenotype demonstrated a significantly marked reduction of glucose uptake compared with average protein function (p <0.0001) (green curves). (F) Highlights the marked separation of the average severe variants compared with the average unaffected curve.
	Figure 4. Comparison of glucose transport across variants seen in the population, mild disease, and severe disease(A) Box and whisker plot for Vmax values obtained from the Michaelis-Menten curves for the unaffected (n = 5), mild (n = 9), and severe (n = 5) variants (single value scatter plots shown in figure e-1, links.lww.com/NXG/A129). (B) Box and whisker plot for the velocity at 100 mM glucose concentration for each group of variants. The variant resulting in NP_006507.2 p.E209D has been positioned as an outlier and not included in the population analysis.

References [+] :

Arsov, Early onset absence epilepsy: 1 in 10 cases is caused by GLUT1 deficiency. 2012, Pubmed