Journal: Human Mutation
Article Title: A Novel Gain‐of‐Function GLUL Variant Is Associated With Developmental and Epileptic Encephalopathy With Enlarged Perivascular Spaces
doi: 10.1155/humu/2799390
Figure Lengend Snippet: Genetic and structural characterization of the GLUL c.522_536dup variant. (a) Pedigree and Sanger sequencing chromatograms. The sequence traces from the proband (upper panel) show a double peak starting from the duplication site, consistent with heterozygosity. (b) Superimposition of the mutant GS dimer (green) onto the WT GS structure (PDB 2QC8, blue). Black dotted lines indicate the active site. The magnified view on the right shows the mutated region, with the five duplicated amino acids (p.Ile175_His179dup) highlighted in magenta. (c) Western blot analysis of GS protein expression in HEK293 cells transfected with WT, c.1021C > T, or c.522_536dup constructs. GAPDH was used as a loading control. (d) GS enzyme activity in HEK293 cells transfected with WT, c.1021C > T, or c.522_536dup constructs, measured across a range of L‐glutamine concentrations (1, 2, 4, 16, 32, and 64 mM). The c.1021C > T variant showed markedly reduced activity at all concentrations except 64 mM. The c.522_536dup variant exhibited significantly higher activity than WT at 1 mM ( p < 0.0001), 2 mM ( p < 0.05), 32 mM ( p < 0.01), and 64 mM ( p < 0.01), with no significant difference at 4 or 16 mM. # p < 0.05; ## p < 0.01; ### p < 0.0001; ns, not statistically significant. (e) Location of the GLUL variants. The variant c.522_536dup identified in this study is indicated in red. Seven variants previously reported in AR GS deficiency are shown against a green background, and six variants associated with AD DEE are shown against a yellow background.
Article Snippet: To evaluate the regulation of GS enzyme activity across different glutamine concentrations, after transfection, the medium was replaced with DMEM media (Servicebio, #G4517) supplemented with 2% penicillin/streptomycin (Gibco, #15140122), 10% FBS (Procell, #164250), and varying concentrations of L‐glutamine (1, 2, 4, 16, 32, and 64 mM) (Servicebio, #GM3031).
Techniques: Variant Assay, Sequencing, Mutagenesis, Western Blot, Expressing, Transfection, Construct, Control, Activity Assay
