Sachie Nakamura1, Hitoshi Osaka2, Shinichi Muramatsu3, Shiho Aoki1, Eriko F Jimbo1, Takanori Yamagata1. 1. Department of Pediatrics, Jichi Medical University, Tochigi, Japan. 2. Department of Pediatrics, Jichi Medical University, Tochigi, Japan. Electronic address: hosaka@jichi.ac.jp. 3. Division of Neurology, Jichi Medical University, Tochigi, Japan; Center for Gene and Cell Therapy, The Institute of Medical Science, The University of Tokyo, Japan.
Abstract
OBJECTIVE: We investigated a correlation between a mutation in the SLC2A1 gene and functional disorders in Glucose transporter I deficiency syndrome (GLUT1DS). METHODS: We performed direct sequence analysis of SLC2A1 in a severe GLUT1DS patient and identified a novel frame shift mutation, c.906_907insG, p.V303fs. We created a plasmid vector carrying the c.906_907insG mutation, as well as A405D or R333W in the SLC2A1, which are found in patients with mild and moderate GLUT1DS severity, respectively. We transiently expressed these mutants and wild type SLC2A1 plasmids in a human embryonic kidney cell line (HEK293), and performed immunoblotting, immunofluorescence, and enzymatic photometric 2-deoxyglucose (2DG) uptake assays. RESULTS: GLUT1 was not detected after transient expression of the SLC2A1 plasmid carrying c.906_907insG by either immunoblotting or immunofluorescence. The degree of glucose transport reduction as determined by enzymatic photometric 2DG assay uptake correlated with disease severity. CONCLUSIONS: Enzymatic photometric 2DG uptake study appears to be a suitable functional assay to predict the effect of SLC2A1 mutations on GLUT1 transport.
OBJECTIVE: We investigated a correlation between a mutation in the SLC2A1 gene and functional disorders in Glucose transporter I deficiency syndrome (GLUT1DS). METHODS: We performed direct sequence analysis of SLC2A1 in a severe GLUT1DSpatient and identified a novel frame shift mutation, c.906_907insG, p.V303fs. We created a plasmid vector carrying the c.906_907insG mutation, as well as A405D or R333W in the SLC2A1, which are found in patients with mild and moderate GLUT1DS severity, respectively. We transiently expressed these mutants and wild type SLC2A1 plasmids in a humanembryonic kidney cell line (HEK293), and performed immunoblotting, immunofluorescence, and enzymatic photometric 2-deoxyglucose (2DG) uptake assays. RESULTS:GLUT1 was not detected after transient expression of the SLC2A1 plasmid carrying c.906_907insG by either immunoblotting or immunofluorescence. The degree of glucose transport reduction as determined by enzymatic photometric 2DG assay uptake correlated with disease severity. CONCLUSIONS: Enzymatic photometric 2DG uptake study appears to be a suitable functional assay to predict the effect of SLC2A1 mutations on GLUT1 transport.