Abstract Details

Title Functional Studies Confirm Relative Pathogenicity of Serine 66 Mutations in Glut1 Deficiency

Topic Child Neurology/Developmental Neurobiology

Presentation(s) P02 - (-)

Poster/Presentation
Number 092

Objective

Background BACKGROUND: Glut1 Deficiency Syndrome (Glut1 DS) was first described by De Vivo et al in 1991.The classic phenotype is characterized by seizures, development delay, acquired microcephaly, and complex movement disorders. Glut1 is the fundamental transporter that facilitates glucose entry into the brain. The defect of glucose transport across the blood brain caused by GLUT1 mutations is the molecular basis for the Glut1 DS.

Design/Methods DESIGN/METHODS: Mutagenesis and cRNA preparation from cDNA templates.S66A, S66F, S66T, S66Y and Wild-type (WT) human GLUT1 mutants were constructed. The c-RNAs were synthesized and injected into Xenopus oocytes for the mutagenesis studies. Zero-trans influx of 3--O-Methyl-D-Glucose (3-OMG), equilibrium exchange influx and zero-trans efflux of 3-OMG in Xenopus oocytes were determined as described previously. The kinetics was calculated with GaphPad Prism 6. Confocal immunofluorescence microscopy and Western blot analysis were performed as described previously. The amount of protein expressed in the oocyte membrane was normalized to WT, which was used to normalize the Vmax values.

Results RESULTS: Under Zero-trans influx, equilibrium exchange influx, and Zero-trans efflux of 3-OMG, the Km values were as follows (in mM): WT 20.2, 139, and 144; S66A 12.9, 58.6, and 160.3; S66F 82.1, 21.0, and 41.8; S66T 9.7, 40.8, and 65.6; S66Y 85.2, 40.8 and 22.8. Vmax were significantly decreased in S66F (9.5%, 1.2% and 0.8%), S66Y (5%, 0.9% and 0.5%) and moderately decreased in S66T (70.1%, 7.4% and 54.1%), variable in S66A (96%, 66.8% and 141%). Confocal studies confirmed efficient membrane expression of the WT and the S66 Glut1 mutant proteins. These findings are consistent with a central role for size rather than hydropathy at position S66 of this mutation.

Conclusions CONCLUSIONS: Expression studies confirm S66F as a disease-causing Glut1 mutation.

Authors/Disclosures
Dong Wang, MD (Neurology Specialts of GA) PRESENTER	No disclosure on file
Hong Yang, MD (Columbia University)	No disclosure on file
Darryl C. De Vivo, MD, FAAN (Columbia University)	Dr. De Vivo has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Biogen and Novartis. Dr. De Vivo has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Aspa Therapeutics.
Michael S. Okun, MD, FAAN (University of Florida)	Dr. Okun has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for NIH. Dr. Okun has received personal compensation in the range of $10,000-$49,999 for serving as an officer or member of the Board of Directors for Parkinson's Foundation. Dr. Okun has received personal compensation in the range of $5,000-$9,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for JAMA Neurology. Dr. Okun has received personal compensation in the range of $500-$4,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for NEJM Journal Watch. The institution of Dr. Okun has received research support from NIH. The institution of Dr. Okun has received research support from Parkinson's Foundation. The institution of Dr. Okun has received research support from Tourette Association of America. The institution of Dr. Okun has received research support from Michael J Fox. Dr. Okun has received publishing royalties from a publication relating to health care.
Jennifer M. Cermak, PhD (Flex Pharma)	No disclosure on file

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