Loss-of-function of an N-acetylglucosaminyltransferase, POMGnT1, in muscle–eye–brain disease
Section snippets
Materials and methods
Construction of POMGnT1 mutants. An expression vector encoding each mutant of POMGnT1 was prepared by site-directed mutagenesis. Template cDNA for site-directed mutagenesis encoding full-length POMGnT1 tagged with the His-tag and Xpress epitope was cloned into pcDNA 3.1 Zeo(+) (Invitrogen), as described previously [7]. Site-directed mutagenesis for missense and frameshift mutants (E223K, C269Y, P493R, H573fs, L611fs, and V626fs) was performed using a QuickChange Site-Directed Mutagenesis Kit
Results and discussion
Previously, we identified 13 mutations in the POMGnT1 gene in patients with MEB [7], [8]. These mutations were all simple point mutations that caused nonsense, missense, frameshift and premature termination, and splicing-site mutation (read-through of intronic sequences and/or skipping of the upstream exon) (Fig. 1). They are dispersed through the entire POMGnT1 gene. MEB patients have either homozygous or compound heterozygous mutations in the gene. Among these mutated enzymes, only two were
Acknowledgements
We thank Dr. James Raymond for editing the manuscript. This study was supported by a Research Grant for Nervous and Mental Disorders (14B-4) from the Ministry of Health, Labour and Welfare of Japan, by a Grant-in-Aid for Scientific Research on Priority Area (14082209) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and grants from The Naito Foundation and Yamanouchi Foundation for Research on Metabolic Disorders.
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