Enzymatic characterization of four new mutations in the glucose-6 phosphatase (G6PC) gene which cause glycogen storage disease type 1a

Glycogen storage disease type la (GSD1a) is caused by mutations in the gene of glucose-6 phosphatase (G6PC), encoding the last enzyme of gluconeogenesis and glycogenolysis. To study the effect of mutations previously identified, but not yet enzymatically characterized, in French GSD1a patients, we used an in vitro expression system of the human glucose-6 phosphatase (hGlc6Pase) cDNA. Wild type hGlc6Pase expressed in COS-7 cells exhibited kinetic features comparable to microsomal Glc6Pase from normal human liver and kidney. Pour new mutations inducing aminoacid changes in the coding sequence, e.g. W77R, A124T, G184E and L211P, were inserted into the Glc6Pase cDNA by site-directed mutagenesis, and studied after transient expression in COS-7 cells. All four mutations totally abolished Glc6Pase activity.