Glycogenosis type II: identification and expression of three novel mutations in the acid alpha-glucosidase gene causing the infantile form of the disease

Glycogenosis type II (GSDII) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). We identified three novel point mutations, C399A, T1064C, and C2104T, in three unrelated Italian patients with the infantile form of the disease. The C399A mutation was present in homozygosity in proband 1. The C >A transition introduces a premature stop signal in exon 2 resulting in no enzyme production that is correlated with the severe clinical phenotype in this patient. The other two nucleotide changes were missense mutations. The T1064C mutation, which changes Leu in position 355 into Pro, was carried in homozygosity by proband 2. The C2104T nucleotide change, which substitutes Arg 702 into Cys, was present in proband 3 in combination with a known severe mutation DeltaI17-18. The in vitro expression in COS-1 cells of T1064C and C2104T constructs demonstrated no enzymatic activity with respect to the negative control cells. Western blot analysis revealed that both T1064C and C2104T mutant proteins produced in COS-1 cells migrated in SDS-PAGE as the GAA inactive precursor of 110kDa. Immunofluorescence detection of mutant alpha-glucosidases showed enzyme localization primarily in the ER-Golgi compartment, suggesting that T1064C and C2104T mutations could affect the normal processing and stability of the enzyme. In vitro studies demonstrated that the same degree of deficiency in T1064C and C2104T mutations, which is in contrast with patient phenotype. A better correlation was observed with the in vivo studies since proband 2, with a less severe phenotype, presented with low residual enzyme activity while in proband 3, with a classic severe infantile onset GSDII, fibroblast enzyme activity was completely absent.