Identification and functional characterization of five novel mutant alleles in 58 Italian patients with Gaucher disease type 1.
Gaucher disease (GD) is the most frequent lysosomal glycolipid storage disorder due to an autosomal recessive deficiency of acid beta-glucosidase characterized by the accumulation of glucocerebroside. In this work we carried out the molecular analysis of the glucocerebrosidase gene (GBA) in 58 unrelated patients with GD type 1. We identified five novel genetic alterations: three missense changes c.187G>A (p.D63N), c.473T>G (p.I158S), c.689T>A (p.V230E), a gene-pseudogene recombinant allele and a non-pseudogene-derived complex allele [c.1379G>A;c.1469A>G] encoding [p.G460D;p.H490R]. All mutant alleles were present as compound heterozygotes in association with c.1226A>G (p.N409S), the most common mutation in GD1. The missense mutant proteins were expressed in vitro in COS-1 cells and analyzed by enzyme activity, protein processing and intracellular localization. Functional studies also included the c.662C>T (p.P221L) mutation recently reported in the Spanish GD population (Montfort et al., 2004). The missense mutant alleles retained an extremely low residual enzyme activity with respect to wild type; the complex allele expressed no activity. Processing of the mutant proteins was unaltered except for c.473T>G which was differently glycosylated due to the exposition of an additional glycosylation site. Immunofluorescence studies showed that protein trafficking into the lysosomes was unaffected in all cases. Finally, the characterization of the novel recombinant allele identified a crossover involving the GBA gene and pseudogene between intron 5 and exon 7.
Miocić
Snjezana
S
Unità di Malattie Metaboliche, I.R.C.C.S. Burlo Garofolo, Trieste, Laboratorio Diagnosi Pre-Postnatale Malattie Metaboliche, I.R.C.C.S.G. Gaslini, Genova and International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Filocamo
Mirella
M
Dominissini
Silvia
S
Montalvo
Anna Lisa E
AL
Vlahovicek
Kristian
K
Deganuto
Marta
M
Mazzotti
Raffaella
R
Cariati
Roberta
R
Bembi
Bruno
B
Pittis
Maria Gabriela
MG
D000328:Adult; D000483:Alleles; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D005260:Female; D005776:Gaucher Disease / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009154:Mutation; D020125:Mutation, Missense; D011544:Pseudogenes; D011995:Recombination, Genetic