title: Identification and functional characterization of five novel mutant alleles in 58 Italian patients with Gaucher disease type 1.
abstract: 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.
authors.affiliation: 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.
authors.forename: Snjezana
authors.initials: S
authors.lastname: Miocić
authors.forename: Mirella
authors.initials: M
authors.lastname: Filocamo
authors.forename: Silvia
authors.initials: S
authors.lastname: Dominissini
authors.forename: Anna Lisa E
authors.initials: AL
authors.lastname: Montalvo
authors.forename: Kristian
authors.initials: K
authors.lastname: Vlahovicek
authors.forename: Marta
authors.initials: M
authors.lastname: Deganuto
authors.forename: Raffaella
authors.initials: R
authors.lastname: Mazzotti
authors.forename: Roberta
authors.initials: R
authors.lastname: Cariati
authors.forename: Bruno
authors.initials: B
authors.lastname: Bembi
authors.forename: Maria Gabriela
authors.initials: MG
authors.lastname: Pittis
mesh_terms: 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