title: Expression and functional characterization of mutated glucocerebrosidase alleles causing Gaucher disease in Spanish patients.
abstract: BACKGROUND
  Gaucher disease (GD) is a heterogeneous disease characterized by an impaired activity of the lysosomal glucocerebrosidase. This heterogeneity is attributed in part to the existence of a large number of mutations in the corresponding gene.
  
  
  METHODS
  To establish genotype-phenotype relationships, we analyzed the residual enzyme activities of six naturally occurring mutations found in Spanish population in the glucocerebrosidase gene [c.160G > A (V15M), c.485T>C (M123T), c.914C>T (P266L), c.1124T>C (L336P), c.1207A>C (S364R) and c.1510-1512delTCT (S465del)]. The mutated genes were subcloned into the mammalian expression vector pCR 3.1 and expressed by transient transfection in COS cells. The enzymatic activity of the expressed protein were measured and compared with the wild-type human glucocerebrosidase cDNA. Also, two previously alleles, c.1226A>G (N370S) and c.1448T>C (L444P), were used for comparative purposes.
  
  
  RESULTS
  The residual activity of the expressed proteins using the synthetic substrate (4-methylumbelliferyl-beta-D-glucopyranoside, 4MU-Glu) ranged from 5.5% (for the 3-bp deletion) to 42.7% (for S364R mutation) of the activity of the wild-type enzyme.
  
  
  CONCLUSIONS
  The present analyses may help to better understand the molecular basis and the pathogenesis of Gaucher disease. However, results of expression of mutated enzymes are necessary but not sufficient to explain the ultimate clinical outcome of Gaucher disease.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza 50009, Saragossa, Spain. palfonso@unizar.es
authors.forename: Pilar
authors.initials: P
authors.lastname: Alfonso
authors.forename: José Carlos
authors.initials: JC
authors.lastname: Rodríguez-Rey
authors.forename: Alberto
authors.initials: A
authors.lastname: Gañán
authors.forename: Juan Ignacio
authors.initials: JI
authors.lastname: Pérez-Calvo
authors.forename: Manuel
authors.initials: M
authors.lastname: Giralt
authors.forename: Pilar
authors.initials: P
authors.lastname: Giraldo
authors.forename: Miguel
authors.initials: M
authors.lastname: Pocoví
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000483:Alleles; D002478:Cells, Cultured; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D005776:Gaucher Disease / Q000235:genetics*; D005962:Glucosylceramidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007223:Infant; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017384:Sequence Deletion; D013030:Spain; D014162:Transfection; D001617:beta-Glucosidase
chemical_list: D001617:beta-Glucosidase; D005962:Glucosylceramidase