title: Functional characterization of the novel mutation IVS 8 (-11delC) (-14T>A) in the intron 8 of the glucocerebrosidase gene of two Italian siblings with Gaucher disease type I.
abstract: Gaucher disease, the most common glycolipid storage disease, can be caused by a large variety of mutations. We report here the identification and characterization of a novel mutation in the human glucocerebrosidase gene, IVS 8 (-11delC) (-14T>A), in two siblings with Gaucher disease type I which occurs within the 3' end of intron 8. Both siblings were compound heterozygotes for the IVS 8 (-11delC) (-14T>A) mutation and for the c.626 G>C (R170P) substitution within exon 6. No mRNA species carrying the IVS 8 (-11delC) (-14T>A) mutation were detected by RT-PCR analysis of the RNA extracted from the patients' fibroblasts. To study the possible effects of the IVS 8 (-11delC) (-14T>A) sequence alteration on the splicing of the proximal exon 9, we have established an in vitro system generating a minigene carrying the genomic region of human glucocerebrosidase spanning from exon 8 to exon 10. Transfections into the human Hep3B cell line of the wild-type construct resulted in the expression of mRNA with the glucocerebrosidase exons correctly spliced. On the contrary, transfections of the construct carrying the IVS 8 (-11delC) (-14T>A) mutation resulted in the expression of mRNA with an 11-bp insertion located between the end of exon 8 and the beginning of exon 9. These results indicated that the 5243T>A substitution created a new 3' splice site 11 bp upstream of the wild-type one, leading to the incorporation into the mRNA of these extra 11 bases. Moreover, the new 3' splice site created by this 5243T>A transversion was preferred over the wild-type one in 100% of cases. The in vitro studies suggest that, in the patients, the 11-bp inclusion causes a shift in the reading frame with the generation of a stop codon after codon 388 which undergoes early degradation.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Padriciano 99, Trieste, 34012, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Romano
authors.forename: G M
authors.initials: GM
authors.lastname: Danek
authors.forename: F E
authors.initials: FE
authors.lastname: Baralle
authors.forename: R
authors.initials: R
authors.lastname: Mazzotti
authors.forename: M
authors.initials: M
authors.lastname: Filocamo
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D001483:Base Sequence; D005091:Exons; D005260:Female; D005776:Gaucher Disease / Q000201:enzymology / Q000235:genetics*; D005962:Glucosylceramidase / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D007438:Introns; D007558:Italy; D008297:Male; D008969:Molecular Sequence Data; D009679:Nuclear Family; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D005962:Glucosylceramidase