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

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 patientsʹ 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.