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 char acterization of a novel mutation in the human glucocerebrosidase gene, NS 8 (-11delC) (-14T>A), in two siblings with Gaucher disease type I which occu rs within the 3' end of intron 8, Both siblings were compound heterozygotes for the TVS 8 (-11delC) (-14T>A) mutation and for the c.626 G>C (R170P) su bstitution within exon 6. No mRNA species carrying the IVS 8 (-11delC) (-14 T>A) mutation were detected by RT-PCR analysis of the RNA extracted from th e patients' fibroblasts. To study the possible effects of the TVS 8 (-11del C) (-14T>A) sequence alteration on the splicing of the proximal exon 9, we have established an in vitro system generating a minigene carrying the geno mic region of human glucocerebrosidase spanning from exon 8 to exon 10. Tra nsfections into the human Hep3B cell line of the wild-type construct result ed in the expression of mRNA with the glucocerebrosidase exons correctly sp liced. On the contrary, transfections of the construct carrying the IVS 8 ( -11delC) (-14T>A) mutation resulted in the expression of mRNA with an Il-bp insertion located between the end of exon 8 and the beginning of exon 9. T hese results indicated that the 5243T>A substitution created a new 3' splic e site 11 bp upstream of the wild-type one, leading to the incorporation in to the mRNA of these extra 11 bases. Moreover, the new 3' splice site creat ed 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 Il-bp i nclusion causes a shift in the reading frame with the generation of a stop codon after codon 388 which undergoes early degradation. (C) 2000 Academic Press.