A novel mutation deep within intron 7 of the GBA gene causes Gaucher disease

Abstract

Background Mutations in the GBA gene that encodes the lysosomal enzyme acid β‐glucocerebrosidase cause Gaucher disease (GD), the most common lysosomal storage disorder. Most of the mutations are missense/nonsense, however, a few splicing mutations within or close to conserved consensus donor or acceptor splice sites have also been described. The aim of the study was to identify the mutation(s) in a Cypriot patient with type I GD. Methods The genomic DNA of the proband was screened for nine common mutations using Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. All exons and exon‐intron boundaries, and the 5’UTR and 3’UTR regions of the GBA gene, were investigated by Sanger sequencing. RNA analysis was performed using standard procedures, and the abnormal transcript was further cloned into pGEM‐T‐Easy plasmid vector and sequenced. The relevant intronic region was further sequenced by the Sanger method to identify the genetic variant. Results A novel point mutation, g.12599C > A (c.999 + 242C > A), was detected deep in intron 7 of the GBA gene. This type of mutation has been previously described for other diseases but this is the first time, as far as we know, that it is described for GD. This mutation creates a new donor splice site leading to aberrant splicing and resulting in the insertion of the first 239nt of intron 7 as a pseudoexon in the mRNA, creating a premature stop codon. Conclusion This study expands the mutation spectrum of GD and highlights the importance of RNA sequencing for the molecular diagnosis of patients bearing mutations in nonexonic regions.

Effect of expression of human glucosylceramidase 2 isoforms on lipid profiles in COS-7 cells

“…Similar increases in GlcCer were seen in the brains of Niemann-Pick Type C model mice, when GBA2 was knocked out or inhibited ( 37) . Glucosylceramides were also significantly higher in lymphoblastoid cells from a patient with a homozygous GBA2 mutation compared to control lymphoblastoid cells ( 20) . So, in general, our observations in overexpression of GBA2 on sphingolipids are opposite to those seen in animal and human cells with GBA2 deficiency, as expected, except that we also detected a significant change in ceramide levels.…”

“…GBA2 enzyme activity was assayed as described elsewhere ( 8,20,21) . Samples were pre-incubated with or without CBE, followed by incubated with the 4-methylumbelliferyl-β-D-glucoside (4MUG) substrate, 3.5 mM final concentration (Sigma-Aldrich) at pH 5.8 and 37 ο C for 30 min.The reactions were terminated by adding 200 µL 1 M of glycine, pH 10.6, then the fluorescent signal was measured in a fluorescence microplate reader with excitation at 355 nm and emission at 460 nm.…”

“…The relevance of alternative splicing is made clear by certain point mutations. For instance, the g. 12599C > A ( c. 999 + 242C > A mutation, found deep in intron 7 of the GBA gene, creates donor splice site 3 nucleotides 5' of this mutation, leading to aberrant splicing that results in the insertion of the first 239nt of intron 7 (20). In contrast, the biological significance of GBA2 alternative splicing in neurological diseases has yet to be reported.…”

Intronic Haplotypes in the GBA Gene Do Not Predict Age at Diagnosis of Parkinson's Disease

“…Nonetheless, some deep intronic variants have been linked directly to genetic disorders, such as Gaucher disease. 6,7 Two common haplotypes were identified, differentiated by three intronic single nucleotide polymorphisms in GBA. These correspond to the previously reported 1.1+ and 1.1− haplotypes.…”

“…6 Deep intronic variants are not commonly regarded as pathogenic as they do not result in amino acid changes in proteins. Nonetheless, some deep intronic variants have been linked directly to genetic disorders, such as Gaucher disease 7,8 . Two common haplotypes were identified, differentiated by three intronic single nucleotide polymorphisms in GBA .…”