title: In silico and functional studies of the regulation of the glucocerebrosidase gene.
abstract: In Gaucher disease (GD), the inherited deficiency of glucocerebrosidase results in the accumulation of glucocerebroside within lysosomes. Although almost 300 mutations in the glucocerebrosidase gene (GBA) have been identified, the ability to predict phenotype from genotype is quite limited. In this study, we sought to examine potential GBA transcriptional regulatory elements for variants that contribute to phenotypic diversity. Specifically, we generated the genomic sequence for the orthologous genomic region ( approximately 39.4kb) encompassing GBA in eight non-human mammals. Computational comparisons of the resulting sequences, using human sequence as the reference, allowed the identification of multi-species conserved sequences (MCSs). Further analyses predicted the presence of two putative clusters of transcriptional regulatory elements upstream and downstream of GBA, containing five and three transcription factor-binding sites (TFBSs), respectively. A firefly luciferase (Fluc) reporter construct containing sequence flanking the GBA gene was used to test the functional consequences of altering these conserved sequences. The predicted TFBSs were individually altered by targeted mutagenesis, resulting in enhanced Fluc expression for one site and decreased expression for seven others sites. Gel-shift assays confirmed the loss of nuclear-protein binding for several of the mutated constructs. These identified conserved non-coding sequences flanking GBA could play a role in the transcriptional regulation of the gene contributing to the complexity underlying the phenotypic diversity seen in GD.
authors.affiliation: Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3708, USA.
authors.forename: Yotam N
authors.initials: YN
authors.lastname: Blech-Hermoni
authors.forename: Shira G
authors.initials: SG
authors.lastname: Ziegler
authors.forename: Kathleen S
authors.initials: KS
authors.lastname: Hruska
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Stubblefield
authors.forename: Mary E
authors.initials: ME
authors.lastname: Lamarca
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Portnoy
authors.forename: Eric D
authors.initials: ED
authors.lastname: Green
authors.forename: Ellen
authors.initials: E
authors.lastname: Sidransky
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D019295:Computational Biology / Q000379:methods*; D017124:Conserved Sequence; D004285:Dogs; D005776:Gaucher Disease / Q000235:genetics / Q000503:physiopathology; D015971:Gene Expression Regulation, Enzymologic; D005962:Glucosylceramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D013045:Species Specificity; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014714:Vertebrates / Q000145:classification / Q000235:genetics
chemical_list: D014157:Transcription Factors; D049409:Luciferases, Firefly; D005962:Glucosylceramidase