title: Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis.
abstract: The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G>T (712-1G>T) and c.2657+5G>A (2789+5G>A) and to assess the applicability of this model in CFTR therapeutic approaches. We performed the relative quantification of CFTR mRNA by reverse transcription quantitative PCR (RT-qPCR) of these splicing mutations in four groups (wild type, CF-F508del controls, CF patients and CF carriers) of individuals. In addition, in vitro assays using minigene constructs were performed to evaluate the effect of a new CF complex allele c.[2657+5G>A; 2562T>G]. Ex vivo qPCR data show that the primary consequence of both mutations at the RNA level is the skipping of their neighboring exon (6 and 16, respectively). The CFTR minigenes results mimicked the ex vivo data, as exon 16 skipping is the main aberrant transcript, and the correctly spliced transcript level was observed in a similar proportion when the c.2657+5G>A mutation is present. In summary, we provide evidence that ex vivo quantitative transcripts analysis using RT/qPCR is a robust technology that could be useful for measuring the efficacy of therapeutic approaches that attempt to achieve an increase in CFTR gene expression.
authors.affiliation: Human Molecular Genetics Group. Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain.
authors.forename: Laia
authors.initials: L
authors.lastname: Masvidal
authors.affiliation: Faculty of Sciences, BioFIG-Centre for Biodiversity, Functional and Integrative Genomics, University of Lisboa, Lisboa, Portugal.
authors.forename: Susana
authors.initials: S
authors.lastname: Igreja
authors.affiliation: Human Molecular Genetics Group. Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain.
authors.forename: Maria D
authors.initials: MD
authors.lastname: Ramos
authors.affiliation: Cystic Fibrosis Adult Unit, Hospital Vall d'Hebron, Barcelona, Spain.
authors.forename: Antoni
authors.initials: A
authors.lastname: Alvarez
authors.affiliation: Cystic Fibrosis Adult Unit, Hospital Vall d'Hebron, Barcelona, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: de Gracia
authors.affiliation: Faculty of Sciences, BioFIG-Centre for Biodiversity, Functional and Integrative Genomics, University of Lisboa, Lisboa, Portugal.
authors.forename: Anabela
authors.initials: A
authors.lastname: Ramalho
authors.affiliation: Faculty of Sciences, BioFIG-Centre for Biodiversity, Functional and Integrative Genomics, University of Lisboa, Lisboa, Portugal.
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
authors.affiliation: Human Molecular Genetics Group. Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Larriba
authors.affiliation: Human Molecular Genetics Group. Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain.
authors.forename: Teresa
authors.identifier: 0000000160782823
authors.initials: T
authors.lastname: Casals
mesh_terms: D000328:Adult; D015153:Blotting, Western; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D005260:Female; D015870:Gene Expression; D005838:Genotype; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D009154:Mutation; D009297:Nasal Mucosa / Q000166:cytology / Q000378:metabolism*; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D012326:RNA Splicing; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator