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.
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Acknowledgements
We thank all participating subjects. SL is sponsored by the Researchers Stabilization Program from the Spanish National Health System (CES09/020). This work was supported by Instituto de Salud Carlos III (FIS/FEDER PI050804 and PI080041) and by Fundación Sira Carrasco from Spain and partially supported by the Portuguese Fundação para a Ciência e Tecnologia (Ciência2008 grant to ASR and PEst-OE/BIA/UI4046/2011 BioFig centre grant). We are grateful to Professor G Cutting (Jonhs Hopkins Hospital, Baltimore, MD, USA) for kindly providing pCDNA5/FTR/CFTR wt mammalian expression vector.
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This study was conceived and designed by TC. Samples and clinical data were selected by AA and JdG. NE samples processing and qPCR experiments was performed by LM. MDR performed qualitative gene expression experiments. Gene expression analysis was supervised by SL. Minigene study design was performed by AR. Minigene experiments was performed by SI and supervised by AR and MA. The manuscript was written by MA, SL and TC. All aspects of the study were supervised by SL and TC.
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Masvidal, L., Igreja, S., Ramos, M. et al. Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis. Eur J Hum Genet 22, 784–791 (2014). https://doi.org/10.1038/ejhg.2013.238
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DOI: https://doi.org/10.1038/ejhg.2013.238
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