Splicing mutations account for approximately 12% of the 1,890 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations described in cystic fibrosis (CF). However, their impact on pre-mRNA processing frequently remains unclear. An interesting opportunity to study CFTR transcripts in vivo involves the use of RNA from nasal brushings. Through this approach we previously identified a deep-intronic mutation (c.1584+18672A>G) that activates a 104-base pair (bp) out-of-frame pseudoexon by creating a donor splice site. The screening of 230 patients with CF identified c.1584+18672A>G in three additional individuals, demonstrating that it is a recurrent, and potentially overlooked, mutation among Italian patients. Haplotype analysis suggests that it originated from at least two independent events. To characterize the mutation further, a genomic region, including the activated pseudoexon and surrounding intronic sequences, was cloned into an expression vector and transfected into HeLa cells. RT-PCR analysis identified two alternative splicing products, produced by the activation of two different cryptic acceptor splice sites. One included the 104-bp pseudoexon (78.7% of transcripts), and the other led to the inclusion of a 65-bp pseudoexon (21.3% of mRNAs). The allele-specific measurement of wild-type and aberrant splicings from the nasal-brushing RNA of the three probands with genotype F508del/c.1584+18672A>G demonstrated: (1) a low level of pseudoexon inclusion in the F508del transcript (not containing the splicing mutation); (2) residual wild-type splicing in the c.1584+18672A>G mRNA; (3) the degradation of aberrant transcripts; and (4) the relative strength of the different cryptic splice sites. Interestingly, the residual wild-type splicing detected in transcripts bearing the c.1584+18672A>G mutation correlates well with the milder clinical phenotype of patients.
Fine characterization of the recurrent c.1584+18672A>G deep-intronic mutation in the cystic fibrosis transmembrane conductance regulator gene / L. Costantino, D. Rusconi, G. Soldà, M. Seia, V. Paracchini, L. Porcaro, R. Asselta, C. Colombo, S. Duga. - In: AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY. - ISSN 1044-1549. - 48:5(2013 May), pp. 619-625.
|Titolo:||Fine characterization of the recurrent c.1584+18672A>G deep-intronic mutation in the cystic fibrosis transmembrane conductance regulator gene|
|Parole Chiave:||RNA splice sites; sequence deletion; adult; alternative splicing; base sequence; cystic fibrosis transmembrane conductance regulator; DNA mutational analysis; female; haplotypes; heLa cells; humans; infant; introns; male; molecular sequence data; mutation; nose; RNA Messenger|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
Settore BIO/13 - Biologia Applicata
Settore MED/38 - Pediatria Generale e Specialistica
|Data di pubblicazione:||mag-2013|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1165/rcmb.2012-0371OC|
|Appare nelle tipologie:||01 - Articolo su periodico|