Fine characterization of the recurrent c.1584+118672A>G deep-intronic mutation in the cystic fibrosis transmembrane conductance regulator gene

Lucy Costantino, Damiana Rusconi, Giulia Soldà, Manuela Seia, Valentina Paracchin, Luigi Porcaro, Rosanna Asselta, Carla Colombo, Stefano Duga

Research output: Contribution to journalArticlepeer-review


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+118672A>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+118672A>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+118672A>G demonstrated: (1) a lowlevel of pseudoexoninclusion inthe F508deltranscript (not containing the splicing mutation); (2) residual wildtypesplicing in the c.1584+118672A>GmRNA; (3) thedegradationof 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+118672A>G mutation correlates well with the milder clinical phenotype of patients.

Original languageEnglish
Pages (from-to)619-625
Number of pages7
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Issue number5
Publication statusPublished - May 2013


  • Cystic fibrosis
  • Deep-intronic mutation
  • In vitro analysis
  • Nasal brushing
  • Pseudoexon

ASJC Scopus subject areas

  • Cell Biology
  • Pulmonary and Respiratory Medicine
  • Molecular Biology
  • Clinical Biochemistry


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