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Transcript analysis of the cystic fibrosis splicing mutation 1525-1G>A shows use of multiple alternative splicing sites and suggests a putative role of exonic splicing enhancers
  1. A S Ramalho1,2,
  2. S Beck2,
  3. D Penque2,
  4. T Gonska3,
  5. H H Seydewitz3,
  6. M Mall3,
  7. M D Amaral1,2
  1. 1Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisboa, Portugal
  2. 2Centre of Human Genetics, National Institute of Health, Lisboa, Portugal
  3. 3University Children’s Hospital, Freiburg, Germany
  1. Correspondence to:
 Dr M D Amaral, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisboa, Campo Grande C8, P-1749-016 Lisboa, Portugal; 

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The autosomal recessive disease cystic fibrosis (CF, MIM 219700) is caused by a wide spectrum of mutations in the gene encoding for the CF transmembrane conductance regulator (CFTR, MIM 602421) protein, a cAMP regulated chloride (Cl) channel located in the apical membrane of secretory epithelial cells.

About 20% of the more than 1000 CFTR mutations reported so far are splicing mutations and generally they represent 15% of all point mutations.1 Although it is generally agreed that mutations at the conserved splice dinucleotide consensus sites GU (donor) and AG (acceptor) disrupt function of the resulting protein product, it is important to know exactly which alternatively spliced mRNA molecules will result in such situations.

Key points

  • The cystic fibrosis (CF) 1525-1G>A mutation abolishes the normal intron 9 acceptor, forcing the spliceosome to use alternative sites. The in frame deletion of exon 10 was proposed to occur and various alternative acceptors in intron 9 present high consensus values (CVs).

  • Here, we studied two CF patients with the 1525-1G>A/F508del genotype. The patients are first cousins, both female, of Afghan descent, with typical CF: raised sweat chloride levels, pancreatic insufficiency (PI), and pulmonary disease.

  • Analysis of CFTR mRNA from the colonic epithelium by RT-PCR and automatic sequencing confirmed the presence of a transcript lacking exon 10 and detected the additional presence of two transcripts using acceptors within exon 10 at positions 1610-1611 (an intense band, exon 10-Alt1) and 1678-1679 (a minor product, exon 10-Alt2).

  • These two acceptors were not among those with the strongest CVs. A possible explanation for their usage may be the presence of two exonic splicing enhancers (ESEs) in exons 10 and 11.

  • CFTR mediated Cl secretion was assessed in rectal biopsies mounted in modified Ussing chambers and was found to be absent in both CF patients.

  • Altogether, these …

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