Characterization of disease-associated mutations affecting an exonic splicing enhancer and two cryptic splice sites in exon 13 of the cystic fibrosis transmembrane conductance regulator gene

• Published in: Human molecular genetics 2003-08, Vol.12 (16), p.2031-2040
• Main Authors: Aznarez, Isabel, Chan, Elayne M., Zielenski, Julian, Blencowe, Benjamin J., Tsui, Lap-Chee
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological and medical sciences; COS Cells; Cystic Fibrosis - genetics; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; Enhancer Elements, Genetic - physiology; Exons; Humans; Medical sciences; Mutation; Neurology; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA Precursors - genetics; RNA Splice Sites - genetics; RNA Splicing; RNA-Binding Proteins; Serine-Arginine Splicing Factors; Trans-Activators - genetics; Trans-Activators - metabolism; Transfection
• ISSN: 0964-6906; 1460-2083; 1460-2083
• DOI: 10.1093/hmg/ddg215

Sequences in exons can play an important role in constitutive and regulated pre-mRNA splicing. Since exonic splicing regulatory sequences are generally poorly conserved and their mechanism of action is not well understood, the consequence of exonic mutations on splicing can only be determined empirically. In this study, we have investigated the consequence of two cystic fibrosis (CF) disease-causing mutations, E656X and 2108delA, on the function of a putative exonic splicing enhancer (ESE) in exon 13 of the CFTR gene. We have also determined whether five other CF mutations D648V, D651N, G654S, E664X and T665S located near this putative ESE could lead to aberrant splicing of exon 13. Using minigene constructs, we have demonstrated that the E656X and 2108delA mutations could indeed cause aberrant splicing in a predicted manner, supporting a role for the putative ESE sequence in pre-mRNA splicing. In addition, we have shown that D648V, E664X and T665S mutations could cause aberrant splicing of exon 13 by improving the polypyrimidine tracts of two cryptic 3′ splice sites. We also provide evidence that the relative levels of two splicing factors, hTra2α and SF2/ASF, could alter the effect on splicing of some of the exon 13 disease mutations. Taken together, our results suggest that the severity of CF disease could be modulated by changes in the fidelity of CFTR pre-mRNA splicing.