Functional analysis of synonymous substitutions predicted to affect splicing of the CFTR gene

J Cyst Fibros. 2012 Dec;11(6):511-7. doi: 10.1016/j.jcf.2012.04.009. Epub 2012 May 14.


Background: Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Over 1800 CFTR mutations have been reported, and about 12% of mutations are believed to impair pre-mRNA splicing. Given that several synthetic, non-splice-junction synonymous substitutions have been reported to alter splicing in CFTR, we predicted that naturally occurring synonymous substitutions may be erroneously classified as functionally neutral.

Methods: Computational tools were used to predict the effect of synonymous substitutions on CFTR pre-mRNA splicing. The functional consequences of selected substitutions were evaluated using a minigene splicing assay.

Results: Two synonymous mutations were shown to have a dramatic effect on CFTR pre-mRNA splicing, and consequently could alter protein integrity and phenotypic outcome.

Conclusions: Traditional methods of mutation analysis overlook splicing defects that occur at internal positions in coding exons, especially synonymous substitutions. We show that bioinformatics tools and minigene splicing assays are a potent combination to prioritize and identify mutations that cause aberrant CFTR pre-mRNA splicing.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Alternative Splicing / genetics*
  • Computational Biology / methods
  • Cystic Fibrosis / genetics*
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics*
  • DNA Mutational Analysis / methods*
  • Databases, Genetic
  • Exons / genetics
  • Humans
  • Open Reading Frames / genetics
  • Phenotype
  • Predictive Value of Tests
  • RNA Precursors / genetics
  • RNA Splice Sites / genetics*


  • CFTR protein, human
  • RNA Precursors
  • RNA Splice Sites
  • Cystic Fibrosis Transmembrane Conductance Regulator