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

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.

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.

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

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.