Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene

Cystic fibrosis (CF) is a common recessive disorder caused by more than 1600 mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. About 13% of CFTR mutations is classified as “splicing mutations”, but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this paper we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses as well as mRNA studies we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nt between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence which is most probably recognized as 5’ and 3’ splice site by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein hnRNPA2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing the activation of the cryptic exon. Our results show that the c.1002-1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of CFTR gene exclusively recognized by the splicing factor SRp75.