Characterization of a Disease-associated Mutation Affecting a Putative Splicing Regulatory Element in Intron 6b of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

Manuela Seia,Alessandra Meloni,Federica Incani,M.Cristina Rosatelli,Denise Corda,A.Maria Baffico,Antonio Cao,Maddalena Masala,Valeria Faà

doi:10.1074/jbc.m109.032623

Abstract

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are 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 work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides 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 are most probably recognized as 5'- and 3'-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing 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 the CFTR gene exclusively recognized by SRp75.

Highlights

Which is involved in a broad spectrum of phenotypes, including male infertility due to congenital bilateral absence of the vas deferens [3, 4], disseminated bronchiectasis [5, 6], and chronic pancreatitis [7, 8]
The analysis showed a transcript with an insertion of 101 bp between exons 6b and 7 that resembles a cryptic exon (Fig. 1)
The 101-bp inserted sequence is part of intron 6b and is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5Ј- and 3Ј-splice sites by the spliceosome

Summary

EXPERIMENTAL PROCEDURES

Patients—This study included three unrelated CF patients of Italian origin affected by a classical form of CF, with one undefined CF allele. Clinical features of affected individuals included pancreatic insufficiency, chronic lung disease with Pseudomonas aeruginosa infections, and a sweat test of Ͼ60 mmol/liter ClϪ These patients were selected after an extensive second level molecular screening (i.e. denaturing HPLC analysis and direct sequencing for searching for point mutations, breakpoint PCR, and multiple ligation-dependent probe amplification for detection of large deletions) carried out on 441 unrelated Italian CF patients. RNA probes were incubated with 5.2 mM HEPES-KOH (pH 7.9), 1 mM MgCl2, 0.8 mM magnesium acetate, 0.52 mM dithiothreitol, 3.8% glycerol, 0.75 mM ATP, 0.5 ␮g/␮l heparin, and 30 ␮g of HeLa nuclear extract in a final volume of 20 ␮l for 20 min at room temperature. The UV cross-linking assay was performed by incubation of the [␣-32P]UTP-labeled RNA probes (1 ϫ 106 cpm/incubation) with 20 ␮g of HeLa nuclear extract in a 20-␮l final volume at 30 °C for 15 min.

RESULTS

Identification of Nuclear Proteins

DISCUSSION