Cystic fibrosis patients with the 3272-26A→G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane

• Published in: Human mutation 1999, Vol.14 (2), p.133-144
• Main Authors: Beck, Sebastian, Penque, Deborah, Garcia, Susana, Gomes, Anita, Farinha, Carlos, Mata, Lucinda, Gulbenkian, Sérgio, Gil-Ferreira, Karin, Duarte, Ângela, Pacheco, Paula, Barreto, Celeste, Lopes, Beatriz, Cavaco, José, Lavinha, João, Amaral, Margarida D.
• Format: Article
• Language: English
• Subjects: Adolescent; Adult; CFTR; Child; cystic fibrosis; Cystic Fibrosis - genetics; Cystic Fibrosis Transmembrane Conductance Regulator - analysis; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Female; Fluorescent Antibody Technique; Frameshift Mutation; Humans; Introns; Male; Mutation; nasal epithelium; Nasal Polyps - genetics; Patch-Clamp Techniques; Portugal; Reverse Transcriptase Polymerase Chain Reaction; RNA Splicing - genetics; RNA, Messenger - analysis; Sequence Analysis, DNA; splicing mutation
• ISSN: 1059-7794; 1098-1004
• DOI: 10.1002/(SICI)1098-1004(1999)14:2<133::AID-HUMU5>3.0.CO;2-T

We characterized the 3272‐26A→G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272‐26A→G/F508del genotype. Besides clinical and haplotype characterization of those patients, we report here results from CFTR transcript analysis in nasal brushings from all five patients. RT‐PCR analysis supports alternative splicing in all patients and carriers, but not in controls. By sequencing, we determined that the alternative transcript includes 25 nucleotides from intron 17a, which predictively cause frameshift and a premature stop codon. The use of this alternative splice site causes a reduction in the levels of normal transcripts from the allele with this mutation and, most probably, of normal protein as well. By immunocytochemistry of both epithelial primary cell cultures and slices from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a high sodium absorption, characteristic of CF. Altogether, the results suggest that the main defect caused by the 3272‐26A→G mutation is a reduction in normal CFTR transcripts and protein and therefore this mutation should be included in class V, according to Zielenski and Tsui. Hum Mutat 14:133–144, 1999. © 1999 Wiley‐Liss, Inc.