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

We characterized the 3272 26A-->G mutation in the cystic fibrosis transmemb rane conductance regulator (CFTR) gene, creating an alternative acceptor sp lice site in intron 17a, that competes with the normal one, although we pre dict from consensus values, with lower efficiency We analyzed five Cystic F ibrosis (CF) Portuguese patients with the 3272-26A-->G/F508del genotype, Be sides 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 an d carriers, but not in controls. By sequencing, we determined that the alte rnative transcript includes 25 nucleotides from intron 17a, which predictiv ely cause frame shift and a premature stop codon, The use of this alternati ve 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 slice s from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a h igh sodium absorption, characteristic of CE 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, (C) 1999 Wiley Liss, Inc.