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

Abstract

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.

Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis

“…3 It has been reported in several genetic disorders that not only splicing mutations but also point mutations can activate/ inactivate these cis and trans elements thereby disrupting the synthesis or function of their encoded proteins. 4,5 Aberrant transcripts that change the open reading frame are eliminated by the cell surveillance machinery resulting in variable levels of residual gene expression. In the end, splicing efficiency affects transcript abundance and consequently the amount of functional protein, the critical point which determines a pathological effect or a predisposition to disease.…”

Cystic Fibrosis F508del Patients Have Apically Localized CFTR in a Reduced Number of Airway Cells

“…Notwithstanding, decreased mRNA levels resulting from the F508del allele or decreased stability of the F508del-CFTR protein at the membrane could also explain our observations. By comparative quantitative analysis of CFTR and F508del-CFTR mRNA from the nasal epithelium of F508del-carriers, no significant reduction of F508del mRNA in comparison with the wt-CFTR product has been observed (Beck et al, 1999a ;1999b;Dö rk et al, 1994;Duarte et al, 1996;Trapnell et al, 1991). On the other hand, it was shown that F508del-CFTR protein is less stable than CFTR by an 8-fold difference when promoted to accumulate in the plasma membrane of heterologous expression systems by lowering the temperature (Lukacs et al, 1993).…”

“…After informed consent, nasal brushings were performed as described before (Beck et al, 1999b). Briefly, an interdental brush with 2.5 to 3 mm bristles (Paro-Isola, Thalwil, Switzerland) was used to scrape along the tip of the inferior turbinate and the adjacent lateral nasal wall.…”

Detection of a large heterozygous deletion and a splicing defect in the CFTR transcripts from nasal swab of a Japanese case of cystic fibrosis

“…This method has been used to detect aberrantly spliced transcripts and reduced mRNA levels that were associated with various types of nonsense, frameshift, stop and splicing mutations. [16][17][18][19] In fact, we detected the CFTR transcripts with or without exon 9 in samples from both the patient and healthy control (Figure 1). The probability of alternative splicing of exon 9 is related to the genotype of poly T and TG repeats in intron 8.…”