Functional Characterization of a Novel CFTR Mutation P67S Identified in a Patient with Atypical Cystic Fibrosis

Abstract

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR serves as a cAMP-stimulated chloride channel in a wide range of epithelial tissues and its dysfunction is a hallmark of CF. Over 1400 mutations in the CFTR gene are known, but functional data exist only for a minority of the mutant channels. The aim of the present study was to functionally characterize a novel CFTR mutation identified in a patient with atypical CF. Full length sequencing of the patient’s CFTR gene revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a P67S amino acid substitution. Mutant and wild-type CFTR were heterologously expressed in Xenopus laevis oocytes. CFTR whole-cell currents were studied using the two-electrode voltage-clamp technique. Channel surface expression was assessed by a chemiluminescence assay. Expression of P67S-CFTR resulted in functional CFTR chloride channels. However, the CFTR chloride conductance observed in oocytes expressing the mutant channel averaged only 24% of that in oocytes expressing wild-type CFTR. Similarly, surface expression of the mutant channel was reduced. In contrast, the mutation did not alter the anion selectivity of the channel, and Western blot analysis indicated a similar protein expression level of mutant and wild-type CFTR. Our findings indicate that the P67S mutation reduces CFTR chloride channel function by reducing channel surface expression. The mild disease phenotype of the patient indicates that the residual function of the mutant channel is sufficient to prevent the development of severe CF symptoms.

Analysis of cystic fibrosis–associated P67L CFTR illustrates barriers to personalized therapeutics for orphan diseases

“…First, P67L has sometimes been incorrectly grouped as a class IV (conduction) defect, perhaps based, in part, on decreased CFTR currents observed previously for a different mutation at the same position, P67S (18). The categorization as class IV argues against usefulness of ivacaftor or lumacaftor and might otherwise be taken as evidence to deter pharmaceutical trials seeking FDA approval and/or third party reimbursement with regard to available CFTR modulators.…”

“…For example, Kraus et al noted diminished ion transport attributable to P67L. Based, in part, on abnormal current/voltage relationship in the closely related P67S defect, P67L may have been incorrectly assigned class IV status (18). Other data from large surveys of rare CF alleles also point to significant processing and/or transport abnormalities associated with P67L (12, 25, 27).…”

A mutation of the epithelial sodium channel associated with atypical cystic fibrosis increases channel open probability and reduces Na+ self inhibition

“…Oocytes were routinely studied 1–2 days after injection using the two‐electrode voltage‐clamp technique (TEVC) as described previously (Rauh et al 2006; Kraus et al 2007; Wielpütz et al 2007). The oocytes were placed in a small experimental chamber and constantly superfused at room temperature with ND96 supplemented with 2 μ m amiloride (Sigma) at a rate of 2–3 ml min −1 .…”

Mutations in the amiloride-sensitive epithelial sodium channel in patients with cystic fibrosis-like disease

“…Oocytes were obtained from adult female Xenopus laevis as described previously [Kraus et al, 2007;Rauh et al, 2006;Wielputz et al, 2007]. Defolliculated stage V-VI oocytes were injected with cRNA for SCNN1A, SCNN1B, and SCNN1G (0.5-1.0 ng/subunit) dissolved in RNase-free water with a total volume of 46 nl per oocyte.…”

“…Whole-cell currents were routinely measured 2 days after injection using the two-electrode voltage clamp technique as described previously [Kraus et al, 2007;Rauh et al, 2006;Wielputz et al, 2007]. The oocytes were superfused with ND96 (see above) and clamped at a holding potential of -60 mV.…”