Clinical Consequences and Functional Impact of the Rare S737F CFTR Variant and Its Responsiveness to CFTR Modulators

Abstract

S737F is a cystic fibrosis (CF) transmembrane conductance regulator (CFTR) missense variant. The aim of our study was to describe the clinical features of a cohort of individuals carrying this variant. In parallel, by exploiting ex vivo functional and molecular analyses on nasal epithelia derived from a subset of S737F carriers, we evaluated its functional impact on CFTR protein, as well as its responsiveness to CFTR modulators. We retrospectively collected clinical data of all individuals bearing at least one S737F CFTR variant and followed at the CF Centre of Tuscany region (Italy). Nasal brushing was performed in cooperating individuals. At study end clinical data were available for 10 subjects (mean age: 14 years; range 1–44 years; three adult individuals). Five asymptomatic subjects had CF, and two were CRMS/CFSPID, and three had an inconclusive diagnosis. Ex vivo analysis on nasal epithelia demonstrated different levels of CF activity. In particular, epithelia derived from asymptomatic CF subjects and from one of the subjects with inconclusive diagnosis showed reduced CFTR activity that could be rescued by treatment with CFTR modulators. On the contrary, in the epithelia derived from the other two individuals with an inconclusive diagnosis, the CFTR-mediated current was similar to that observed in epithelia derived from healthy donors. In vitro functional and biochemical analysis on S737F-CFTR expressed in immortalized bronchial cells highlighted a modest impairment of the channel activity, which was improved by treatment with ivacaftor alone or in combination with tezacaftor/elexacaftor. Our study provide evidence towards the evaluation of CFTR function on ex vivo nasal epithelial cell models as a new assay to help clinicians to classify individuals, in presence of discordance between clinical picture, sweat test, and genetic profile.

Laboratory Tools to Predict CFTR Modulator Therapy Effectiveness and to Monitor Disease Severity in Cystic Fibrosis

“…Data from IVA-promoted CFTRmediated chloride transport in HNE cells also correlated well with alterations in SCC and ppFEV 1 of PwCF carrying either p.Arg117His (legacy: R117H) or p.Gly551Asp (legacy: G551D) [41]. Responses of CFTR function to modulator drugs in HNE cultures also demonstrated a good correlation with alterations in SCC, ICM, and lung function (measured as percent predicted forced expiratory volume in one second [ppFEV 1 ]) of PwCF carrying rare genotypes [40,[43][44][45]76] or homozygous for p.Phe508del [25,77]. Furthermore, modulator-promoted responses in HNE cultures have been assessed to identify non-eligible responders for compassionate use [45].…”

Beyond Kaftrio : mechanistic insights to maximize N1303K-CFTR rescue

“…NCL-SG3 human eccrine sweat gland cell line was provided by Thibault Kervarrec (Faculté de Pharmacie, Tours, France) (27). Human Nasal Epithelial cells (HNECs) were obtained by nasal brushing and cultured as previously reported (28)(29)(30).…”

“…CFTR expression level and profile were assessed by Western Blot (28)(29)(30). CFTR cell surface expression was quantified using the nanoluciferase complementation assay in HEK293 cells (31).…”

“…Culture inserts with differentiated HNECs were mounted in Ussing chambers. Short-circuit current (Isc) was measured after short-circuiting the trans-epithelial ion flux with a voltageclamp (14,(28)(29)(30). The following activators or inhibitors were added sequentially: amiloride (10μM) at the apical side to block sodium reabsorption; CPT-cAMP (100μM) or alternatively Forskolin/IBMX 10µM/100µM at both apical and basolateral sides to activate CFTR; IVA (1μM) at the apical side to potentiate the channel gate; API (25μM) at the apical side to copotentiate IVA; and the CFTR specific inhibitor-172 (Inh-172) (20μM) at the apical side (all from Sigma Aldrich).…”

“…CFBE41o-cells stably expressing the halide-sensitive yellow fluorescent protein (HS-YFP) were transfected using Lipofectamine 2000 with vectors encoding WT, N1303K or F508del-CFTR (28)(29)(30). YFP quenching rate was used to assess CFTR activity.…”