CFTR Modulators Rescue the Activity of CFTR in Colonoids Expressing the Complex Allele p.[R74W;V201M;D1270N]/dele22_24

Abstract

An Italian, 46-year-old female patient carrying the complex allele p.[R74W;V201M;D1270N] in trans with CFTR dele22_24 was diagnosed at the Cystic Fibrosis (CF) Center of Verona as being affected by CF-pancreatic sufficient (CF-PS) in 2021. The variant V201M has unknown significance, while both of the other variants of this complex allele have variable clinical consequences, according to the CFTR2 database, with reported clinical benefits for treatment with ivacaftor + tezacaftor and ivacaftor + tezacaftor + elexacaftor in patients carrying the R74W-D1270N complex allele, which are currently approved (in USA, not yet in Italy). She was previously followed up by pneumologists in northern Italy because of frequent bronchitis, hemoptysis, recurrent rhinitis, Pseudomonas aeruginosa lung colonization, bronchiectasis/atelectasis, bronchial arterial embolization and moderately compromised lung function (FEV1: 62%). Following a sweat test with borderline results, she was referred to the Verona CF Center where she presented abnormal values in both optical beta-adrenergic sweat tests and intestinal current measurement (ICM). These results were consistent with a diagnosis of CF. CFTR function analyses were also performed in vitro by forskolin-induced swelling (FIS) assay and short-circuit currents (Isc) in the monolayers of the rectal organoids. Both of these assays showed significantly increased CFTR activity following treatment with the CFTR modulators. Western-blot analysis revealed increased fully glycosylated CFTR protein after treatment with correctors, in line with the functional analysis. Interestingly, tezacaftor, together with elexacaftor, rescued the total organoid area under steady-state conditions, even in the absence of the CFTR agonist forskolin. In conclusion, in ex vivo and in vitro assays, we measured a residual function that was significantly enhanced by in vitro incubation with CFTR modulators, especially by ivacaftor + tezacaftor + elexacaftor, suggesting this combination as a potentially optimal treatment for this case.

Variability of Clinical Presentation in Patients Heterozygous for the F508del Cystic Fibrosis Variant: A Series of Three Cases and a Review of the Literature

“…Three of the variants in case two, R74W, V201M, and D1270N were previously found to be in cis as a complex allele p.(R74W;V201M;D1270N). This complex variant is associated with various clinical features, including CF and CFTR-RD, and is frequently found in patients with CFTR-RD [ 4 , 18 ]. Notably, this patient is the only one in this case series to be diagnosed with CF, which was specified to be "mild" in his clinical notes.…”

Theratyping of the Rare CFTR Genotype A559T in Rectal Organoids and Nasal Cells Reveals a Relevant Response to Elexacaftor (VX-445) and Tezacaftor (VX-661) Combination

“…Crypts isolation and human intestinal organoids were obtained as described previously [ 44 ]. Briefly, human rectal biopsies recovered with colon forceps were immediately stored at 4 °C in surgical medium (RPMI-1640, glutamax 1X, HEPES 10 mM, P/S 1%, gentamicin 10 µg/mL and ciprofloxacin 20 µg/mL).…”