Description of rare alleles of the CFTR gene in cystic fibrosis using functional tests and forskolin-induced swelling assay in rectal organoids

Abstract

Cystic fibrosis (CF) is a systemic hereditary disease caused by a mutation of the CFTR gene and characterized by damage to the glands of external secretion and severe dysfunctions of the respiratory system. The “severe” genotypes are associated with more pronounced and early clinical manifestations. The “mild” genotypes are associated with a delayed onset of clinical manifestations.Methods. An analysis of the medical history of patients with asymptomatic CF from the Russian CF centers was carried out. Rectal biopsy specimens were used for intestinal current measurement (ICM) and forskolin-induced swelling assay in intestinal organoids. DNA for sequencing was isolated from the venous blood leukocytes.Results. 2 CF patients with genotypes F508del/c.1584+18672A>G (patient 1, 12 years old) and F508del/G509D-E217G (patient 2, 3 years old) were examined. The diagnosis was based on neonatal screening. The ICM assay showed a reduced function of the CFTR channel: ΔISC for forskolin was 12.33 ± 2.35 μA/cm2 in patient 1 and 25 ± 3.37 μA/cm2 in patient 2. The negative response to carbachol and histamine reflects the entry of potassium ions into the cells, which is typical for CF. The assay in intestinal organoids showed that the amount of functional protein on the apical membrane of the intestinal epithelium increases both under the action of the potentiator and the corrector in both patients. The greatest effect is observed when the VX-770 and VX-809 are used together.Conclusion. The ICM functional assay and the forskolin-induced swelling assay in intestinal organoids are sensitive enough to determine the residual functional activity of the CFTR channel and clarify the diagnosis in difficult cases. The studied genetic variants c.1584+18672A>G and G509D-E217G belong to class IV – VI mutations. The clinical picture corresponds to the manifestations of “mild” mutations. The studied patients still attend the Russian cystic fibrosis centers.

Personalized Selection of a CFTR Modulator for a Patient with a Complex Allele [L467F;F508del]

“…This case also demonstrates the use of modern functional methods for assessing the function of the chloride channel—the determination of intestinal current measurement (ICM) and using a forskolin-induced swelling assay on intestinal organoids to assess the effectiveness of CFTR modulators. These methods have been used in the Russian Federation since 2018 at the Research Centre for Medical Genetics to study the pathogenicity of CFTR gene mutations not described in international databases in complex diagnostic cases, as well as to evaluate the effectiveness of targeted therapy for carriers of rare pathogenic variants [ 7 , 8 , 9 ]. Forskolin-induced swelling (FIS) assay of patient-derived organoids has been used to assessment of residual functional activity of the CFTR protein and to study the effects of CFTR modulators.…”

“…Forskolin activated the CFTR channel located at the apical membrane of the epithelial cells. Organoid swelling occurs through excretion chloride ions and H 2 O into the organoid lumen [ 7 ]. The swelling of organoids in response to forskolin is directly dependent on the functional activity of CFTR [ 8 ].…”