In the cohort of Russian patients with cystic fibrosis, the p.[Leu467Phe;Phe508del] complex allele (legacy name [L467F;F508del]) of the CFTR gene is understudied. In this research, we present the results of frequency evaluation of the [L467F;F508del] complex allele in the Russian Federation among patients with a F508del/F508del genotype, its effect on the clinical course of cystic fibrosis, the intestinal epithelium ionic channel function, and the effectiveness of target therapy. The frequency of the [L467F;F508del] complex allele among patients with homozygous F508del was determined with multiplex ligase-dependent probe amplification followed by polymerase chain reaction and fragment analysis. The function of ionic channels, including the residual CFTR function, and the effectiveness of CFTR modulators was analyzed using intestinal current measurements on rectal biopsy samples and the forskolin-induced swelling assay on organoids. The results showed that the F508del/[L467F;F508del] genotype is present in 8.2% of all Russian patients with F508del in a homozygous state. The clinical course of the disease in patients with the F508del/[L467F;F508del] genotype is severe and does not vary from the course in the cohort with homozygous F508del, although the CFTR channel function is significantly lower. For patients with the F508del/[L467F;F508del] genotype, we can recommend targeted therapy using a combined ivacaftor + tezacaftor + elexacaftor medication.
Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.
Nearly 30 synthetic nucleosides were tested with human recombinant poly(ADP-ribose) polymerase 1 as potential inhibitors of this enzyme. The most active compounds were some disaccharide analogues of thymidine: 3'-O-β-D-ribofuranosyl-5-iodo-dUrd (2d; IC₅₀ = 45 μM), 3'-O-β-D-ribofuranosyl-2'-deoxythymidine (2e; IC₅₀ = 38 μM), and 3'-O-β-D-ribofuranosyl-2'-deoxythymidine oxidized (4; IC₅₀ = 25 μM). These compounds also reduced H₂O₂-induced synthesis of poly(ADP-ribose) in cultured human ovarian carcinoma (SKOV-3) cells in a dose-dependent manner. Furthermore, compounds 2d or 2e until a concentration of 1 mM did not affect growth of SKOV-3 cells, whereas dialdehyde compound 4, as well as thymidine, exhibited a significant cytotoxicity.
The presence of complex alleles in the CFTR gene can lead to difficulties in diagnosing cystic fibrosis and cause resistance to therapy with CFTR modulators. Tezacaftor/ivacaftor therapy for 8 months in a patient with the initially established F508del/F508del genotype did not lead to an improvement in her condition—there was no change in spirometry and an increase in the patient’s weight, while there was only a slight decrease in NaCl values, measured by a sweat test. The intestinal current measurements of the patient’s rectal biopsy showed no positive dynamics in the rescue of CFTR function while taking tezacaftor/ivacaftor. The assumption that the patient had an additional mutation in the cis position was confirmed by sequencing the CFTR gene, and the complex allele [L467F;F508del] was identified. Based on the rescue of CFTR function by elexacaftor/tezacaftor/ivacaftor obtained using forskolin-induced swelling on intestinal organoids, the patient was prescribed therapy with this targeted drug. The use of elexacaftor/tezacaftor/ivacaftor for 7 months resulted in a significant improvement in the patient’s clinical condition.
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