Pathogenic Variants and Genotypes of the CFTR Gene in Russian Men with Cystic Fibrosis and CBAVD Syndrome.

Pathogenic CFTR variants cause cystic fibrosis (CF), and CF-related disorders (CF-RD), including bilateral aplasia of the vas deferens (CBAVD). The spectrum of clinical manifestations depends on the CFTR genotype. The frequency and spectrum of the CFTR variants vary between populations and clinical groups. CFTR variants and genotypes were analyzed in Russian men with CF (n = 546) and CBAVD syndrome (n = 125). Pathogenic variants were detected in 93.95% and 39.2% of the CF and CBAVD alleles, respectively. The most frequent c.1521_1523del (F508del; p.Phe508del) variant was found in 541 (49.5%) CF alleles. A total of 162 CFTR genotypes were revealed in CF patients, including 152 homozygous and 394 compound-heterozygous. The most common CF-genotype was F508del/F508del (24.9%). Other frequent CF-genotypes were F508del/3849+10kbC>T, F508del/CFTRdele2,3, and F508del/E92K. CF-causing variants and/or 5T allele were found in 88% of CBAVD patients: 5T/CFTRmut (48.0%), CFTRmut/N (17.6%), CFTRmut/CFTRmut (6.4%), 5T/5T (10.4%), 5T/N (5.6%) and N/N (12.0%), with the most common CBAVD-genotype being F508del/5T (29.6%). The allele frequencies of F508del, CFTRdele2,3 394delTT, and 3849+10kbC>T were significantly higher in CF patients. L138ins/L138ins, 2184insA/E92K, and L138ins/N genotypes were found in CBAVD, but not in CF patients. The results indicate certain differences in the frequency of some CFTR variants and genotypes in Russian CF and CBAVD patients.

Clinical and Genetic Characteristics of a Patient with Cystic Fibrosis with a Complex Allele [E217G;G509D] and Functional Evaluation of the CFTR Channel.

The intricate nature of complex alleles presents challenges in the classification of CFTR gene mutations, encompassing potential disease-causing, neutral, or treatment-modulating effects. Notably, the complex allele [E217G;G509D] remains absent from international databases, with its pathogenicity yet to be established. Assessing the functionality of apical membrane ion channels in intestinal epithelium employed the intestinal current measurements (ICM) method, using rectal biopsy material. The effectivity of CFTR-targeted therapy was evaluated using a model of intestinal organoids of a patient harboring the genotype F508del/[E217G;G509D]. ICM analysis revealed diminished chloride channel function. Remarkably, [E217G;G509D] presence within intestinal organoids correlated with heightened residual CFTR function. Employing CFTR modulators facilitated the restoration of the functional CFTR protein. This multifaceted study intertwines genetic investigations, functional analyses, and therapeutic interventions, shedding light on the intricate interplay of complex alleles within CFTR mutations. The results highlight the potential of targeted CFTR modulators to restore functional integrity, offering promise for advancing precision treatments in cystic fibrosis management.

L138ins Variant of the CFTR Gene in Russian Infertile Men.

(1) Introduction: Pathogenic variants in the CFTR (Cystic Fibrosis Transmembrane conductance Regulator, OMIM: 602421) gene cause Cystic Fibrosis (CF, OMIM: 219700) and CF-related disorders (CF-RD), often accompanied by obstructive azoospermia due to congenital bilateral aplasia of vas deferens (CBAVD, OMIM: 277180) in male patients. The L138ins (c.413_415dup; p. (Leu138dup)) is a mild variant in the CFTR gene that is relatively common among CF-patients in Slavic populations. The frequency of this variant in Russian infertile men has not been sufficiently studied; (2) Materials and Methods: The sample consisted of 6033 Russian infertile men. The patients were tested for 22 common in Russian populations pathogenic variants of the CFTR gene and the IVS9Tn-polymorphic locus of the intron 9. Molecular-genetic studies were performed using amplified fragment length polymorphism (AFLP-PCR), multiplex ligation-dependent probe amplification (MLPA), and nested PCR (for analysis of the IVS9Tn-polymorphic locus); (3) Results: Pathogenic variants in the CFTR were detected in 3.9% of patients. The most frequent variants were F508del and CFTRdele2.3(21kb), accounted for 61.0% and 7.1% of detected variants, respectively. The L138ins variant was detected in 17 (0.28%) individuals: one of them was homozygous, 10 patients were heterozygous, and 6 patients were compound-heterozygous (F508del/L138ins, n = 4; L138ins/N1303K, n = 1; L138ins/5T, n = 1). Two pathogenic CF-causing variants in the CFTR gene were detected in 8 patients, including 7 compound heterozygous (F508del/L138ins, n = 4; F508del/N1303K, n = 1; 2184insA/E92K, n = 1; 3849+10kbC>T/E92K, n = 1) and one homozygous (L138ins/L138ins). The L138ins variant was found in 7 out of 16 (43.75%) chromosomes in six of these patients. The most common pathogenic variant, F508del, was identified in five out of them, in 5 of 16 (31.25%) chromosomes. The allele frequency (AF) of the L138ins variant in the sample has been found to be 0.0014.; (4) Conclusions: The L138ins variant of the CFTR gene is the third most common variant after F508del and CFTRdele2.3(kb) among Russian infertile men.

Clinical and Functional Characteristics of the E92K CFTR Gene Variant in the Russian and Turkish Population of People with Cystic Fibrosis.

The pathogenic variant E92K (c.274G > A) of the CFTR gene is rare in America and Europe, but it is common for people with cystic fibrosis from Russia and Turkey. We studied the effect of the E92K genetic variant on the CFTR function. The function of the CFTR channel was studied using the intestinal current measurements (ICM) method. The effects of CFTR modulators on the restoration of the CFTR function were studied in the model of intestinal organoids. To assess the effect of E92K on pre-mRNA splicing, the RT-PCR products obtained from patients' intestinal organoid cultures were analyzed. Patients with the genetic variant E92K are characterized by an older age of diagnosis compared to homozygotes F508del and a high frequency of pancreatic sufficiency. The results of the sweat test and the ICM method showed partial preservation of the function of the CFTR channel. Functional analysis of CFTR gene expression revealed a weak effect of the E92K variant on mRNA-CFTR splicing. Lumacaftor (VX-809) has been shown to restore CFTR function in an intestinal organoid model, which allows us to consider the E92K variant as a promising target for therapy with CFTR correctors.

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

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.

Evaluation of the Complex p.[Leu467Phe;Phe508del] CFTR Allele in the Intestinal Organoids Model: Implications for Therapy.

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.

Health Characteristics of Patients with Cystic Fibrosis whose Genotype Includes a Variant of the Nucleotide Sequence c.3140-16T>A and Functional Analysis of this Variant.

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease, associated with pathogenic variants in the CFTR gene. The splicing variant c.3140-16T>A (3272-16T>A) has been described previously and, according to the Russian CF Patients Registry, occurs with a frequency of 0.34%. The phenotypic features of CF patients with the c.3140-16T>A variant were compared with those of patients with the genotype F508del/F508del. Patients with the allele c.3140-16T>A had higher average age and age at diagnosis, and the allele was present in a greater proportion of adults. Patients carrying the c.3140-16T>A allele were characterised by better physical development indicators, both in adults and in children, had preserved pancreatic function, as well as the absence of a number of complications, and required pancreatic enzyme replacement therapy less often than patients with the F508del/F508del genotype. Sweat test values also were lower in patients with the c.3140-16T>A genotype. According to the results of clinical and laboratory studies, the phenotype of patients with the genetic variant c.3140-16T>A can be considered "mild". Functional CFTR protein activity in the presence of c.3140-16T>A was evaluated using intestinal current measurements (ICM) and the forskolin-induced swelling assay on organoids obtained from patients' rectal biopsies. c.3140-16T>A had high residual CFTR channel activity and was amenable to effective pharmacological correction with thea VX-770 potentiator. To evaluate the effect of the variant on CFTR pre-mRNA splicing we performed a minigene assay, as well as RT-PCR analysis of RNA isolated from the nasal epithelium and rectal biopsy of patients. We showed that the c.3140-16T>A variant creates a novel acceptor AG dinucleotide within CFTR intron 19, resulting in a 14-nucleotide extension of exon 20. This frameshift produces a premature termination codon and triggers mRNA degradation by the nonsense-mediated decay (NMD) mechanism. Moreover, we observed that the c.3140-16T>A allele could produce a residual amount of normally spliced transcript, thus explaining the patient's mild phenotype.