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.


Introduction
Infertility is a common medico-social problem affecting 10-15% of married couples."Male factor" is revealed in about half of all infertile couples, with genetic abnormalities responsible for at least 50% of cases of severe infertility [1].Common genetic causes of male infertility include sex chromosome abnormalities (Klinefelter syndrome, X/XY mosaicism, disomy Y, and others), the Y chromosome microdeletions in the AZF (azoospermia factor) locus, and CFTR gene variants [2][3][4].
CBAVD syndrome (OMIM# 277180) is an autosomal recessive disease, considered as "genital form" of CF in male patients.In adult male patients, CBAVD is one of the manifestations of Cystic Fibrosis, but it may be independent of CF disease without extragenital symptoms [4].Some patients with CBAVD syndrome show extragenital symptoms of CF (chronic pancreatitis, bronchitis, sinusitis), which are usually mild and develop mainly in adulthood.Therefore, differential diagnosis and identification of mild ("atypical") forms of CF and other CFTR-RD in azoospermic patients, especially the heterozygosity for CFTR gene variants, is highly relevant.CBAVD is a common cause of obstructive azoospermia, accounting for 25% of its cases, and is diagnosed in 1-2% of infertile men from various populations [6,7].However, the actual frequency of congenital bilateral and unilateral aplasia of vas deferens (CBAVD and CUAVD, respectively) is rather underestimated.It is due to a number of problems in differential diagnosis CAVD from other forms of obstructive azoospermia and oligozoospermia [8].Overall, in 80% patients, CBAVD syndrome is resulted from CF-causing variants/5T allele of the CFTR gene; rare cases of this disorder are caused by pathogenic variants in the ADGRG2 or other candidate genes (SLC9A3, SCNN1B, and CA12) [8].
The CFTR gene is located on chromosome 7 (7q31.2),has a size of about 189 Kb, and consists of 27 exons and 26 introns [9].CFTR protein is a member of the ATP-binding cassette (ABC) transporter superfamily, which functions as a chloride channel, controlling ion and water secretion and absorption in epithelial tissues [9].To date, more than 2000 pathogenic variants in the CFTR gene have been identified [9].The severity and spectrum of clinical manifestations depend on the severity of the CFTR genotype, which determines the degree of defect/preservation of the encoded protein.
Numerous studies show that the genotypes of patients with CF and CBAVD syndrome differ significantly, with the combination of two "severe" pathogenic variants in transposition, i.e., on both CFTR alleles, leads to severe forms of CF [10].Commonly, CBAVD syndrome is characterized by genotypes with "mild" CFTR mutations in combination with the 5T allele (IVS-9T5 polymorphic variant), which is detected in 40-50% of CBAVD patients and in 4-5% individuals of Caucasian origin [11,12].The frequency of CBAVD syndrome varies in different regions (in European populations, it is slightly higher than in Asian populations), which is probably due to the different frequencies of pathogenic CFTR gene variants in different regions and ethnic groups [10,11].
The frequency and spectrum of the CFTR gene variants significantly vary between different populations, ethnic and clinical groups, particularly CF and CF-RD.The spectrum of the CFTR gene variants and genotypes in Russian CF patients has been studied in detail, including differences in various age groups (children and adults), as well as in different regions of the Russian Federation [13].However, to date, no combined study of Russian CF patients and men with CBAVD syndrome has been performed.Therefore, the aim of the present study was a comparative analysis of common pathogenic CFTR gene variants and genotypes in Russian men with cystic fibrosis and Russian patients with CBAVD syndrome.

Discussion
The results of the present study indicate a high prevalence of CFTR gene mutations in Russian men with CBAVD syndrome.A comparative analysis of the prevalence of various CFTR pathogenic variants in the two evaluated groups indicates a similar distribution of their occurrence among both adult men with CF and CBAVD syndrome from the Russian Federation.According to our recent research, ten following CF-causing variants, F508del, CFTRdele2,3, L138ins, W1282X, 1677delTA, 3849+10kbC>T, E92K, 2143delT, G542X, and 2184insA, are more common in Russian infertile men [14].Their allelic frequency (AF) is 0.0005 or more, and the proportion of each variant is 2% or more of all detected pathogenic CFTR gene variants in both Russian infertile men and CBAVD patients.
F508del (c.1521_1523delCTT; p.Phe508del) variant is the most common CFTR gene mutation both in Russian men with cystic fibrosis and patients with CBAVD syndrome (44.4% and 52.0% of all detected mutations in these groups, respectively).Previously, we examined CFTR genotypes in a sample of 72 Russian patients with CBAVD [15].The data of the evaluation of CFTR variants and genotypes in a larger sample of CBAVD patients are consistent with data on a smaller sample of patients.
All examined CF patients are characterized by the presence of one or two pathogenic CFTR gene variants, either in a homozygous or in compound heterozygous state.While in the sample of CBAVD patients, two mutations are rare (<5%), CBAVD is characterized by "mild" CFTR genotypes [11].Severe CF-causing variants in the homozygous state were not detected among the examined CBAVD patients.Out of 125 patients, only 8 patients had two CF-causing variants (Table 3).One individual was found to have a mutation in the homozygous state (L138ins/L138ins), and 7 patients were compound-heterozygous for the combination of "severe" (F508del) variants and one of the "mild" CFTR gene variants, or two "mild" CFTR gene variants.It should be noted that at least some of them may have or develop "atypical" cystic fibrosis with age.
According to the literature, men from European populations with "isolated CBAVD", CBAVD without diagnosed cystic fibrosis, homozygotes for "severe" pathogenic CF-causing variants were also not revealed, but compound heterozygotes for a "severe" mutation with the R117H (c.350G>A) variant of the CFTR gene are often found in patients with CBAVD from Western European populations [10,11,16].The R117H variant is a "mild" mutation, the allelic frequency of which in men with CBAVD averages 3%, of which 4.0-11.3% in Caucasian patients, while the F508del/R117H genotype is found in 4-6% of patients.This variant of the CFTR gene is not typical for other populations [10,11], including Russian CF patients [17].Thus, we did not detect any R117H variant among patients from both samples.
Interestingly, 394delTT is one of the common CFTR gene variants detected in 17 (3.1%)Russian CF patients (including in homozygous, n = 4, and heterozygous, n = 13), but not in Russian men with CBAVD syndrome.This variant is the second (4%) of the most frequent "Nordic" CFTR mutations in CF patients from Danish, Swedish, Norwegian, and Finnish populations [18].In West-European populations, this variant has also been reported in a few patients with CBAVD/CUAVD syndromes [8].According to the RUSeq database [http://ruseq.ru/accessed on 20 September 2023], in the European part of Russia, the allelic frequency (AF) of the 394delTT (rs121908769) variant is 0.0002134.This variant is the second most common CFTR gene variant (3.5% of all detected mutations) in CF patients from Bashkortostan [19].In participants (n = 642) from the population-based cohort study ESSE-Vologda, the 394delTT (rs121908769) variant was found to have 0.0047 alleles [20].This variant of the CFTR gene was found in a large cohort of 6,033 Russian infertile men [14].However, a heterozygous 394delTT variant was detected in 2 of the 2,146 Russian infertile men examined by Solovyova et al. (2018) [21].Both of these patients had a 394delTT/N, 7T/9T genotype; one of them was normozoospermic; the spermatological diagnosis of the second patient was not provided; and the presence of CBAVD was not indicated.
Overall, the CFTR gene mutation and/or 5T allele was found in 88% of Russian infertile men with CBAVD syndrome.This is consistent with our results and data from other researchers, who found that 80-85% of CBAVD patients of Caucasian origin have CFcausing variants and/or 5T allele of the CFTR gene [3].In the spectrum of CFTR genotypes, CFTRmut/5T was the most common in our cohort (29.6%) and in other samples of CBAVD patients, especially in a large cohort of patients from France (28.44%) [3].The second most common CFTR genotype in our cohort of Russian men with CBAVD syndrome was 5T/5T homozygosity, revealed in 10.4% of patients.Recently, we reported the results of an analysis of the CFTR gene on a smaller sample of Russian CBAVD patients (n = 72) and they were generally similar to the data on a larger sample [15].
Two CF-causing mutations in the CFTR genotype were found in 8 (6.4%) patients, while the L138ins variant was revealed in 7 out of 16 alleles, and the proportion of this mutation was 7.1% of all detected mutations.Thus, L138ins was found to be the second most common CFTR gene variant in Russian patients with CBAVD syndrome, and the third in prevalence after F508del and CFTRdele2.3(kb) in Russian infertile men.The allele frequency (AF) of this variant in Russian infertile men (n = 6033) was 0.0014 [14].The L138ins variant was found as compound heterozygous F508del/L138ins in four CBAVD patients (Table 3).This genotype was also found in 3 CF patients.Notable, that these CBAVD patients did not have "classic" forms of CF, but at least some had adult-onset pancreatitis or respiratory disease.The possibility of undiagnosed CF or CFTR-RD should be taken into account in the examination of infertile men, especially in patients with obstructive azoospermia.
It should be noted that our study is not without drawbacks.Not all patients underwent sequencing and analysis of major rearrangements or copy number variants, CNVs (for example, deletions and duplications) in the CFTR gene.A comprehensive analysis of the nucleotide variants could identify up to 99% of pathogenic alleles.So, Petrova et al. (2018) identified up to 99% and found 10 (9.6%) intergenic CNVs of 104 Russian CF patients.Taulan et al. have identified pathogenic CFTR variants (two variants-82.4% and one variant-9.3%)in 91.7% of CBAVD patients from European populations.Subsequently, they have detected two large heterozygous complex deletions (one encompassing exon 2, the other removing exons [22][23][24] in the CFTR gene in two CBAVD patients, and both individuals had pathogenic variants on the other allele [22].Ma et al. detected 5 CNVs (4 partial deletions and 1 partial duplication) in the CFTR gene in 1.9% (5/263) Chinese CBAVD patients [23].Although their frequency would be low, we cannot exclude rare nucleotide variants or intergenic in some CF and CBAVD patients from our sample, which had CFTR alleles considered "normal" or unknown CF-causing variants.Screening for large genomic rearrangements in the CFTR gene is beneficial not only in CF but also in CBAVD patients, especially for those azoospermic men who are carriers of a CFTR "mild" mutation/5T allele.
Modern methods of assisted reproductive technologies (ART), especially the IVF/ICSI procedure, allow having children in patients with various forms of male infertility, including obstructive azoospermia due to CF/CBAVD [25].The high frequency of the CFTR gene variants in patients with CBAVD/obstructive azoospermia should be taken into account; therefore, their children are at increased risk for both CF and CF-RD patients.In this regard, genetic counseling and screening for pathogenic CFTR gene variants are very important for these patients and their spouses.Couples at high risk should be offered preimplantation genetic testing (PGT) or prenatal DNA diagnosis.

Materials and Methods
A cohort of examined individuals consisted of 671 unrelated Russian men, including 125 patients with CBAVD syndrome and 546 adult male CF patients.The patients with CBAVD syndrome had no diagnosed cystic fibrosis.All CF patients had one or two pathogenic CFTR gene variants.Informed consent for the study was obtained from all participants.
Molecular study was performed on genomic DNA extracted from peripheral blood leukocytes using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA).
A classification of the genotypes into "severe" or "moderate" is based on the severity of the negative effect of different pathogenic CFTR variants on the function of the encoded protein in accordance with the generally accepted classification [3].
Statistical analysis was performed using the STATISTICA software, version 13.0 (Stat-Soft Inc., Tulsa, OK, USA).A significant difference in the frequencies of the CFTR gene alleles and genotypes was evaluated using the χ 2 criterion.Results were considered reliable if the level of the index did not exceed 0.05 (p < 0.05).

Conclusions
Russian adult male patients with cystic fibrosis and CBAVD syndrome share a similar spectrum of pathogenic CFTR gene variants; however, some of the variants and genotypes dramatically differ between these patients' groups.So, the "mild" CF-causing 3849+10kbC>T variant characterized for PS-CF is common in adult men with cystic fibrosis, including some azoospermic CF patients having CBAVD, but this variant is extremely rare in isolated congenital bilateral aplasia of the vas deferens.394delTT is one of the common CF-causing variants in Russian CF patients, but not in CBAVD patients.In contrast, some other CFTR gene variants (L138ins, 2143delT, and W1282X) were detected 3-4 times more common in CBAVD, than in CF patients.Total frequency of CF-causing variants and 5T allele, as well as the structure of CFTR genotypes in Russian men with CBAVD syndrome, is very similar to the patients from European populations.

Table 1 .
CF-causing CFTR gene variants in Russian men with CF and CBAVD syndrome.

Table 3 .
CFTR genotypes in 125 Russian patients with CBAVD syndrome.