Human Reproduction, Vol. 15, No. 7, 1476-1483,
July 2000
© 2000 European Society of Human Reproduction and Embryology
Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens
1 Medical and Molecular Genetics Center-IRO, Hospital Duran i Reynals, 2 Andrology Department, Fundació Puigvert, 3 Service of Reproductive Medicine, Institut Universitari Dexeus and 4 Centro de Patología Celular, Barcelona, Spain
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Abstract |
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Congenital absence of the vas deferens (CAVD) is a heterogeneous disorder, largely due to mutations in the cystic fibrosis (CFTR) gene. Patients with unilateral absence of the vas deferens (CUAVD) and patients with CAVD in association with renal agenesis appear to have a different aetiology to those with isolated CAVD. We have studied 134 Spanish CAVD patients [110 congenital bilateral absence of the vas deferens (CBAVD) and 24 CUAVD], 16 of whom (six CBAVD, 10 CUAVD) had additional renal anomalies. Forty-two different CFTR mutations were identified, seven of them being novel. Some 45% of the CFTR mutations were specific to CAVD, and were not found in patients with cystic fibrosis or in the general Spanish population. CFTR mutations were detected in 85% of CBAVD patients and in 38% of those with CUAVD. Among those patients with renal anomalies, 31% carried one CFTR mutation. Anomalies in seminal vesicles and ejaculatory ducts were common in patients with CAVD. The prevalence of cryptorchidism and inguinal hernia appeared to be increased in CAVD patients, as well as nasal pathology and frequent respiratory infections. This study confirms the molecular heterogeneity of CFTR mutations in CAVD, and emphasizes the importance of an extensive CFTR analysis in these patients. In contrast with previous studies, this report suggests that CFTR might have a role in urogenital anomalies.
Key words: CAVD/cystic fibrosis/obstructive azoospermia/renal agenesis/vas deferens
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Introduction |
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Cystic fibrosis (CF) is a common severe autosomal recessive disease that affects 1 in 2500 individuals in Caucasian populations. The disease is characterized by abnormal flux of chloride in the apical membrane of epithelial cells, leading to a wide variability in clinical presentation (pancreatic insufficiency, progressive lung disease, meconium ileus, elevated sweat electrolytes and male infertility) (Welsh et al., 1995
). CF is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR) (Kerem et al., 1989). More than 800 mutations in the CFTR gene have been reported (http://www.genet.sickkids.on.ca), which determine different phenotypes: CF (Welsh et al., 1995); congenital absence of the vas deferens (CAVD) (Anguiano et al., 1992; Chillón et al., 1995); bronchiectasis (Pignatti et al., 1995); and pancreatitis (Cohn et al., 1998; Sharer et al., 1998).
Congenital bilateral absence of the vas deferens (CBAVD) occurs in 1–2% of infertile men (Jequier et al., 1985). Obstruction of the Wolffian duct results in the absence or atrophy of the vas deferens, epididymal body and tail, seminal vesicles and the ejaculatory ducts (Taussig et al., 1972). Obstructive azoospermia is present in more than 95% of CF males. Different studies have shown a high frequency of CFTR mutations in CBAVD patients (Casals et al., 1995; Costes et al., 1995; Rave-Harel et al., 1995; Dörk et al., 1997; Mak et al., 1999). The 5T allele in intron 8 of the CFTR gene leads to a higher proportion of mRNA transcripts lacking exon 9 than the two other alleles, 7T and 9T. Consequently, the 5T variant produces abnormally low levels of CFTR protein. The 5T variant is the most frequent mutation associated to the CBAVD phenotype (Chillón et al., 1995).
A lower frequency of CFTR mutations has been detected in patients with unilateral absence of the vas deferens (CUAVD) (Casals et al., 1995; Mickle et al., 1995). Between 11% and 26% of patients with CAVD have renal agenesis in association (Schlegel et al., 1996), and initial negative results in the analysis of CFTR mutations in these patients suggested that urogenital anomalies have a different aetiology to isolated CAVD (Augarten et al., 1994; Casals et al., 1995; Schlegel et al., 1996).
We have performed an extensive CFTR gene analysis in 134 Spanish CAVD patients, 16 of whom had renal malformations, and have carried out a correlation with clinical features. The study confirms the high molecular heterogeneity for CAVD, emphasizes the importance of extensive CFTR screening, and also suggests that CFTR might have a role in urogenital anomalies.
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Materials and methods |
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Patients and clinical evaluation
A total of 134 consecutive men with a diagnosis of CAVD were studied. These men had been referred by different centres within Spain, and provided a good representation of all regions of the country. None of the men had been diagnosed with CF (Welsh et al., 1995
). In all cases, the initial evaluation included anamnesis, a scrotal examination and a semen analysis. Overall, CBAVD was found in 110 men, while 24 men had CUAVD which affected either the right (n = 11) or left (n = 13) side. All these individuals had consulted for couple infertility, except for two fertile men with CUAVD who were diagnosed during screening for vasectomy.
Although molecular analysis of CFTR was performed in the 134 patients with CAVD, 86 patients (64 CBAVD, 22 CUAVD) were studied clinically in detail as they had been referred from centres which had resident teams of experienced andrologists. In the remaining cases, the clinical information was scarce. Within this subset of 86 patients, complete clinical data concerning infertility were obtained and features of CF were excluded (Welsh et al., 1995). The diagnosis of CAVD was based on physical examination, when one or both vasa deferentia were non-palpable in the scrotal portion. Semen analysis included volume, pH, sperm count and motility, in accordance with WHO guidelines (WHO, 1992). Concentrations of fructose and citrate in seminal plasma were measured with commercial kits (FructoScreen, CitricScreen, Bioscreen Inc., New York, USA). Alpha-glucosidase activity in seminal plasma (EpiScreen, Fertipro NV, Beernem, Belgium) was measured in those cases studied recently. Transrectal ultrasonography was performed using a Toshiba Sonolayer SSA-250-A, with a 7.5 MHz linear transducer (PVL 625-RT); this allowed studies to be made of the morphology and size of the seminal vesicles, prostate and ejaculatory ducts. Scrotal ultrasonography was performed if physical examination revealed testis atrophy, cystic masses or reflux of spermatic veins. Abdominal ultrasonography was performed in order to evaluate the pelvis and the upper urinary tract. Excretory urograms were also performed in some cases to confirm ultrasound findings. Vasograms were performed in selected cases of CUAVD (n = 6) to characterize the morphology of the preserved seminal tract when transurethral resection of ejaculatory ducts was foreseeable. Testicular biopsy was carried out under local anaesthesia by open incision, and the specimens fixed in Bouin's solution and processed for histological analysis. Sweat chloride analysis was performed in 59 individuals (Gibson and Cooke, 1959). Preliminary data of some patients (30 CBAVD, 10 CUAVD) were reported previously (Casals et al., 1995).
CFTR gene analysis
Molecular analysis of the CFTR gene was performed in all 134 patients. Genomic DNA samples were isolated from peripheral blood lymphocytes using standard methods. Mutations 
F508 and G542X (Kerem et al., 1989, 1990) were analysed in all patients, as they are the most common mutations in Spanish CF patients, 53% and 8% respectively (Casals et al., 1997). The haplotypes obtained with three CFTR microsatellites (IVS8CA, IVS17bTA and IVS17bCA) allowed us to identify other less common mutations (Morral et al., 1996). Recently, direct analysis of 31 CFTR mutations (PCR/OLA Cystic Fibrosis Assay; Perkin Elmer, Foster City) was performed in 30 of these infertile men. An extensive CFTR screening was carried out in all samples by multiplex denaturing gradient gel electrophoresis (DGGE) (Costes et al., 1993) for 15 exons and by single-strand conformation polymorphism analysis (SSCP) (Chillón et al., 1994) (Multiphor; Amersham Pharmacia Biotech, Bucks, UK) for the other 12 exons, the combination of these techniques giving a mutation detection level of 97% (Casals et al., 1997; also T.Casals, unpublished results). The DNA fragments were visualized by ethidium bromide staining in DGGE analysis, or by silver staining in SSCP gels. The abnormally migrating fragments were characterized by sequencing with the DyeDeoxyTM chain terminator method on an ABI 377 sequencer. The 5T variant in the polymorphic region IVS8-6(T) was analysed as described previously (Chillón et al., 1995). The M470V polymorphism (Cuppens et al., 1994) was analysed in 82 available samples. The same CFTR gene analysis was performed in 50 individuals from the general population (Lázaro et al., 1999). In order to compare the frequencies of F508, L997F, 3732delA and 5T mutations, a total of 200 control subjects was studied.
Statistical analysis
Differences between proportions were tested by the 
2 statistic. A paired Pearson's coefficient was calculated to study correlation between continuous variables. Differences between groups of discrete variables were analysed by one-way analysis of variance (ANOVA) with the SSPS program (SPSS Hispanoportuguesa SL, Madrid, Spain) for personal computers (version 6).
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Results |
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CFTR mutations in CAVD
Forty-two different CFTR mutations were identified in the 134 CAVD patients. Nineteen CFTR mutations (45%) were detected only in patients with CAVD, and were found neither in Spanish patients with CF (Casals et al., 1997; T.Casals, unpublished results) nor in the general population (Lázaro et al., 1999
). A significant difference in the detection level was observed between CBAVD and CUAVD patients, with 38 mutations in CBAVD (accounting for 71% of alleles; 156/220), and six mutations in CUAVD (accounting for 29% of alleles; 14/48; 2 = 8.01, P = 0.004). Twenty-nine of the 42 mutations were found only once, and seven novel mutations were detected (Table I). None of these novel mutations was found among the general population.
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Among the mutations identified, the 5T variant was the most common in both groups of patients, accounting for 23% of CBAVD alleles (50/220) and 12% of those with CUAVD (6/48).
F508 and G542X were the most frequently identified CF mutations, but at lower frequencies than in CF patients (Casals et al., 1997) [43% versus 53% (2 = 156.44, P < 0.001) and 6% versus 8% (2 = 6.56, P < 0.02) respectively]. In contrast, mutations L206W and R117H, each causing a mild CF phenotype (Dean et al., 1990; Desgeorges et al., 1995), were found with a higher frequency in CAVD than in CF patients [4.0% versus 0.6% (2 = 20.09, P < 0.001) and 3.6% versus 0.3% (2 = 28.45, P < 0.001) respectively].
Only 13 mutations were found more than once, accounting for a total of 83% of the mutated alleles, while 29 other mutations were detected in single patients (Table II). IVS8-6(5T), F508, G542X, L206W and R117H are the most common mutations in CAVD, each with a frequency over 5% of the mutated alleles.
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CFTR genotypes in CBAVD and CUAVD
The different genotypes found in the patients with CBAVD are shown in Table III
. CFTR mutations were identified in 85% of these patients (56% with two CFTR mutations, and 29% with one) after extensive CFTR gene analysis. In four patients, two CFTR mutations were found in cis, being the 5T variant and another mutation (S50P, 2751+3A
G, A1006E and F1074L). Except for the S50P mutation, which is associated to 5T and 7T alleles in CAVD patients, the other three mutations were always found with the 5T allele in both CAVD and CF phenotypes. The most common genotype was the combination of any CFTR mutation and the 5T allele (30%). We detected only three homozygous patients (one for V232D and two for 5T). Table IV shows the CUAVD genotypes; CFTR mutations were identified in 38% patients (21% with two mutations, and 17% with one).
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Among the patients of this study, 16 had renal agenesis associated (six CBAVD, 10 CUAVD). Five of these patients (31%) carried one CFTR mutation, three being
F508, L997F or 3732delA, and two the 5T variant. This frequency was significantly higher (2 = 9.95, P = 0.001) than that expected in the general population for these CFTR mutations that cause either CF or CAVD (7.5%). The specific frequencies for each of these mutations in the general Spanish population (200 samples not CF) are F508 2%, L997F 0.5%, 3732delA 0%, and 5T 5% (Chillón et al., 1995; Casals et al., 1997; Lázaro et al., 1999).
CFTR polymorphisms
A total of 21 different polymorphisms were identified. The M470V variant in exon 10 was analysed in 82 patients (98 alleles M470 and 66 V470), (TTGA)n in intron 6a which presented the higher frequency of seven repeats (129/272 alleles), T854T in exon 14a (72/272 alleles), 4521GA in exon 24 (62/272) and 3601-65C/A in intron 18 (48/272) were the most frequent. Six polymorphisms: 125G/C, 1525-61A/G, 1898+152T/A, 1716G/A, G576A and 875+40A/G presented frequencies of between 2.5% and 4.0%. Another four with frequencies of 1–2% were 1816G/A, 4404C/T, 1001+11C/T and R668C. Finally, six polymorphisms were found each in one patient: 104G/T, 296+128G/C, 741C/T, 3195A/T, 3212T/C and 4029A/G. The five new polymorphisms identified are described in Table I
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Clinical features
Two of the patients had relatives with known CAVD, and one patient had a sister with CF. Ten patients (five CUAVD, five CBAVD) had siblings who died during infancy of respiratory infections. Seven of these men had at least one CFTR mutation.
A number of renal anomalies were observed in the CAVD patients (Table V). Unilateral renal agenesis was diagnosed in 41% of CUAVD and in 5.4% of CBAVD patients (2 = 12.4, P < 0.001). Renal agenesis predominated in men without CFTR mutations, but three patients with CUAVD and two with CBAVD showed co-existing renal agenesis and one CFTR mutation (three patients CF/–, two patients 5T/–). Frequencies of other clinical conditions, such as history of cryptorchidism, inguinal hernia, nasal polyps, rhinosinusitis and varicocele, are shown in Table V. Two men with CUAVD had unilateral cryptorchidism associated with ipsilateral inguinal hernia. In the remaining patients hernia was contralateral to the maldescended testis, or occurred in subjects with bilateral cryptorchidism. Nasal pathology was more frequent in CBAVD patients with mutations (36%) than in those without mutations (8.3%). None of the patients showed pulmonary or gastrointestinal symptoms of CF. However, repeated respiratory infections or bronchitis were common in both groups, often associated with heavy smoking habit (>20 cigarettes per day). One patient with CUAVD and three with CBAVD (all with CFTR mutations) suffered from asthma.
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Anomalies of seminal vesicles—including agenesis, hypoplasia and cystic dysplasia—were very common among CAVD individuals. Unilateral abnormalities (typically on the same side of the absent vas deferens) predominated in CUAVD, whereas bilateral dysplasia was more common in CBAVD (Table V
). Total length of seminal vesicles measured by transrectal ultrasonography was significantly smaller in CBAVD than in CUAVD (F = 8.1, P = 0.005). Dilatation of ejaculatory ducts, often resembling utricular cysts, was demonstrable also in some men, all of whom were azoospermic (Figure 1). Vasograms performed in six patients with CUAVD not only confirmed ultrasonographic findings, but also showed additional abnormalities at various levels of the seminal tract. The volume and consistency of testes was normal, except in patients with other concomitant pathologies, such as cryptorchidism (n = 6), trauma (n = 2), orchitis (n = 2) or tumour (n = 1). The prostate gland showed normal size and morphology in all patients.
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Semen volume, pH, sperm concentration and fructose were significantly different in CUAVD compared with CBAVD (Table VI
). Azoospermia was more frequent in CUAVD patients with CFTR mutations (7/9) than in those without mutations (8/13), and sperm concentration was higher in the latter group. However, the differences were not statistically significant. Concentrations of citrate in seminal plasma showed no significant differences between groups. In a small group of patients, alpha-glucosidase activity was 18.5 ± 2.7 mU/ml in CUAVD (n = 4), and 26.7 ± 5.5 mU/ml in CBAVD (n = 7). After reclassification of the patients according to the presence of zero, one or two mutations, none of the variables showed significant differences either in CUAVD or CBAVD (not shown). As expected, fructose and citrate concentrations were highly correlated with seminal volume (r = 0.70, r = 0.58 respectively; P < 0.001). The length of seminal vesicles showed significant correlation with fructose in seminal plasma (r = 0.52, P < 0.001), semen pH (r = 0.47, P < 0.001) and with chloride in sweat (r = –0 35, P = 0.02).
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Chloride concentrations in sweat were higher in CBAVD patients with CFTR mutations than in those without mutations (F = 3.4, P = 0.07), and approached statistical significance. Chloride concentration was higher in CUAVD men without mutations (n = 5) than in those with mutations (n = 5), but the number of cases tested was low and most likely not representative. Discrimination analysis showed that the single best variable to predict the presence of CFTR mutations was the type of CAVD, followed by the pH of semen and the sperm concentration. A canonical discriminatory function including all three variables was able to classify correctly in 80% of the cases.
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Discussion |
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This report and previous studies (Chillón et al., 1995
; Costes et al., 1995; Rave-Harel et al., 1995; Dörk et al., 1997; Mak et al., 1999) highlight the heterogeneity that exists for mutations in the CFTR gene in patients with CAVD, and indicate that the spectrum of CFTR mutations is different in CAVD than in CF patients. Less than 60% of the mutations detected here are found in patients with CF (Casals et al., 1997). Although only 11 CFTR mutations account for a total of 83% of the mutated alleles in CAVD, the presence of 31 other different mutations indicates a high heterogeneity in the CFTR gene in CAVD. This suggests that a complete characterization of the CFTR gene is needed in many patients with CAVD in order to identify one or two mutations that they might have. Thus, the OLA/PCR mutation detection system that covers 31 mutations in patients with CF, only allows the identification of 26% of the CFTR mutations in CAVD patients. Despite the fact that CFTR microsatellites facilitate mutation analysis in CF patients (Morral et al., 1996), the heterogeneity and the different spectrum of mutations in CAVD patients indicate that microsatellite studies before mutation analysis are not helpful in identifying mutations in patients with CAVD. Nineteen of the mutations detected in patients with CAVD were not previously identified in over 700 unrelated Spanish patients with CF (T.Casals, unpublished data). Most of these new mutations correspond to amino acid changes, in addition to some splice site mutations, and it is likely that they cause a mild CFTR dysfunction, as expected for an incomplete CF phenotype.
Genetic counselling is especially difficult in couples with infertility due to CAVD. The wide spectrum of CFTR mutations, some of them with unpredicted clinical consequences, suggest that the CFTR analysis should not only be focused on mutations common in CF patients. A complete characterization of the CFTR gene in these couples would be desirable, especially as some of the patients carry only one CFTR mutation, which is not present in those patients with CF.
One interesting finding was the strong association of the 5T allele with the valine at position 470 (71%) (2 = 13.67, P < 0.001). This result is in agreement with a previous report (De Meeus et al., 1998), suggesting that the M470V locus could contribute to the variable expression of the 5T allele, with valine being involved in lower CFTR protein levels.
The high proportion of cryptorchidism (18% in CUAVD and 4.7% in CBAVD) confirms the increased prevalence of this alteration in CAVD described previously (Schlegel et al., 1996), and suggests an association between CAVD and testicular descent. The prevalence of inguinal hernia was also significant in patients with CAVD, even after excluding the two cases with ipsilateral cryptorchidism. Both conditions can be observed at high rate in men with CF (Holsclaw et al., 1971).
Anomalies of the seminal vesicles were very common in CAVD patients. Unilateral alterations predominated in CUAVD, whereas bilateral abnormalities were found more frequently in CBAVD. Overall, 84% of CUAVD and 60% of CBAVD patients showed some dysplasic seminal vesicles, with either agenesis, hypoplasia or cystic dysplasia. Additional explorations, such as vasography performed in some CUAVD patients, revealed new malformations in these men that were not detectable by transrectal ultrasonography. Previous reports found variable frequencies of changes in seminal vesicles, ranging from 36% to 92% in CBAVD (Goldstein and Schlossberg, 1988; Marmar et al., 1993; Jarvi et al., 1998; Taille et al., 1998), and 85% in CUAVD (Mickle et al., 1995; Schlegel et al., 1996). Ultrasonographic measurement of seminal vesicles showed diminished length in CBAVD compared with CUAVD, and sweat chloride concentration showed a significant negative correlation with the size of seminal vesicles. These data suggest indirectly that variable phenotypic expression of CFTR could result in genital manifestations with corresponding degrees of severity.
Although none of the CAVD patients had symptoms of CF, nasal polyps and/or rhinosinusitis were associated with the presence of CFTR mutations, especially in patients with CBAVD. Frequent respiratory infections and bronchitis were also noted in CAVD individuals, though no clear relationship with genotype could be established, as other risk factors (e.g. heavy smoking habit) were likely to be implicated. Nevertheless, these observations suggest minor, often under-reported, clinical manifestations of some particularly sensitive epithelial tissues in our patients.
Seminal variables, such as volume, pH and fructose were more clearly affected in CBAVD than in CUAVD, and showed good correlation with the size of seminal vesicles. This is in keeping with the hypothesis that the different degrees of hypoplasia observed in CAVD exhibit progressive functional impairment. While all CBAVD subjects were azoospermic, 32% (7/22) of CUAVD patients had some spermatozoa in their semen. Three of these men (one of whom was fertile) had CFTR mutations. It was also indicated (Mickle et al., 1995) that men with CUAVD, who had a patent contralateral seminal duct, showed no CFTR mutations; these workers concluded that two distinct subpopulations with different aetiologies could be established, based upon the mutational status of the CFTR gene. Our results add more complexity to this hypothesis, and suggest that some CUAVD patients with CFTR mutations have spermatozoa in their semen and may be fertile. The discrepancy may be due to the fact that our patients were not exclusively infertile, and CFTR analysis was more complete.
Measurement of neutral glucosidase has been proposed for diagnosis of obstructive lesions of the epididymis and the vas deferens (Guerin et al., 1986). Previous studies have suggested that the different methods currently used for determination of glucosidase are suitable for clinical purposes in men (Mahmoud et al., 1998). Our results indicate that the EpiScreen method used in this study measures the activity of other acidic 
-glucosidase isoenzymes, most likely an isoenzyme contained in the prostatic secretion, and probably leads to inaccurate results.
We have found that about one-third (5/16) of patients with CAVD and renal agenesis have mutations in CFTR (most of these patients being CUAVD). These results contrast with those reported previously of a low proportion of CFTR mutations in such patients (Anguiano et al., 1992; Augarten et al., 1994; Schlegel et al., 1996; Taille et al., 1998). We attribute this discrepancy to the reduced number of samples of these previous studies, which were focused on the most frequent CFTR mutations, without performing a complete analysis of the whole CFTR gene. Although the sample in the present study is small (16 patients), these findings should encourage other investigators to characterize fully those cases of CAVD and renal agenesis. These studies should help to define the proportion of cases of CAVD and renal agenesis that are due to mutations in CFTR.
It is still unclear how CFTR is involved in the development of CAVD. Moreover, the putative relationship between CFTR and renal agenesis (found here in 31% of cases) is even more intriguing. It has been proposed that when renal anomalies co-exist with CAVD, a defect in the Wolffian duct is produced at the time of, or before, formation of the ureteral bud, resulting in malformation of the entire Wolffian duct and subsequent vasal agenesis (Dumur et al., 1995; Schlegel et al., 1996). The involvement of CFTR in both CAVD and renal agenesis can be understood from a polygenic/multifactorial point of view. Mutations in CFTR in conjunction with variants in genes involved in renal formation could participate in a synergistic action in renal and vas deferens alterations. The identification of the genetic and environmental factors that participate in renal development will require the analysis of a larger number of cases and the use of genomic analysis approaches.
In summary, our results have confirmed the high molecular heterogeneity for CAVD and the different spectrum of CFTR mutations when compared with CF patients. The study has also highlighted the importance of an extensive CFTR molecular characterization of CAVD patients in order to provide a better understanding of the molecular basis of this disorder. Finally, our findings suggest that CFTR mutations might also have a role in urogenital anomalies.
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Acknowledgments |
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We thank Helena Kruyer for her help with the manuscript, and the ECCACF for providing primers for DGGE analysis. These studies were supported by grants from Fondo de Investigaciones Sanitarias (96/2005 and 99/0654), Fundació La Marató de TV3 (980410), the Institut Català de la Salut, and the Associació Catalana de Fibrosi Quística.
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Notes |
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5 Present address: Andrology Unit, Hospital General Universitario, Alicante, Spain
6 To whom correspondence should be addressed at: Medical and Molecular Genetics Center-IRO, Hospital Duran i Reynals, Barcelona, Spain. E-mail: tcasals{at}iro.es
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Submitted on December 22, 1999; accepted on March 27, 2000.
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