1 Department of Genetics, Faculty of Medicine, 4 Laboratory of Cell Biology, ICBAS, University of Porto, 3 Centre for Reproductive Genetics Alberto Barros, Porto, Portugal and 2 Department of Biochemistry and Genetics, Henri Mondor Hospital, Creteil, France
5 To whom correspondence should be addressed at: Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Lg. Prof. Abel Salazar 2, 4099-003 Porto, Portugal. Email: msousa{at}icbas.up.pt
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Abstract |
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Key words: azoospermia/CAVD/CFTR gene mutations/IVS8 poly(T)/male infertility
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Introduction |
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The CFTR gene appears highly susceptible to mutations due to its large size and misprocessing errors (Riordan et al., 1989). More than 1000 genetic alterations were described in the CFTR gene; most of them are disease-causing mutations, about half are the result of amino acid substitutions (missense mutations), 20% are due to splicing errors, and the remainder appear to be caused by nonsense and frameshift (including small deletions and insertions) or promoter mutations (Cystic Fibrosis Mutation Database, 2003
). The most frequent disease-causing mutation, F508del, causes deletion of a single amino acid, phenylalanine, at position 508 of the polypeptide. This mutation accounts for
70% of CF chromosomes worldwide, although the estimated prevalence varies from region to region, being as high as 80% in Northern Europe and falling to <50% in countries around the Mediterranean basin and in the Middle East. In Portugal, the estimated frequency is
50% (Duarte et al., 1990
; Estivill et al., 1997
).
The predominant manifestations of CF are due to impaired chloride conduction across epithelial cells in the respiratory, digestive and reproductive tracts. Patients with CF have an elevated sweat chloride concentration (>60 mmol/l) that confirms the diagnosis. Respiratory tract manifestations are the main cause of morbidity and mortality among CF patients, with airway obstruction being caused by thick, sticky mucus, with subsequent infections, especially by Pseudomonas species. Other CF manifestations include the gastrointestinal tract, such as pancreatic insufficiency (85%) as a result of pancreatic duct obstruction, meconial ileus (510%) due to fetal intestinal obstruction, and liver disease (25%) (Rosenstein and Cutting, 1998; Welsh et al., 2001
).
Nearly 98% of all CF males are also infertile due to obstructive azoospermia (OAZ). The spectrum of the observed Wolffian duct abnormalities includes atrophy, fibrosis or absence of the epididymis body and tail, vas deferens, seminal vesicles and/or ejaculatory ducts (Chillón et al., 1995). On the contrary, in the general population, congenital absence of the vas deferens (CAVD) is responsible for
2% of male infertility cases and for 6% of the cases with OAZ (Oates and Amos, 1994
). A genetic link between CF and CAVD was suggested when an increased frequency of heterozygotes for the F508del mutation was reported. In 1992, the first observation of compound heterozygosity for the CFTR gene in a patient with congenital bilateral absence of the vas deferens (CBAVD) was described and, from the observation that many CBAVD men had mutations in their CFTR gene, it was proposed that CBAVD may be a primary genital form of CF (Anguiano et al., 1992
). These results were confirmed subsequently and, in a review of 420 CBAVD cases, 19% were shown to carry two CFTR mutations, 47% a single CFTR mutation and 34% had no identified CFTR mutation (Lissens et al., 1996
).
The most common CFTR mutations in CAVD are the F508del, R117H and the 5T allele (Chillón et al., 1995; De Braekeleer and Feréc, 1996
). The poly(T) sequence located in the splicing acceptor site of intron 8 [IVS8 poly(T)] exists in three variants, with five, seven or nine thymidines (the 5T, 7T and 9T alleles, respectively) (Kiesewetter et al., 1993
). The 7T and 9T alleles predominantly generate normal mRNA transcripts, whereas the 5T variant results in reduced levels of normal mRNA due to deletion of exon 9. The protein product of the CFTR transcript lacking exon 9 is devoid of cAMP-activated chloride conductance, and, therefore, the 5T allele is considered to be a mild mutation with an incomplete penetrance (Chu et al., 1993
; Teng et al., 1997
).
In Portugal, the type and rate of CFTR mutations and the frequency of the IVS8-5T allele in the CAVD population remain to be evaluated. In the present study, we have screened 38 Portuguese infertile males with CAVD, and the type and frequency of mutations were compared with another 39 infertile patients with conserved spermatogenesis, 16 with secondary OAZ and 23 with non-obstructive azoospermia (NOAZ). As the prevalence of the IVS8 poly(T) variants is also unknown in the Portuguese general population, the allelic frequency of the poly(T) variants was also determined in 114 fertile males.
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Materials and methods |
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Screening of CFTR gene mutations
DNA samples were obtained from peripheral blood lymphocytes of patients using a salting-out method (Miller et al., 1988). All patients were screened for 31 CFTR gene mutations that include the 24 mutations more commonly associated with CF within the Caucasian population (Estivill et al., 1997
), by using a commercial kit that has a sensitivity value of 0.99 (Cystic Fibrosis Diagnostic System, Applied Biosystems, Foster City, CA). This technique enables amplification of several DNA fragments simultaneously using PCR, and the oligonucleotide ligation assay (OLA) uses different probes to detect the presence of normal and mutant CFTR alleles. The fluorescent DNA fragments were separated and sorted based on their size and colour in an ABI PRISM 310 Genetic Analyzer using the appropriate software (Genotyper, Applied Biosystems).
Screening of the full gene by denaturing gradient gel electrophoresis (DGGE) was performed in CAVD patients with only one identified mutation or absence of mutations. DGGE is a highly sensitive scanning method widely used in CF diagnosis, which detects >95% of CF mutations in our population and whose sensitivity in our laboratories was thus estimated to be 9698% (Fanen et al., 1992; Costes et al., 1993
). PCR products were obtained with specific primers for each of the 27 exons and their flanking intron sequences (CFTR DGGE, Ingeny, The Nertherlands). PCRs were performed in a 30 µl reaction volume with 2 µl of 10x PCR buffer (100 mmol/l TrisHCl pH8.3, 500 mmol/l KCl; MBI Fermentas, Foster City, CA), 0.8 µl of MgCl2 (25 mmol/l; MBI Fermentas), 2 µl of dNTP mix (12.5 pmol of each dNTP/ml; Invitrogen, Foster City, CA), 10 pmol of each primer, and 0.25 µl of recombinant Taq DNA polymerase (5 U/µl; MBI Fermentas). PCR cycling conditions were as follows: 40 cycles with a pre-soak at 94°C for 3 min, denaturation at 94°C for 1 min, annealing at 50 or 55°C (dependent on primer sequence) for 1 min, a subsequent polymerization step at 72°C for 1 min, and a final extension step at 72°C for 10 min. The amplification was confirmed on 2.5% agarose gels stained with ethidium bromide. DGGE was performed in a denaturing gradient gel of 2065% prepared with 9% acryl/bisacryl (37.5:1). The running conditions were 1800 V/h at 58°C. DNA samples with an abnormal migration pattern were amplified by PCR using the same primers and sequenced with the Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems), and run in an ABI PRISM 310 Genetic Analyzer using the appropriate software (Sequencing Analysis, Applied Biosystems) for the identification of the mutations.
Screening of poly(T) variants at intron 8
Identification of the poly(T) variants at intron 8 was performed by PCR amplification, using primers I9D9 (FAM-labelled) and E9R2 (Chillón et al., 1995). PCRs were performed in a 25 µl reaction volume containig 2.5 µl of 10x PCR buffer, 1 µl of MgCl2, 2 µl of dNTP mix, 0.5 µl of each primer (12.5 pmol/µl) and 0.2 µl of recombinant Taq DNA polymerase. Cycling conditions were as follows: 30 PCR cycles with a pre-soak for 10 min at 95°C, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, with a subsequent polymerization step at 74°C for 40 s, and a final extension step at 72°C for 10 min. The amplification was confirmed on 2.5% agarose gels stained with ethidium bromide and products then analysed in an ABI PRISM 310 Genetic Analyzer with the appropriate software (GeneScan, Applied Biosystems).
Statistical analysis
Proportions and 95% confidence intervals (CIs) were calculated as described (Newcombe and Altman, 2000). Proportions between groups were compared by using the difference between two proportions test (Statistica v5·1), with the significance of the P-value being set at 0.05.
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Results |
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Of the 31 CBAVD patients with normal renal development, the initial screening identified 16 CFTR mutations (five different mutations) in 14 (45.2%) cases. Biallelic mutations were found in two (6.5%) cases, whereas monoallelic mutations were found in 12 (38.7%) patients (Figure 1; Table I). DGGE analysis performed in patients with only one or no identified mutations revealed nine further mutations (seven different mutations), four in previous heterozygotes and five in four other patients (two mutations in cis and three single mutations). This increased to 25 the number of CFTR mutations detected (12 different mutations) and to 18 out of 31 (58.1%) the number of CBAVD patients with at least one CFTR mutation detected. In total, 6 out of 31 (19.4%) cases had two CFTR mutations (five compound heterozygotes and one homozygote), whereas 12 out of 31 (38.7%) patients carried a single CFTR mutation (Figure 1, Table I). The study of IVS8 poly(T) splicing variants (Table II) revealed 14 out of 31 (45.2%) cases with the 5T variant, 3 out of 31 (9.7%) homozygous (5T/5T) and 11 of 31 (35.5%) heterozygous (5T/). The association of CFTR mutations with the 5T allele thus increased to 22 out of 31 (71%) the number of CBAVD patients with at least one mutation detected. Overall, in the 31 CBAVD patients (31 x 2=62 alleles), the 5T allele was the most frequent mutation found, which accounted for 17 of the 62 (27.4%) total alleles. The F508del mutation was the second more common mutation and represented 8 out of the 62 (12.9%) total alleles. The frequency of the other mutations was: four of 62 (6.5%) for R334W, two of 62 (3.2%) for R117H, P205S and G576A, and one of 62 (1.6%) for D614G, V562I, R668C, 2789-5GA, S1235R, I507del and 3272-26A
G.
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Infertile males with secondary OAZ showed two out of 16 (12.5%) cases with mutations, one (6.3%) patient with a heterozygous CFTR mutation and one (6.3%) case with a heterozygous 5T variant (Table I). The large majority of the patients had normal semen volume and pH (12 out of 16, 75%). The patient with F508del (7T/9T) had a reduced semen volume and pH, whereas the patient with 5T/7T had a normal semen volume and pH, was previously oligozoospermic and became azoospermic following orquiepididymitis. The other three patients (7T/9T, 2x 7T/7T) with low semen volume and pH were also previously oligozoospermic and became azoospermic following orquiepididymitis (one after tuberculosis).
Of the 22 NOAZ patients with conserved spermatogenesis and normal renal development, there were seven (31.8%) patients with mutations, two (9.1%) cases with heterozygous CFTR mutations and six (27.3%) cases with the 5T allele, one (4.5%) in homozygous and five (22.7%) in heterozygous, of which one was associated with F508del. The large majority of the patients had normal semen volume (19 out of 22, 86.4%) and pH (21 out of 22, 95.5%). The patient with the 3659del mutation (7T/7T) had normal semen volume and pH, the case with the F508del mutation (5T/9T) had reduced semen volume (0.5 ml) and pH (6.8), and all cases with the 5T allele had normal semen pH and volume, with the exception of the 5T/5T case that had a normal semen pH (7.4) but reduced semen volume (0.7 ml).
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Discussion |
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In CBAVD patients, a high frequency of compound heterozygosity with severe/mild or mild/mild mutations has been reported (Anguiano et al., 1992). In the Portuguese CBAVD population, there were 19.4% compound heterozygotes, and 38.7% heterozygotes. F508del was the most frequent mutation (12.9%), an allelic frequency similar (1214%) to Brazil (Bernardino et al., 2003
) and Greece (Kanavakis et al., 1998
), but lower than that found (2133%) in Canada (Jarvi et al., 1998
; Mak et al., 1999
), the USA (Oates and Amos, 1994), Germany (Dork et al., 1997
), France (Claustres et al., 2000
) and Spain (Casals et al., 2000
). Contrary to all other countries, the second most frequent mutation found in Portugal was R334W (6.5%), whereas no differences from other countries were found regarding the third most frequent mutations, R117H, P205S and G576A (3.2%), with the exception of Germany regarding R117H (11%) (Dork et al., 1997
).
In CBAVD patients, one of the most frequent genotypes found is the combination of the 5T allele with CFTR mutations (Chillón et al., 1995). This suggests that the vas deferens is one of the most susceptible tissues to the effect of changes in CFTR activity (Anguiano et al., 1992
). In fact, although there might be enough CFTR protein to prevent disease in other organs, the 5T variant may specifically affect the reproductive tract in CBAVD as the splicing efficiency of exon 9 in the vas deferens is lower and results in the production of a non-functional protein (Chillón et al., 1995
; Rave-Harel et al., 1997
; Teng et al., 1997
). In Portuguese CBAVD patients, there were 45.2% of cases with the 5T allele, which gives an allelic frequency of 27.4%. This frequency is similar to that found (2127%) in Canada (Jarvi et al., 1995
, 1998
; Mak et al., 1999
), Brazil (Bernardino et al., 2003
), Spain (Casals et al., 2000
), Croatia (Sertic et al., 2001
) and Italy (Gelfi et al., 1998
), higher than that found (1018%) in the USA (Wang et al., 2002
), The Netherlands (Dohle et al., 1999
), Germany (Dork et al., 1997
), France (Claustres et al., 2000
) and Greece (Kanavakis et al., 1998
), and lower than that (4456%) in Israel (Rave-Harel et al., 1997
) and Egypt (Lissens et al., 1999
). In the Portuguese CBAVD population, 71% of the patients had at least one CFTR or 5T mutation, 51.6% with two mutations (25.8% of compound heterozygotes each for severe/mild and mild/mild mutations) and 19.4% with only one mutation, whereas 29% had no mutation detected. The high rate of patients with only one or no mutations (48.4%) thus suggests that other changes not determined by DGGE and IVS8 poly(T) variant analysis might be located at non-screened sites (introns, promoter or regulatory elements), resulting in a CFTR protein with normal structure but with low levels of expression, thus changing the development of susceptible tissues such as the vas deferens and epididymis (Anguiano et al., 1992
; Dohle et al., 1999
).
In common with other countries, a reduced number of patients with CUAVD were studied using full CFTR screening. In Portugal, the rate of CFTR mutations was higher (75%) than (2027%) in Canada (Mak et al., 1999) and Spain (Casals et al., 2000
), but similar (60100%) to Germany (Dork et al., 1997
) and France (Jézéquel et al., 2000
). This frequency was due to the allelic frequency of G542X (25%) in Portuguese CUAVD males, which is higher than in other countries where it was also found (717%), France (Jézéquel et al., 2000
) and Spain (Casals et al., 2000
).
Portuguese CAVD patients were compared with secondary OAZ males. Although four patients presented with reduced semen pH, it was not possible to know if this was caused by past genital infections or if it corresponds to discrete congenital hypoplasia of the seminal vesicles. As expected, although CFTR mutations were found in OAZ (6.3%), the frequency did not differ significantly from that (4%) observed in the general European Caucasian population (Welsh et al., 2001). This frequency appears similar to France (7%) (Boucher et al., 1999
; Jézéquel et al., 2000
), but is higher than in Brazil (0%) (Bernardino et al., 2003
), and lower than (1630%) in Canada (Jarvi et al., 1995
; Mak et al., 2000), The Netherlands (Dohle et al., 2002
) and Greece (Kanavakis et al., 1998
). The allelic frequency of the 5T variant was also not significantly different between OAZ (3.1%) and the fertile male population (3.5%). This frequency was similar (5%) to France (Boucher et al., 1999
; Jézéquel et al., 2000
), Spain (Chillón et al., 1995
) and Greece (Kanavakis et al., 1998
), higher than in Brazil (0%) (Bernardino et al., 2003
) and lower than in Canada (12%) (Jarvi et al., 1995
; Mak et al., 1999
).
CAVD patients were also compared with NOAZ males with conserved spermatogenesis and histopathological signs of tubular obstruction, but without any evidence of excretory genital duct obstruction. As expected, although CFTR mutations were found in NOAZ (9.1%), the frequency did not differ significantly from that (4%) observed in the general European Caucasian population (Welsh et al., 2001). A similar rate (4%) has also been reported in Canada (Jarvi et al., 1995
; Mak et al., 2000) and Germany (Jakubiczka et al., 1999
). However, and even if the F508del (5T/9T) patient is not considered due to the reduced semen pH, the 5T allelic frequency in NOAZ was found here to be significantly increased (14.3%), corresponding to 23.8% of the NOAZ patients with normal semen pH. This suggests the presence of a non-detected (intratesticular) obstruction in this particular azoospermic population that deserves further investigation.
In conclusion, we have characterized the type and frequency of CFTR mutations and of IVS8 poly(T) variants in Portuguese CAVD males with absence of CF clinical symptoms. About 58% of CBAVD and 75% of CUAVD patients showed CFTR mutations. The increased allelic frequency of R334W in CBAVD (6.5%) and of G542X in CUAVD (25%) appears particular to the Portuguese population. In comparison with the Portuguese fertile population (3.5%), the allelic frequency of the 5T allele also appeared increased in CBAVD (27.4%) and CUAVD (25%) patients.
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Acknowledgements |
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References |
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Submitted on December 17, 2003; resubmitted on April 29, 2004; accepted on July 16, 2004.
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