Mol. Hum. Reprod. Advance Access originally published online on May 19, 2006
Molecular Human Reproduction 2006 12(7):469-473; doi:10.1093/molehr/gal046
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Molecular analysis of the IVS8-T splice variant 5T and M470V exon 10 missense polymorphism in Iranian males with congenital bilateral absence of the vas deferens
1Department of Reproductive Genetics, 2Department of Male Infertility and 3Department of Stem Cell, Reproductive Biomedicine Research Center, Royan Institute, Tehran, Iran
4 To whom correspondence should be addressed at: Department of Reproductive Genetics, Reproductive Biomedicine Research Center, Royan Institute, PO Box 19395-4644, Tehran, Iran. E-mail: rradpour{at}royaninstitute.org
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Abstract |
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Congenital bilateral absence of the vas deferens (CBAVD) is responsible for 2–6% of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. To investigate CBAVD at the molecular level in Iran, we have characterized the mutations in the CFTR gene in 106 patients with this condition. None had clinical manifestations of cystic fibrosis (CF). We also analysed a DNA variant (the 5T allele) in a noncoding region of CFTR, which causes reduced levels of the normal CFTR protein and M470V exon 10 missense polymorphism. Five of the 106 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Eighty-five patients had a mutation in at least one copy of CFTR, and of these patients, 46 had one 5T allele (in 11 cases, two alleles and in 35 cases, just one allele of 5T was detected). In 21 patients, no CFTR and 5T mutations were found (19.81%). 5T/M470 genotype was found in 19 patients, 5T/V470 was found in 3 and 5T with heterozygote form of M470V was found in 24 CBAVD patients. In CBAVD patients, 28 F508del carriers were identified. Most of our patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a CF mutation in the other copy is the most common cause of CBAVD in Iran. The 5T allele mutation has a wide range of clinical presentations and revealed a high frequency, occurring in patients with CBAVD or moderate forms of CF and infertile men.
Key words: CBAVD/CFTR/IVS8-5T/male infertility
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Introduction |
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Male infertility as a result of isolated congenital bilateral absence of the vas deferens (CBAVD) is a recognized primary genital form of cystic fibrosis (CF). When CBAVD is not associated with renal malformations, extensive analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene allows mutations to be identified in up to 85% of men (Claustres et al., 2000
). CBAVD is also a common feature in males with CF, an autosomal recessive disease primarily characterized by respiratory tract disease (Zielenski et al., 1991). CBAVD is caused by mutations in CFTR gene that contains 27 exons encompassing 
180 kb of DNA on chromosome 7q31.2. Over 1000 mutations have been described (Cystic Fibrosis Mutation Consortium, 2006)—mutations that are clustered in six different classes including defective CFTR biosynthesis, defective protein processing, alteration in CFTR regulation, disruption of the pore activity, alteration of CFTR localization and genesis of unstable CFTR (Claustres, 2005). There is a core of 25 most common mutations designated by the CF Steering Committee in 2001, which occur with a European frequency of 
0.1%. Some mutations are clearly associated with a mild phenotype (Daudin et al., 2000). Other attempts to link mutations in CFTR to disease severity have not been successful, suggesting an influence of non-CFTR gene modifiers and environmental factors (Jezequel et al., 2000; Wu et al., 2005a).
In the majority of cases, CBAVD can be considered as a genital form of CF, presenting without the other clinical features of CF. Generally, 20% have two CFTR mutations, 60% have one mutation and 20% have no CFTR mutations (Claustres, 2005).
An intervening sequence 8 splice variant, called 5T (IVS8-5T), is also frequently observed in CBAVD and seems to be specific for this condition and probably also for other CF-related disease presentations without the major features of CF (Chillon et al., 1995; Grangeia et al., 2004). Length variants of a polypyrimidine tract within the splice acceptor site at the end of intron 8 of the CFTR gene, named IVS8(T)n, lead to alternative splicing which results in two types of mRNA transcripts, one with and the other without exon 9. Of the three IVS8(T)n alleles (9T, 7T and 5T), the shortest (5T) is associated with the highest rate of incomplete transcripts. mRNA without exon 9 results in CFTR proteins that will not mature and will therefore not function as chloride channels in the apical membrane of epithelial cells. The 5T allele is therefore classified as a CBAVD mutation, although with incomplete penetrance because identical genotypes can be found both in CBAVD and in non-CBAVD individuals (Chillon et al., 1995).
Molecular diagnosis is based on CFTR mutation screening. However, extensive allelic heterogeneity frequently makes difficult the rate of detection and therefore the value of the genotypic diagnosis. Several mutation scanning methods have been applied to the detection of sequence variations in the entire coding region of CFTR, such as heteroduplex analysis and restriction enzyme analysis (Chillon et al., 1995; Claustres, 2005; Mennicke et al., 2005), single-strand conformation polymorphism analysis (SSCP) (Liechti-Gallati et al., 1999), denaturing gradient-gel electrophoresis (DGGE) method (Culard et al., 1994) and denaturing high-performance liquid chromatography (DHPLC) (Ravnik-Glavac et al., 2002).
We have studied 106 Iranian males with CBAVD from Iran, with the aim to determine the frequency of CF mutations and the 5T variant in these patients. Because no data are available on either the frequency of CBAVD or the mutations leading to CBAVD in Iran, we have included the most common mutations seen in CF worldwide (Cuppens and Cassiman, 2004).
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Materials and methods |
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Samples
Blood samples were collected from 106 unrelated Iranian males with azoospermia and CBAVD visiting the Royan Institute, Iran. The diagnoses of CBAVD were initially suggested by the clinical observation of impalpable vasa in the patients and were subsequently confirmed by transrectal and transabdominal ultrasonography (Table I). Each patient had a sperm count of zero. We studied 43 fertile males from the general population in Iran as control subjects. We also studied 7 patients with congenital unilateral absence of the vas deferens (CUAVD) and 10 patients with obstructive azoospermia not due to CBAVD or CUAVD (obstructive azoospermia was due to vasectomy and epididymal obstruction affected by infections, i.e. chlamydia or gonorrhoea). Cytobiochemical parameters of semen (including pH, ejaculate volume and total sperm count) were analysed for each sample according to WHO criteria (World Health Organization, 1999
) (Table II).
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CFTR mutations
Genomic DNA was isolated from peripheral blood lymphocytes according to standard procedures (Mennicke et al., 2005). All samples (patients and controls) were analysed for the 29 common mutations by ElucigeneTM CF29 multiplex ARMS kit (Tepnel Diagnostics Ltd, UK) as a rapid screening according to the manufacturer’s instructions. Missense polymorphism M470V in exon 10 was typed by HphI restriction enzyme analysis. During the course of the study, samples shown to have only one or none of the 29 common mutations were selected for further investigation, and all exons were amplified using the published primer pairs for sequencing (Zielenski et al., 1991) and studied by DGGE (Culard et al., 1994) or by SSCP (Liechti-Gallati et al., 1999) and results confirmed by sequencing. Sequencing of PCR products was carried out by VBC-Genomics (VBC-Genomics Bioscience Research) using 50 ng (2 µl) of PCR product and 4 pmol/l (1 µl) of non-fluorescent primer (forward and reverse separately), 4 µl of BigDye Terminator ready reaction kit (Perkin Elmer) and 3 µl of double-distilled water to adjust the volume to 10 µl. Sequencing results were compared with the sequence of wild-type CFTR gene published on Cystic Fibrosis Mutation Database.
Genomic analysis of IVS8-5T allele
To evaluate the frequency of the 5T allele of intron 8 in CBAVD and infertility, we studied the frequency of heterozygosity for the allele in patients with CBAVD, patients with CUAVD, patients with azoospermia but not CBAVD and the general population. Amplification and sequencing of the polypyrimidine tract in front of exon 9 was performed using primers 9i-5 and 9i-3 (Chillon et al., 1995). Nested PCR was performed to amplify the polypyrimidine sequence with modified primers RF9 (5'-CCGCTGTGTGTGTGTGTGTGTTTTT-3') and RR9 (5'-GGATCCAGCAACCGCCAA-3'). The nested PCR conditions were as follows: denaturation at 95°C for 30 s, annealing at 54°C for 30 s and extension at 74°C for 40 s, for 35 cycles. The reaction mixture contained 5 µl of PCR buffer, 200 µmol/l each of dNTPs, 20 pmol of each primer and 1 unit of Taq DNA polymerase in a final volume of 50 µl, containing 1 µl of the first PCR product. Nested PCR products finally were digested with XmnI enzyme and visualized on 12% nondenaturing polyacrylamide gel after electrophoresis for 4–5 h at 230 V (Figure 1).
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Results |
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CFTR mutations in studied groups
Patients and normal subjects were first screened using a standard panel of 29 different CF mutations (CF29 Tepnel kit), and if there were not two known mutations, we performed genome scan by SSCP or DGGE, followed by sequencing. The analysis of the entire coding sequence allowed us to identify 16 different mutations in CBAVD patients, 13 mutations identified by kit and 3 mutations identified by genome scan (R347H, R553X and 1540A/G) (Table III). Most of the mutations have been described previously in patients with CF, but others have been detected specifically in patients with CBAVD. Of these, 46 cases were homozygous or compound heterozygous (+/+), 39 were positive for only one mutation (+/–) and 21 cases were negative for both mutations (–/–). In CUAVD patients, we identified two different CFTR mutations. In the control group, no CFTR mutations were identified (Table IV).
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Frequencies of the IVS8-5T variant and M470V missense mutation
Study of the polypyrimidine tract in front of exon 9 revealed a high frequency of the 5T allele in the CBAVD males (Table V). Eleven males were homozygous for 5T, whereas 35 males were heterozygotes with a 7T or a 9T on the other allele. In contrast, none of the normal males were found to carry a 5T allele. The 5T allele distribution was 25.94%, 54.25% for 7T and 19.81% for 9T in the CBAVD males. We evaluated the frequency of the 5T allele in men with various types of infertility. In our studied population, the percentage of patients with CBAVD who had this allele was significantly higher than the general population (
2 = 11.4, P < 0.05), whereas the proportion of patients with CUAVD who had the 5T allele (21.43%) was higher than, but not significantly different from, the patients with obstructive azoospermia. In most patients with CBAVD, the 5T allele was strongly associated with the presence of a CF mutation in the other copy of the CFTR gene. The association between the various CFTR mutations and the 5T allele in the patients with CBAVD was analysed by studying the transmission of the mutations within families. Seven CFTR mutations were associated with the 5T allele in our population (Table III), whereas all the others were associated with the 7T or the 9T allele, confirming that in each patient with CBAVD, the 5T allele corresponded to the chromosome that did not carry the CFTR mutation. Levels of normal CFTR mRNA depend on the genotype determining the length of the thymine sequence in intron 8 of CFTR. When both CFTR genes bear the 5T allele (the 5T/5T genotype), the proportion of normal CFTR mRNA is reduced to 8 to 12%, indicating that the shorter the sequence of thymines in intron 8, the higher the proportion of CFTR mRNA in which exon 9 is lacking. Also, analysis of IVS8-5T with M470V showed that 5T/M470 was found in 19 CBAVD patients, 5T/V470 was found in 3 and 5T with heterozygote form of M470V was found in 24 CBAVD patients (Table III). In CUAVD patients, 5T was found with heterozygote form of M470V in two cases (Table IV).
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In summary, we report that in 25.94% of cases, the CBAVD phenotype results from the combined action of the 5T allele and a CF mutation on the other chromosome. Moreover, the presence of only one CFTR mutation is identified in 36.79% of patients.
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Discussion |
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In order to study the possible involvement of CFTR dysfunction in CBAVD males from Iran, a country with presumed low frequency of CF, we analysed the CFTR gene in 106 patients for the presence of CFTR mutations (all exons and their flanking regions studied by DGGE or by SSCP and results confirmed by sequencing) and the IVS8-5T variant, a variant frequently found in Iranian CBAVD males. A high frequency of the IVS8-5T variant was found in these patients: 55 of 212 alleles or 25.94% of the alleles (Table V). The frequency of IVS8-5T in the Iranian patients was similar to data published in Portuguese (27.4%) (Grangeia et al., 2005
) and Taiwanese (29.2%) (Wu et al., 2004) but higher than Turkish patients (19.6%) (Dayangac et al., 2004). Therefore, our results support the preliminary conclusion of studying a few CBAVD patients of Lebanese and Vietnamese origin that the IVS8-5T variant is involved in many cases of CBAVD, including countries where CF is rare (Grangeia et al., 2004; Disset et al., 2005).
Most patients with CBAVD in this study (80.19%) had a mutation in at least one of their CFTR genes, but 43.4% had mutations on both chromosomes, with at least one of the two mutations being mild. Inability to identify the second mutation in most patients with CBAVD, even after all 27 CFTR exons were analysed, suggests that mutations could be located elsewhere in the noncoding regions of CFTR. These mutations may result in a CFTR protein with a normal structure but with low levels of expression (Wong et al., 2004; Stuppia et al., 2005), which may cause diseases only in the organs most sensitive to CFTR dysfunction, such as the vas deferens (Kolettis and Sandlow, 2002).
The reduced levels of normal CFTR mRNA due to the deletion of exon 9 depend on the presence of the 5T allele sequence in intron 8. This nonfunctional CFTR mRNA accounts for up to 92% of the total mRNA when both CFTR genes have the 5T allele (Anzai et al., 2003; Grangeia et al., 2004). We have found a significant proportion of men with CBAVD who have the 5T allele, as compared with men in the general population, which suggests that this allele functions as a disease mutation in CBAVD. Similarly, the proportion of Iranian males with CUAVD who have the 5T allele was higher than in the fertile males but lower than among men with CBAVD, whereas the proportion of patients with CUAVD who had the 5T allele was higher than, but not significantly different from, the patients with obstructive azoospermia (Table V). Because CFTR mutations have also been found in patients with CUAVD (Table IV), that condition could be an incomplete form of CBAVD (Claustres et al., 2000; Ravnik-Glavac et al., 2000).
Additional information about the importance of the 5T mutation was obtained by screening 106 patients with CBAVD. We identified 14 adults with the F508del/5T genotype who had CBAVD but no pancreatic disease. Because persons with a CF mutation and the 5T allele may have levels of normal CFTR mRNA below the range of 8–12% (the minimal level for a normal phenotype) but above the range of 1–3% (the level below which severe CF occurs) (Wong et al., 2004; Disset et al., 2005; Wu et al., 2005b), a wide clinical variation is expected in them, depending on the variability of levels of normal CFTR mRNA. These clinical forms should include CBAVD, moderate CF and the absence of fertility problems.
Previous studies suggested that when IVS8-5T is combined with additional activity-reducing variations, such as the higher number of IVS8(TG)m and the V470 variant, it shows higher disease penetrance (Cuppens et al., 1998). Although (TG)m was not analysed in this study, the results of the M470V polymorphism support this notion. Notably, a common polymorphism of M470V appears to affect the intensity of the disease association. Between the two haplotypes having IVS8-5T, the 5T-V470 haplotype showed higher disease association than the 5T-M470 haplotype. A larger number of 5T CFTR genes need to be studied before significant conclusions about the involvement of M470V on phenotypic expression in Iranian CBAVD patients can be drawn.
In summary, we report the following findings: First, that the 5T allele in intron 8 of CFTR has clinical effects related to male infertility. Second, that in 25.94% of cases, the CBAVD phenotype results from the combined action of the 5T allele and a CF mutation on the other chromosome. Moreover, the presence of only one CFTR mutation in 36.79% of patients suggests that other undetected changes in CFTR may be involved in CBAVD. Furthermore, the relatively high proportion of patients with CBAVD who do not have CFTR mutations (19.81%) allows us to propose that another gene or genes could be responsible for CBAVD. Finally, CUAVD could be an incomplete form of CBAVD.
A large number of CF mutations have been discovered during the past 5 years, and it seems that we are now better prepared to understand how mutations combine to cause diseases. The combination of the 5T allele with a CF mutation in the other CFTR gene is the most common cause of CBAVD, but it also has other clinical presentations. Our report on CFTR mutations in patients with CBAVD indicates that CBAVD and CF may be extreme forms of a wide nosologic spectrum of conditions that have a common molecular basis.
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Electronic websites for data in this article are as follows: Online Mendelian Inheritance of Man (OMIM), http://www.ncbi.nlm.nih.gov/omim/ (for CBAVD [MIM #277180], CFTR [MIM *602421] and CYSTIC FIBROSIS [MIM #219700]).
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Acknowledgements |
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We are indebted to the CBAVD patients for their cooperation. This research was supported by grants from the Reproductive Biomedicine Research Center of Royan Institute, Iran.
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Submitted on March 22, 2006; accepted on April 22, 2006.
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