Molecular Human Reproduction Vol.12, No.7 pp.

469–473, 2006
Advance Access publication May 19, 2006 doi:10.1093/molehr/gal046

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

Ramin Radpour1,4, Mohamad Ali Sadighi Gilani2, Hamid Gourabi1, Ahmad Vosough Dizaj2
and Sepideh Mollamohamadi3

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1
Department 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@royaninstitute.org

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 manifes-
tations 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 carri-
ers 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

Introduction In the majority of cases, CBAVD can be considered as a genital

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 mal-
formations, 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 com-
mon feature in males with CF, an autosomal recessive disease prima-
rily 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 pheno-
type (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).
form of CF, presenting without the other clinical features of CF. Gen-
erally, 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. How-
ever, extensive allelic heterogeneity frequently makes difficult the
rate of detection and therefore the value of the genotypic diagnosis.

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R.Radpour et al.

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 confor-
mation 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
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 pub-
lished 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

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and Cassiman, 2004). To evaluate the frequency of the 5T allele of intron 8 in CBAVD and infertil-
ity, we studied the frequency of heterozygosity for the allele in patients with
CBAVD, patients with CUAVD, patients with azoospermia but not CBAVD
Materials and methods and the general population. Amplification and sequencing of the polypyrimi-
Samples dine tract in front of exon 9 was performed using primers 9i-5 and 9i-3
Blood samples were collected from 106 unrelated Iranian males with azoosper- (Chillon et al., 1995). Nested PCR was performed to amplify the polypyrimi-
mia and CBAVD visiting the Royan Institute, Iran. The diagnoses of CBAVD dine sequence with modified primers RF9 (5′-CCGCTGTGTGTGTGTGTGT-
were initially suggested by the clinical observation of impalpable vasa in the GTTTTT-3′) and RR9 (5′-GGATCCAGCAACCGCCAA-3′). The nested PCR
patients and were subsequently confirmed by transrectal and transabdominal conditions were as follows: denaturation at 95°C for 30 s, annealing at 54°C
ultrasonography (Table I). Each patient had a sperm count of zero. We studied for 30 s and extension at 74°C for 40 s, for 35 cycles. The reaction mixture
43 fertile males from the general population in Iran as control subjects. We contained 5 μl of PCR buffer, 200 μmol/l each of dNTPs, 20 pmol of each
also studied 7 patients with congenital unilateral absence of the vas deferens primer and 1 unit of Taq DNA polymerase in a final volume of 50 μl, contain-
(CUAVD) and 10 patients with obstructive azoospermia not due to CBAVD or ing 1 μl of the first PCR product. Nested PCR products finally were digested
CUAVD (obstructive azoospermia was due to vasectomy and epididymal with XmnI enzyme and visualized on 12% nondenaturing polyacrylamide gel
obstruction affected by infections, i.e. chlamydia or gonorrhoea). Cytobio- after electrophoresis for 4–5 h at 230 V (Figure 1).
chemical 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).
Results
CFTR mutations in studied groups
CFTR mutations Patients and normal subjects were first screened using a standard panel
Genomic DNA was isolated from peripheral blood lymphocytes according to of 29 different CF mutations (CF29 Tepnel kit), and if there were not
standard procedures (Mennicke et al., 2005). All samples (patients and con- two known mutations, we performed genome scan by SSCP or DGGE,
trols) were analysed for the 29 common mutations by Elucigene™ CF29 multi- followed by sequencing. The analysis of the entire coding sequence
plex ARMS kit (Tepnel Diagnostics Ltd, UK) as a rapid screening according to allowed us to identify 16 different mutations in CBAVD patients, 13
the manufacturer’s instructions. Missense polymorphism M470V in exon 10
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
Table I. Transabdominal and transrectal ultrasonography results of patients detected specifically in patients with CBAVD. Of these, 46 cases were
and fertile controls
homozygous or compound heterozygous (+/+), 39 were positive for
Study groups n Ultrasonography results only one mutation (+/–) and 21 cases were negative for both mutations
(–/–). In CUAVD patients, we identified two different CFTR mutations.
CBAVD 106 Absence of the vas deferens and seminal In the control group, no CFTR mutations were identified (Table IV).
vesicles
CUAVD 7 Absence of one of the vas deferens and
seminal vesicles
Obstructive azoospermia 10 Epididymal obstruction (8 cases) and renal Frequencies of the IVS8-5T variant and M470V missense
cysts (2 cases) mutation
Fertile males 43 Normal
Study of the polypyrimidine tract in front of exon 9 revealed a high
CBAVD, congenital bilateral absence of the vas deferens; CUAVD, congenital frequency of the 5T allele in the CBAVD males (Table V). Eleven
unilateral absence of the vas deferens. males were homozygous for 5T, whereas 35 males were heterozygotes

Table II. Semen analysis of patients and fertile controls

Study groups Age (years) Cytobiochemical parameters of semen [mean ± SD (range)]

pH* Ejaculate volume (ml) Total sperm count (/ml) Duration of abstinence (days)

CBAVD 22–51 6.5 ± 0.9 (6–6.8) 0.5 ± 1.8 (0.1–1.1) <3 × 10 6

7 ± 1.8 (6–10)
CUAVD 27–60 6.5 ± 1.3 (6.2–7) 1.3 ± 1.0 (0.8–2) 3–19 × 106 6 ± 0.8 (4–9)
Obstructive azoospermia 24–55 6.6 ± 1.5 (6.5–6.8) 0.7 ± 1.6 (0.5–1.2) 0 × 106 9 ± 1.2 (8–11)
Fertile males 23–56 7.6 ± 0.8 (7.5–7.8) 3.7 ± 1.4 (2–4.5) >40 × 106 7 ± 1.6 (4–9)

CBAVD, congenital bilateral absence of the vas deferens; CUAVD, congenital unilateral absence of the vas deferens.
*Semen pH range of 7.5–8.1 was considered as normal.
470
 Molecular analysis of the IVS8-5T variant and M470V missense polymorphism

Table IV. CFTR gene mutations in CUAVD patients and other control groups

Different groups n CFTR M470V IVS8-5T

mutations
M470 M/V V470

CUAVD 1 R334W/? 1 – – 0/2 (allele)

2 R117H/? – 2 – 1/4 (allele)
4 No mutation 1 2 1 2/8 (allele)
Obstructive azoospermia 10 No mutation 6 4 – 4/20 (allele)
Fertile males 43 No mutation 23 19 1 0/86 (allele)
Figure 1. Poly T alleles in the intron 8 of the cystic fibrosis transmembrane CFTR, cystic fibrosis transmembrane conductance regulator; CUAVD, congenital
conductance regulator (CFTR) gene. unilateral absence of the vas deferens.

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Table III. Cystic fibrosis transmembrane conductance regulator (CFTR) gene
mutations in patients with congenital bilateral absence of the vas deferens CBAVD who had this allele was significantly higher than the general
population (c2 = 11.4, P < 0.05), whereas the proportion of patients
Mutation geno types IVS8-PolyT M470V n (%) with CUAVD who had the 5T allele (21.43%) was higher than, but not
significantly different from, the patients with obstructive azoosper-
Two mutations detected
F508del/R117H 9T/9T M/M 1 (0.94) mia. In most patients with CBAVD, the 5T allele was strongly associ-
F508del/621+1G>T 7T/7T V/V 1 (0.94) ated with the presence of a CF mutation in the other copy of the CFTR
1540A/G/1540A/G 7T/7T M/M 2 (1.89) gene. The association between the various CFTR mutations and the 5T
R347H/R117H 9T/7T M/V 1 (0.94) allele in the patients with CBAVD was analysed by studying the trans-
G551D/IVS8-5T 7T/5T M/V 2 (1.89)
F508del/IVS8-5T 7T/5T M/V 8 (7.55) mission of the mutations within families. Seven CFTR mutations were
9T/5T M/M 6 (5.67) associated with the 5T allele in our population (Table III), whereas all
1717–1G>A/IVS8-5T 7T/5T M/V 4 (3.77) the others were associated with the 7T or the 9T allele, confirming that
R117H/IVS8-5T 7T/5T M/V 2 (1.89) in each patient with CBAVD, the 5T allele corresponded to the chro-
621+1G>T/IVS8-5T 7T/5T M/V 3 (2.83)
mosome that did not carry the CFTR mutation. Levels of normal
9T/5T M/M 2 (1.89)
1540A/G/IVS8-5T 7T/5T M/V 2 (1.89) CFTR mRNA depend on the genotype determining the length of the
R553X/IVS8-5T 7T/5T M/V 1 (0.94) thymine sequence in intron 8 of CFTR. When both CFTR genes bear
IVS8-5T/IVS8-5T 5T/5T V/V 3 (2.83) the 5T allele (the 5T/5T genotype), the proportion of normal CFTR
5T/5T M/M 8 (7.55) mRNA is reduced to ∼8 to 12%, indicating that the shorter the
One mutation detected
G85E/– 7T/7T V/V 2 (1.89) sequence of thymines in intron 8, the higher the proportion of CFTR
G551D/– 9T/7T V/V 1 (0.94) mRNA in which exon 9 is lacking. Also, analysis of IVS8-5T with
621+1G>T/– 7T/7T M/M 2 (1.89) M470V showed that 5T/M470 was found in 19 CBAVD patients, 5T/
9T/7T M/V 1 (0.94) V470 was found in 3 and 5T with heterozygote form of M470V was
R334W/– 7T/7T M/V 1 (0.94)
found in 24 CBAVD patients (Table III). In CUAVD patients, 5T was
F508del/– 7T/7T M/V 7 (6.60)
9T/7T M/M 3 (2.83) found with heterozygote form of M470V in two cases (Table IV).
9T/9T M/V 2 (1.89) In summary, we report that in 25.94% of cases, the CBAVD pheno-
IVS8-5T/– 5T/7T M/M 3 (2.83) type results from the combined action of the 5T allele and a CF muta-
5T/9T M/V 2 (1.89) tion on the other chromosome. Moreover, the presence of only one
1717–1G>A/– 7T/7T M/V 3 (2.83)
9T/7T M/V 2 (1.89) CFTR mutation is identified in 36.79% of patients.
R117H/– 7T/7T M/M 2 (1.89)
9T/7T M/V 1 (0.94) Discussion
2789+5G>A/– 7T/7T M/M 1 (0.94)
3120+1G>A/– 9T/7T M/V 2 (1.89) In order to study the possible involvement of CFTR dysfunction in
R560T/– 9T/7T M/V 1 (0.94) CBAVD males from Iran, a country with presumed low frequency of
N1303K/– 9T/7T V/V 1 (0.94) CF, we analysed the CFTR gene in 106 patients for the presence of
1651A/G/– 7T/7T M/V 1 (0.94)
R553X/– 9T/7T M/V 1 (0.94) CFTR mutations (all exons and their flanking regions studied by DGGE
No mutation detected or by SSCP and results confirmed by sequencing) and the IVS8-5T var-
–/– 7T/7T M/M 12 (11.32) iant, a variant frequently found in Iranian CBAVD males. A high fre-
–/– 9T/9T M/M 3 (2.83) quency of the IVS8-5T variant was found in these patients: 55 of 212
–/– 9T/7T M/V 6 (5.66)
alleles or 25.94% of the alleles (Table V). The frequency of IVS8-5T in
Mutation genotypes were designated following the recommended the Iranian patients was similar to data published in Portuguese (27.4%)
nomenclature (Beaudet et al., 1993). The M470V alleles were designated M (Grangeia et al., 2005) and Taiwanese (29.2%) (Wu et al., 2004) but
for nucleotide A at position 1540 and V for nucleotide G at that position. higher than Turkish patients (19.6%) (Dayangac et al., 2004). There-
Genotype frequencies are given as numbers and, in brackets, as percentages of fore, our results support the preliminary conclusion of studying a few
total cohort (106 patients).
CBAVD patients of Lebanese and Vietnamese origin that the IVS8-5T
variant is involved in many cases of CBAVD, including countries
with a 7T or a 9T on the other allele. In contrast, none of the normal where CF is rare (Grangeia et al., 2004; Disset et al., 2005).
males were found to carry a 5T allele. The 5T allele distribution was Most patients with CBAVD in this study (80.19%) had a mutation
25.94%, 54.25% for 7T and 19.81% for 9T in the CBAVD males. We in at least one of their CFTR genes, but 43.4% had mutations on both
evaluated the frequency of the 5T allele in men with various types of chromosomes, with at least one of the two mutations being mild. Ina-
infertility. In our studied population, the percentage of patients with bility to identify the second mutation in most patients with CBAVD,

471
R.Radpour et al.
Table V. Frequencies of IVS8 poly (T) variants in the Persian population
n Allele frequency (%)
5T
CBAVD 106 55/212 (25.94)
CUAVD 7 3/14 (21.43)
Obstructive azoospermia 10 4/20 (20)
Fertile males 43 0/86 (0)
CBAVD, congenital bilateral absence of the vas deferens; CUAVD, congenital unilateral absence of the vas deferens. IVS8 Poly (T) frequencies are given as numbers
and, in brackets, as percentages of total cohort (212 alleles—106 patients).
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 gen-
eral 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 pan-
creatic 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 hav-
ing IVS8-5T, the 5T-V470 haplotype showed higher disease associa-
tion than the 5T-M470 haplotype. A larger number of 5T CFTR genes
need to be studied before significant conclusions about the involve-
ment 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 infertil-
ity. 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 muta-
tion in 36.79% of patients suggests that other undetected changes in
472
Genotype frequency (%)
7T 9T 5T/5T
115/212 (54.25) 42/212 (19.81) 11/106 (10.38)
6/14 (42.86) 5/14 (35.71) 0/7 (0)
11/20 (55) 5/20 (25) 1/10 (10)
48/86 (55.81) 38/86 (44.19) 0/43 (0)
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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 under-
stand 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.
Acknowledgements
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.
Electronic databases
Electronic websites for data in this article are as follows: Online Men-
delian 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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Submitted on March 22, 2006; accepted on April 22, 2006
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