Download Mutations in the cystic fibrosis gene in men with congenital bilateral

Molecular Human Reproduction vol.2 no.9 pp. 669-677, 1996
Mutations in the cystic fibrosis gene in men with congenital
bilateral absence of the vas deferens
Marc De Braekeleer13 and Claude Ferec2
'Cystic Fibrosis Research Laboratory, Departement des Sciences Humaines, Universite du Quebec a Chicoutimi et
Clinique de Fibrose Kystique, Hopital de Chicoutimi, 555 Boulevard de I'Universite, Chicoutimi, Quebec G7H 2B1,
Canada, and 2£tablissement de Transfusion Sanguine de Bretagne Occidentale (ETSBO), Brest, France
•'To whom correspondence should be addressed
This paper reviews the relationship between mutations in the cystic fibrosis (CF) gene (CFTR mutations) and
congenital bilateral absence of the vas deferens (CBAVD). Two CFTR mutations were identified in 14.5% of
the 449 men with CBAVD thus far reported in the literature while one CFTR mutation was found in another
48.1%. CBAVD appears to be a heterogeneous genetic condition, many cases being mild forms of cystic
fibrosis, others having no relationship with CF. The 5T allele has also been found in 46% of men with CBAVD,
but is not associated by the 'classical' picture of cystic fibrosis. The role of the CFTR gene presumably extends
beyond a normal development of the vas deferens, possibly playing a role in spermatogenesis. The detection
of CFTR mutations in CBAVD had considerable implications in genetic counselling. Couples requesting
microsurgical epididymal sperm aspiration/in-vrtro fertilization and those in which the man has CF should be
offered CFTR mutations screening if CBAVD is the cause of the male infertility.
Key words: CBAVD/CFTR/cystic fibrosis/gene mutations/review
Introduction
The causes of male infertility have been extensively studied
over the past decades (Hotchkiss, 1944; Amelar, 1966; Girgis
et al, 1969; Dubin and Amelar, 1971; Lipschultz, 1980;
Chandley, 1984; De Braekeleer and Dao, 1991; Asch and
Silber, 1992; Bhasin et al, 1994; Skakkebaek et al, 1994). In
one of the largest series involving 1294 consecutive cases,
Dubin and Amelar (1971) noted the absence of the vas deferens
in 1.8% of the cases (Dubin and Amelar, 1971). Girgis et al.
(1969) reported that the congenital bilateral absence of vas
deferens (CBAVD) and/or epididymis represented 13.4% of
the 299 men with obstructive azoospermia. In North America,
of the 7.5% of the males with fertility problems, 8% have
azoospermia, 2/3 of them because of an obstructive cause
(Lipschultz, 1980; Jequier et al, 1985).
CBAVD is a distinct clinical entity (Michelson, 1949;
Amelar and Hotchkiss, 1965; Charny and Gillenwater, 1965;
Rubin, 1975; Jequier et al., 1985). The diagnosis is easily
made by scrotal examination. Indeed, the vas deferens is
a cord-like structure which can be easily palpated in the
normal scrotum.
Disturbances in the development of the human embryo are
responsible for the absence of the vas deferens. The testis and
the globus major of the epididymis arise from the genital ridge
while the other structures of the male reproductive excretory
system, including the vas deferens, derive from the
mesonephric duct (Michelson, 1949; Campbell, 1963; Charny
and Gillenwater, 1965; Mickle et al, 1995). The extent of the
abnormalities depends upon the time at which the mesonephric
duct stops developing. If it occurs early in embryonic life
(around the fourth week), the ureter and kidney are also absent.
© European Society for Human Reproduction and Embryology
However, if the disturbances happen later, the kidney and
ureter are present, although their development could be abnormal (Valman and France, 1969; Holsclaw et al, 1971; Sarto
and Simpson, 1978).
Several families in which CBAVD was transmitted as an
autosomal recessive trait have been reported in the literature
(Schellen and van Stratten, 1980; Budde et al, 1984; Czeizel,
1985; Kleczkowska et al, 1989; Gilgenkrantz et al., 1990;
Martin et al, 1992; McKusick, 1992). This led several authors
to postulate that some genetic factor might be responsible for
CBAVD, at least in some families.
CBAVD in cystic fibrosis
Cystic fibrosis (CF) is one of the most common autosomal
recessive disorders in the European populations and those of
European extraction (Boat et al, 1989; Daigneault et al, 1992;
De Braekeleer and Daigneault, 1992). The clinical picture is
dominated by a progressive lung disease consisting of recurrent
episodes of infection, colonization by micro-organisms and a
reduced pulmonary function responsible for most of the deaths.
Pancreatic insufficiency is present in the majority of the
patients. The disease is characterized by an elevated concentration of sweat electrolytes in most of the patients (Boat
etal, 1989).
Infertility, or at least subfertility, in males with cystic fibrosis
was first suspected in the 1960s when it was realized that
several CF men were childless after several years of marriage
(Denning et al, 1968; Stern et al, 1977, 1982; Brugman and
Taussig, 1984; Seale et al, 1985; Kaufman et al, 1986). In
1968, based on 25 CF men > 17 years of age with azoospermia,
669
M.De Braekeleer and C.Firec
Kaplan et al. attributed their infertility to the absence of vas
deferens (Kaplan et al, 1968). Some workers postulated that
the vas deferens could be obliterated, as it is the case for
pancreatic ducts (Di Sant'Agnese, 1968; Oppenheimer and
Esterly, 1969), while, for others, the absence of the vas deferens
in the surgical or pathological specimens resulted from their
lack of development (Gracey et al., 1969; Landing et al.,
1969; Olson and Weaver, 1969; Valman and France, 1969;
Wang et al., 1970; Holsclaw et al., 1971).
Abnormalities in the semen parameters have also been
identified in males with cystic fibrosis. They include azoospermia, reduced volume, increased acidity, and absent or low
concentrations of fructose (Denning et al., 1968; Kaplan et al.,
1968; Rule et al, 1970; Holsclaw et al, 1971).
Not all male CF patients are infertile, a few of them having
fathered children. The first case described in the literature was
reported by Feigelson et al. (1969). A few other cases are
mentioned in the literature, though their semen was not
examined (Bumbalo, 1969; Taussig et al, 1972; Barreto et al,
1991). Data on fertility on 117 male CF patients was collected
from a survey of 78 CF centres. Two males proved to be
fertile by semen studies (normal sperm counts and volume of
ejaculate, one of whom had fathered two children) (Taussig
et al, 1972). It is now believed that 2-3% of CF males are
fertile (Boat et al, 1989). More recently, it was shown that at
least some CF individuals carrying the 3849 + 10 kb C->T
mutation are fertile and have no CBAVD (Passero, 1995; Stem
et al, 1995; Dreyfus et al, 1996).
Genotype-phenotype
fibrosis
correlations
in
cystic
The gene responsible for cystic fibrosis, called cystic fibrosis
transmembrane conductance regulator (CFTR) gene, was identified in 1989 (Kerem et al, 1989; Riordan et al, 1989;
Rommens et al., 1989). The most common mutation responsible
for CF is the deletion of a base triplet coding for a phenylalanine
amnioacid in position 508 (AF508) of the resulting protein
(which contains 1480 aminoacids) (Kerem et al, 1989). It
accounts for ~70% of the CF chromosomes, though population
and regional variations exist (Cystic Fibrosis Genetic Analysis
Consortium, 1990, 1994; De Braekeleer and Daigneault, 1992).
Over the last 6 years, more than 500 mutations and polymorphisms have been reported to the Cystic Fibrosis Genetic Analysis
Consortium.
Analyses of the correlation between phenotype and genotype
showed that the CFTR mutations could be grouped in two
categories, mild or severe, with respect to pancreatic function
(Kristidis et al, 1992). The severe mutations are associated
with pancreatic insufficiency whereas the mild mutations,
leading to a higher residual CFTR activity, confer pancreatic
sufficiency (Kristidis et al, 1992). A CF patient is likely to
be pancreatic sufficient if he has one or two mild mutations.
Numerous studies have shown that some genotypes are
associated with a severe phenotype, while others have a milder
clinical picture and evolution (see, for example, (Kerem
et al, 1990; Shoshani et al, 1992; Cystic Fibrosis GenotypePhenotype Consortium, 1993; Highsmith et al, 1994; De
670
Braekeleer et al, 1995; Stern et al, 1995; Zielenski and Tsui,
1995; Patrizio and Zielenski, 1996). However, high variability,
mostly in the pulmonary function, was observed in each given
genotype or between sibs, suggesting that other factors, genetic
or environmental, may affect the severity and/or the progression
of the disease (Tizzano and Buchwald, 1995).
In the early 1970s, it was suggested that CBAVD was a
mild form of cystic fibrosis (Holsclaw et al, 1971). However,
only a few cases presenting a family history of CF and a
positive sweat test have been documented (Stem et al, 1977,
1982; Kaufman et al, 1986). The development of molecular
techniques to identify CFTR mutations now allows testing of
this hypothesis, which was done in several studies over the
past few years. They are the subject of this review.
CBAVD and CFTR mutations
Table I shows the distribution of the 572 patients with CBAVD
thus far reported according to their genotype. Five individuals
(0.9%) were found to be homozygotes (HH) for one CFTR
mutation and 86 (15%) were compound heterozygotes (hh),
i.e. carriers of two different CFTR mutations. In 279 individuals
(48.8%), only one CFTR mutation was identified (heterozygotes, h). No mutation was found in the remaining 202
individuals (normal, N) (35.3%). Altogether, at least one CFTR
mutation was identified in 64.7% of the 572 individuals with
CBAVD.
This estimate should be considered as a minimum since a
DNA sequencing of the coding sequences and the splice
junctions was not performed in all the studies (Maddalena and
Sherins, 1992; Patrizio et al, 1993; Meschede et al, 1993b;
Williams et al, 1993; Vazquez-Levin et al, 1994). Failure to
identify a second CFTR mutation in these men does not
exclude the possibility of mutations in the promoter region of
the CFTR gene, or at other regulatory sites. Furthermore, only
a few studies have gone to the extreme of sequencing the
whole coding region of the CFTR gene and, even when
all the 27 exons were covered, the denaturing gradient gel
electrophoresis (DGGE) technique, which is not 100% errorproof, was used. Also, the analysis of rnature mRNA could
reveal the presence of a splicing abnormality due to intronic
sequence variation not detected by other methods.
The frequency of individuals with CBAVD heterozygous
for one CFTR mutation (excluding the homozygotes and
compound heterozygotes) is much higher than the expected
carrier rate in the general population (1 in 2 individuals versus
1 in 25 inhabitants). The CF carrier rate is usually calculated
from the Hardy-Weinberg equilibrium equation based on
the incidence of CF patients, therefore carrying two CFTR
mutations severe enough to induce clinical problems leading
to the diagnosis of cystic fibrosis.
Recently concerns have been raised that inferring the carrier
rate from the incidence of CF might introduce biases. Several
groups have found higher differences in allele frequencies
between healthy carriers and CF patients (Kalman et al, 1994).
Indeed, studies on genotype-phenotype have now shown that
some mutations and/or genotypes are associated with mild
phenotypes (Dean et al., 1990; Strong et al, 1991; Varon
CBAVD and cystic fibrosis gene mutations
Table I. Distribution of patients with congenital bilateral absence of the vas deferens (CBAVD) according to their genotype
References
No. of
patients
No. of
homozygotes (HH)
No. of compound
heterozygotes (hh)
No. of
heterozygotes (h)
No. of
normal (N)
1
25*
0
3
16
6
2/3
10
0
3
2
5
4
18
0
1
9
8
5/6
59
0
4
27
28
0
3
3
2
7/8/9
8**
10/11
44
0
8
23
13
12/13/14/29
33***
0
6
15
12
15/16
36"
3
2
13
18
17/18/30
70
1
8
40
21
19/24
67"
1
15
28
23
20/24
28°
0
3
19
6
21
1
0
0
5
2
22
23
35
26d
0
0
5
15
7
15
16
31
35
0
1
18
16
32
26
0
6
14
6
33
45
0
15
25
5
572
1
4
1
1
5
86
1
3
279
202
TOTAL
25
26
27
28
3
1
1
Remarks
Most common mutations in the studied
population
Most common mutations in the studied
population
Most common mutations in the studied
population
Most common mutations in the studied
population
DNA sequencing of the coding regions
splice junctions
DNA sequencing of the coding regions
splice junctions
DNA sequencing of the coding regions
splice junctions
DNA sequencing of the coding regions
splice junctions
Most common mutations in the studied
population
DNA sequencing of the coding regions
splice junctions
DNA sequencing of the coding regions
splice junctions
Most common mutations in the studied
population
Only tested for AF508 and R117H
DNA sequencing of the coding regions
splice junctions
Most common mutations in the studied
population
Most common mutations in the studied
population
DNA sequencing of the coding regions
splice junctions
Case
Case
Case
Case
and the
and the
and the
and the
and the
and the
and the
and the
report
report
report
report
•Including one congenital unilateral absence of the vas deferens (CUAVD).
••Excluding one CUAVD and three with congenital bilateral absence of the epididymis (CBAE).
•"Excluding five congenital absence of the vas deferens (CAVD) with unilateral renal agenesis.
"Excluding 10 CBAVD with renal malformations.
"Excluding CUAVD and CBAVD with renal abnormalities.
c
Excluding two with renal agenesis and 10 with CUAVD.
d
Including one with a horseshoe kidney and two with unilateral kidney.
References to Table I
(1) Jezequel el ai, 1994 (2) Meschede el ai, 1993b (3) Meschede el ai, 1993c (4) Maddalena and Shenns, 1992 (5) Bey-Omar el ai, 1994 (6) Duneu el ai,
1995 (7) Desgeorges el ai, 1994 (8) Culard et ai, 1994b (9) Culard et ai, 1994a (10) Anguiano el al., 1992 (11) Amos et al., 1992 (12) Dumur el ai, 1990
(13) Rigot el al., 1991 (14) Gervais el al., 1993 (15) Augarten et al., 1994 (16) Rave-Harel el al., 1995 (17) Patrizio el al., 1993 (18) Zielenski et al., 1995
(19) Mercier el al., 1995b (20) Casals et al., 1995 (21) Vazquez-Levin et al., 1994 (22) Williams et ai, 1993 (23) Osborne el ai, 1993 (24) Chillon et ai,
1995 (25) Goshen el ai, 1992 (26) Mickle el ai, 1992 (27) Martins et ai, 1993 (28) Meschede el ai, 1993a (29) Dumur et ai, 1996 (30) Jarvi et ai, 1995a
(31) Schlegel et ai, 1995a (32) Le Lannou et ai, 1995 (33) Costes et ai, 1995.
et al., 1995), even with a normal sweat test (Strong et ai,
1991; Highsmithe/a/., 1994; Stern et al., 1995; Stewart et al.,
1995) or with no CF symptoms at all (Lee et al, 1992).
Therefore, some CFTR genotypes may not be associated with
pulmonary, nor gastrointestinal problems; thus the individuals
carrying these genotypes may not be identified as having
cystic fibrosis.
All the homozygote and compound heterozygote individuals
with CBAVD for whom the information was available had no
or just a few CF signs whereas some of them had an elevated
chloride concentration (>60 mmol/1) at the sweat test. These
individuals would not have been identified had they not sought
counselling for infertility.
The CFTR genotype distribution of 90 patients with CBAVD
carrying two mutations (HH and hh) is given in Table II.
Three individuals were homozygous for the D1152H mutation
and two for R117H. Among the compound heterozygotes,
AF50S7R117H was the most frequently found genotype (36
out of 90, 40%). In fact, R117H was found on 47 of the
180 chromosomes (26.1%) and AF508 in 67 of the 180
chromosomes (37.2%).
Table III shows the distribution of the CFTR mutations
671
M.De Braekeleer and C.F6rec
Table II. CFTR genotype distribution of the individuals with congenital
bilateral absence of the vas deferens (CBAVD) carrying two mutations
Table III. Distribution of the CFTR mutations found in individuals with
congenital bilateral absence of the vas deferens (CBAVD)
Genotypes
Mutations
No. chromosomes
AF5O8
R117H
W1282X
R347H
G542X
N1303K
D1152H
R668C
D1270N
G551D
R553X
R75Q
1717-1G->A
621 + 1G->T
F508C
R1070W
R117C
1677delTA
G576A
R75L
S1235R
S549N
1078delT
1525-1G->A
1716+12T->C
185+4>T
2184delA+A-»G
2322delG
244"
54
37
9
8
8
7
6
4"
4
4
4
3
3
3
3
3
2
2
2
2C
2
D1152H/D1152H
R117H/R117H
R117H/2322delG
R117H/712-1G->T
R117H/G551D
R117H/R347P
R117H/R553X
R117H/W1282X
R117H/2347AG
R117H/AF508
AF508/R347H
AF508/G576A
AF508/R117C
AF508/R668C
AF508/R74W + D1270N
AF508/R75L
AF508/A1067 V
AF508/R1070W
AF508/185+4A->T
AF508/D1152H
AF508/D1270N
AF508/F508C
AF508/G628R + S1235R
AF508/L206W
AF508/other
AF508/R258G
AF508/1R75Q
AF508/S5OY
AF508/N1303K
AF508/P574H
AF508/D1154G
AF508/R668C + D443Y
2622+lG->A/D1377H
3272-26 A->G/P99L
E193K/N1303K
G149R/R668C
I556V/Q1352H
Q1411X/R668C + D443Y
R117C/W1282X
R347E1/R1066H
R553X/R668C
S549N/RI070Q
W1282X3849+10kb C-»T
No. patients
36
3
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
I
1
1
found on chromosomes from individuals with CBAVD. No
difference was made, whether the individuals were homozygotes, compound heterozygotes, or heterozygotes. The
AF508 mutation was found on 244 of the 464 CF chromosomes
identified (52.6%) while R117H was found on 54 chromosomes
(11.6%) and W1282X on 37 chromosomes (8%). The other
66 different mutations occurred on <2% of each of the CFTR
chromosomes, most of them having been identified on a sole
chromosome. The majority of these rare mutations have not
been identified in patients with cystic fibrosis. No screening
for most of these mutations has been performed in large
randomly selected populations; their frequencies in the general
population remain unknown.
All the studies on CFTR mutations in CBAVD were conducted in Europe and North America. The frequencies of the
AF508 and R117H mutations on the CFTR chromosomes were
compared to those based on patients with typical cystic fibrosis
and reported by workers from these two continents to the
Cystic Fibrosis Genetic Analysis Consortium (Cystic Fibrosis
672
2347AG
2622+ 1G->A
2789+5G->A
3272-26 A->G
3849+10kb C->T
4173delC
712-1G->T
876-10 del 8
A1067 V
A800G
D1154G
D1377H
D443Y
D614G
E193K
E60X
F1052V
G1069R
G149R
I556V
K1060T
L206W
P574H
P99L
Q1352H
Q1411X
Q493X
R1066H
R1070Q
R1162X
R258G
R334W
R347P
S50Y
V232D
V562I
Y1092X
AE115
"Including one chromosome AF5O8 + A1067T.
•"Including two chromosomes D127ON + R74W.
Including one chromosome S1235R + G628R.
CBAVD and cystic fibrosis gene mutations
Genetic Analysis Consortium, 1994). Of the 38 873 CFTR
chromosomes identified, 25773 (66.3%) carried the AF508
mutation and 126 (0.3%) the R117H mutation. These figures
are very different from the 37.2% and 26.1% observed in
homozygotes and compound heterozygotes with CBAVD. The
genotypes including at least a copy of the AF508 mutation
usually lead to signs suggestive of cystic fibrosis, which allows
the diagnosis to be made (Kerem et al, 1990; Kristidis
et al, 1992).
The high frequency of R117H was unexpected since this
mutation has rarely been described in patients with cystic
fibrosis (Dean et al, 1990; Kristidis et al, 1992). However,
the R117H mutation was found in 11% of the 54 carriers
identified during a pilot project to assess the validity of
CF carrier screening in the USA (Handelin et al., 1992).
Furthermore, the R117H mutation has been described in
clinically distinguishable CF patients (Dean et al., 1990),
males with CBAVD and asymptomatic individuals (Lee et al,
1992). It now appears that the R117H mutation is carried
on two different chromosomal backgrounds consisting of a
variation of the polypyrimidine tract in the splice acceptor site
in intron 8 of the CFTR gene (Chu et al., 1991).
Three length variants, called 5T, 7T, and 9T (polyT variants),
have been identified; they are associated with variable degrees
of exon 9 skipping. The 5T allele causes reduced levels of
normal CFTR mRNA and a non-functional protein (Chu et al.,
1993). Two mutations, A800G and R117H, have thus far been
found on chromosomes bearing the 5T variant. The R117H
mutation is also carried on chromosomes having the 7T variant
(Kiesewetter et al., 1993). A CF phenotype was observed in
genotypes including the R117H mutation carried either on the
5T variant chromosomes or the 7T variant chromosomes
whereas CBAVD was only associated with the R117H on 7T
variant chromosomes. Therefore, the genetic context in which
a mutation occurs can play a significant role in determining
the phenotype (Kiesewetter et al., 1993).
The finding that variations in the polypyrimidine tract in
intron 8 lead to different degrees of exon 9 skipping led several
workers to investigate whether there could be a relation
between the polyT variants, more particularly the 5T variant,
and CBAVD (Chillon et al., 1995; Costes et al, 1995;
Garnerone et al, 1995; Jarvi et al, 1995b; Zielenski et al,
1995).
A total of 275 males with CBAVD have now been studied
for the presence of the 5T variant in intron 8. Of these, 95
individuals (34.6%) had a CFTR mutation on one chromosome
and the 5T variant on the other. Five individuals (1.8%),
including one who had also the A800G mutation,were homozygous for the 5T variant whereas 26 (9.5%) were heterozygous
for the 5T variant. In another study involving 11 patients with
CBAVD, six of the 22 chromosomes (27.3%) carried the 5T
variant (Gamerone et al, 1995). Therefore, 137 (24%) of the
572 chromosomes thus far analysed carried the 5T variant.
The frequency of the 5T allele, which shows some differences between the several populations reported (ranging from
3.1-5.4%), is estimated at 4.4% (37 out of 836) in the general
population (Kiesewetter et al, 1993; Cuppens et al, 1994;
Chillon et al, 1995; Dork et al, 1994; Garnerone et al, 1995;
Zielenski et al, 1995). The proportion of the 5T variant in
individuals with CBAVD is significantly higher than that of
the general population (P <0.0001). Of the 140 CBAVD men
who carried a CFTR mutation on one chromosome, but not
on the other, 95 (67.9%) had the 5T variant on the chromosome
not carrying the CFTR mutation (Chillon et al, 1995; Costes
et al, 1995; Jarvi et al, 1995a; Zielenski et al, 1995; Dumur
et al, 1996).
~
Although the data are still sparse, it is possible that differences in the frequency of the 5T allele may exist between
sexes (Chillon et al, 1995; Zielenski et al, 1995). Six of the
290 (2.1%) non CF chromosomes (considered as normal) in
males having had a child with cystic fibrosis carried the 5T
variant while 4.7% (seven out of 150) of the non CF chromosomes in females did. The meaning of this observation is
not known.
The data published in the literature indicate that the 5T
variant is possibly involved, in some way, in male infertility.
It is evident that the 5T variant alone is not sufficient to induce
a typical cystic fibrosis (Chu et al, 1992); it appears to be a
specific allele for CBAVD. Indeed, it is possible that different
requirements in the concentration of the CFTR protein are
needed to assume a normal physiology in different tissues.
Chillon et al. (1995) suggested that azoospermia not due to
CBAVD was unrelated to CFTR. Their assumption was based
on a population of only 10 azoospermic patients without
CBAVD, the 5T variant having been identified on a sole
chromosome. Men heterozygous for one CFTR mutation may
be at a higher risk of being infertile. Screening for 13 CFTR
mutations most prevalent in the Chicago area was performed
on 127 healthy males attending infertility clinics (van der Ven
et al, 1996). No mutation was identified among 26 males with
normal semen parameters. Out of 80 men, 14 (17.5%) with
infertility due to reduced sperm quality and two out of 21
men (9.5%) with azoospermia had one CFTR mutation. One
azoospermic male was a compound heterozygote G551D/
R117H. These frequencies are higher than the frequency of
4% of CF carriers expected in the same population. However,
CBAVD could not be completely ruled out (exclusion made
by measurements of fructose concentrations and total semen
volume).
A CFTR mutation analysis was conducted among 167 sperm
donors from the Nebraska genetic semen bank to determine
their carrier status (Traystman et al, 1994). Eight different
mutations, including AF508, R117H, W1282X, G551D,
R553X, and G542X, were searched. Of the 167 donor males,
13 (7.8%) carried one CFTR mutation but differences were
found between the several categories of donors. Seven of the
56 infertile men (12.5%), none of them having CAVD, were
heterozygotes; six carried the AF508 mutation and one the
G542X mutation. The carrier frequency in this group is twice
that expected assuming a carrier rate of one in 25 of the
general population. A CFTR mutation was found in six of the
remaining 111 men (5.4%) who were normal healthy semen
donors, prevasectomy patients and prechemotherapy or preradiation cancer patients. Two men carried the AF508 mutation
and four the Rl 17H mutation; the semen analysis was normal
in five individuals, including all with the R117H mutation. It
673
M.De Braekeleer and C.F6rec
appears that the CFTR protein may be involved in the
spermatogenesis and/or sperm maturation. Heterozygosity for
one CFTR mutation may not be sufficient to induce infertility,
but rather be a contributing factor (Mercier et ai, 1995b).
CFTR haplotype analysis also shows that brothers with or
without CBAVD sometimes carry the same two CFTR alleles
(Rave-Harel et ai, 1995). Therefore, CBAVD may sometimes
be unrelated to the CFTR genotype. It may be a simplistic
view to postulate that all those men with CBAVD in whom
only one CFTR mutation or no mutation was identified have
one or two yet unknown and undetected CFTR mutation(s).
All the individuals considered here have congenital bilateral
absence of the vas deferens without renal abnormalities. CFTR
genotyping in 11 individuals with congenital unilateral absence
of the vas deferens (CUAVD) without renal abnormalities has
also been reported (Culard et ai, 1994a; Casals et ai, 1995).
Two CFTR mutations were found on the 22 chromosomes
(9.1%), which is about twice the expected carrier frequency.
Schlegel et al. (1995b) also reported that, among a sample of
72 men with congenital absence of the vas deferens (CAVD),
CFTR mutations were detected in 25% of the males with
CUAVD. Mickle et al. (1995) studied 21 infertile men with
CUAVD, analysing 37 CFTR mutations. None of the five
males with renal anomalies had a CFTR mutation identified.
No mutation was found in the eight individuals who had
CUAVD with one anatomically complete and patent vas
deferens and no renal anomalies. All the eight males with
congenital absence of one vas deferens and occlusion of the
other at the inguinal or pelvic level carried a CFTR mutation
(AF508 in four men, R117H in two, G551D and N1303K in
one each). CFTR mutations have also be searched in 34 men
with CAVD associated with renal abnormalities (renal agenesis,
horseshoe kidney) (Gervais et al., 1993; Augarten et ai, 1994;
Casals et al., 1995; Mickle et al., 1995; Schlegel et al,
1995b). None of the men with renal abnormalities had CFTR
mutations detected.
CFTR gene and development of the vas deferens
Given the fact that CFTR mutations are detected in men with
CBAVD and, with a lesser frequency, in men with CUAVD,
it appears that a normal amount of a functional CFTR protein
is required to ensure proper development of the vas deferens.
However, the lack of CFTR mutations detection in CAVD
with renal abnormalities also suggests that the mesonephric
duct plays an important role in the development of the vas
deferens. Therefore, one can hypothesize that the CFTR protein
is required at some specific embryonic stage of the development
of the vas deferens.
The mechanism(s) by which CFTR mutations contribute to
the pathogenesis of CBAVD is (are) still unknown. Studies on
the cellular localization of CFTR mRNA and expression of
the CFTR gene are underway in humans and other species
(Harris et ai, 1991; Tizzano et ai, 1993, 1994; Trezise et ai,
1993a,b). Trezise et ai (1993b) studied the CFTR expression
in the reproductive organs of rodents. In rats, CFTR mRNA
is present in the round spermatids during the cycle of the
seminiferous epithelium in the testis. CFTR expression occurs
674
throughout the cycle of the seminiferous epithelium in the
mouse testis, indicating that there might be differences in
CFTR expression between species. Indeed, Tizzano et al.
(1994) failed to identify CFTR expression during human
spermatogenesis. Significant expression was detected in the
epithelium of the epididymis and vas deferens in humans
(Tizzano et ai, 1994), but not in vas deferens in rats (Trezise
et ai, 1993b).
The cellular localization of CFTR mRNA and expression
of the CFTR gene have also been studied in human fetal
tissues (Tizzano et ai, 1993; Trezise et ai, 1993a). CFTR
mRNA was detected in fetal epididymis at 18 weeks gestation
and was specifically localized to the duct epithelium (Trezise
et ai, 1993a). Tizzano et ai, (1993) studied human fetuses
from 10-33 weeks gestation; low CFTR expression was present
in the epithelium of the epididymis at all stages. The detection
of CFTR mRNA in human fetuses suggests that CFTR acts
as a chloride channel early in the development of the reproductive tissues.
CFTR mutations, CBAVD and genetic counselling
CBAVD had usually been viewed as an untreatable case of
male sterility until it was shown that spermatozoa that have
not gone through the epididymis could fertilize eggs in vitro
(Silber ef a/., 1990). Men with CBAVD can undergo microsurgical epididymal sperm aspiration (MESA). The technique is
then followed by in-vitro fertilization (IVF) with or without
micromanipulation (intracytoplasmic sperm injection; ICSI)
and embryo transfer (Silber et ai, 1990; Asch and Silber,
1992). The MESA/IVF technique has now been successfully
attempted in a few couples in which the man had cystic fibrosis
with congenital bilateral absence of the vas deferens (Mellinger
et ai, 1992; Hamberger et ai, 1992). In these conditions, the
woman should be screened for CFTR mutations so that proper
genetic counselling can be offered. Detection of a CFTR
mutation in the spouse's patient indicates a high risk situation
whereas failure to detect such a mutation indicates a lower
risk situation depending on the number of mutations screened
and their frequencies in the population. Because the spectrum
of CFTR mutations is markedly different among populations
(Cystic Fibrosis Genetic Analysis Consortium, 1994), the
ethnic background of the patients and their spouses should be
carefully checked to ensure that the most prevalent mutations
in that particular population are included in the screening
panel. CFTR mutations screening should also be offered to
any couple requesting MESA/IVF in which CAVD is the cause
of the male infertility (F6rec et ai, 1996). If a CFTR mutation
is detected in a man with CAVD, genetic counselling could
be offered to his close relatives, since they will be at a higher
risk of having a child with cystic fibrosis.
Conclusions
CBAVD is a heterogeneous genetic condition. In many cases,
it is a mild form of cystic fibrosis. Another proportion of cases
has no relationship to cystic fibrosis and is presumably due to
mutations in other developmental genes. Mutations in one CF
CBAVD and cystic fibrosis gene mutations
allele are detected in many patients with CBAVD. At present,
it cannot be excluded that heterozygosity at the CFTR locus
can interact with other environmental factors or genes to cause
CBAVD, and even infertility not related to CBAVD but due
to a reduced sperm quality. The role of the CFTR gene
presumably extends beyond the normal development of the
vas deferens, possibly playing a role in spermatogenesis. The
detection of CFTR mutations in CBAVD had considerable
implications in genetic counselling. Couples requesting MESA/
IVF should be offered CFTR mutations screening if CBAVD
is the cause of the male infertility.
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Received on April 11, 1996; accepted on July 27, 1996
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