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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. 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