1 Laboratoire de Génétique Moléculaire, Institut de Biologie, CHU, CNRS IGH UPR 1142, 2 Hôpital Arnaud de Villeneuve, Laboratoire de Biologie de la Reproduction, 34060 Montpellier Cedex, France
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Abstract |
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Key words: cystic fibrosis mutations/CFTR mutant polyvariants/oligoasthenoteratozoospermia
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Introduction |
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The analysis of CFTR gene mutations has been extended to other forms of sterility such as bilateral ejaculatory duct obstruction (BEDO), isolated anomalies of the seminal vesicles (IASV), congenital unilateral absence of the vas deferens (CUAVD) (Meschede et al., 1997) or Young syndrome (Le Lannou et al., 1995
). Most men with BEDO and a proportion of men with CUAVD without renal anomalies are carriers of CFTR mutations; however, IASV and Young syndrome do not seem to be associated with alterations in the CFTR gene (Meschede et al., 1998
).
Moreover, studies suggested that CFTR protein, in addition to its role in the development of Wolffian duct-derived structures, could also be involved in sperm maturation (Trezise et al., 1993). First, CFTR is expressed in the post-natal human epididymis, and the CFTR protein is localized in the luminal border of the human cauda epididymal epithelium (for review, see Wong, 1998). Second, it has been shown that the CFTR protein contributes to the secretion of electrolytes and water by the epididymal epithelium, a process which is important in the formation of an optimal fluid microenvironment for sperm maturation and transport (Wong, 1998
). A recent paper reported that CF mutations are present at higher than expected frequency (up to 17.5%) in healthy non-CBAVD men with reduced sperm quality (van der Ven et al., 1996
). However, these findings were not confirmed by a recent study (Tuerlings et al., 1998
). As only the most common CF mutations had been searched in both investigations, we undertook the complete analysis of coding and flanking sequences in patients with severe oligoasthenoteratozoospermia (OAT) in order to obtain further insight into a putative involvement of CFTR dysfunction in male sterility. As it has been shown recently (Cuppens et al., 1998a
) that some of the more common polymorphisms in the CFTR gene affect expression and function of the CFTR protein, we also analysed the distribution of three intragenic markers [TGn and Tn in intron 8, and 1540A/G (M470V) in exon 10] in men with OAT and in controls. The main objective of this study was to compare the complete CFTR genotypes of 56 men with OAT and 50 controls from the same population background, in order to evaluate a putative involvement of CFTR severe or mild mutants and/or variants in infertility due to altered spermatogenesis.
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Materials and methods |
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CFTR gene analysis
All 27 exons and flanking regions of the CFTR gene were screened for mutations and polymorphisms by using denaturing gel gradient electrophoresis (DGGE) assay on genomic DNA amplified by polymerase chain reaction (PCR), a procedure performed in our laboratory since 1992 (Claustres et al., 1993; Culard et al., 1994
). Each PCR product with an abnormal DGGE pattern was directly sequenced in order to identify the mutation or the sequence variation that caused abnormal migration. In addition, we searched for mutations 1811+1.6kbA/G in intron 11 and 3849+10kb in intron 19 by restriction analysis after amplification with appropriate primers, as they have significant frequencies in our population (Des Georges et al., 1998
). The IVS8(T)n acceptor site polymorphism was analysed using an improved procedure derived from a previously described method (Chillon et al., 1995
). The alleles at intron 8 (TG)n preceding the (T)n were determined using an appropriate DGGE procedure and/or by direct DNA sequencing.
Statistical analysis
2 test was used to compare differences between proportions. The statistical analyses were performed with SAS statistical software. P-values < 0.05 were considered to indicate statistical significance.
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Results |
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In the control group of 50 individuals from the general population, we detected the deletion F508 twice, and two possibly but as yet unconfirmed disease-causing missense mutations. G622D has been reported previously (Zielenski et al., 1996
) in a patient with oligozoospermia, and V562L was identified in a CF patient (Hughes et al., 1995
).
Other CFTR sequence variations
A total of 18 different sequence changes have been identified in this study, including 11 missense mutations in the coding sequence, six intronic variations and one nucleotide change in the 5' untranslated region (5'UTR) of CFTR (Table II). We detected two double mutant alleles, 1859G/C associated in cis (on the same gene) with 2134C/T, and 3041-71G/C associated in cis with 4002A/G. We did not find significant differences in the frequencies of variants between controls, OAT, and a group of 50 CBAVD patients whose genotypes had been previously analysed in our laboratory. Six variants present on OAT alleles, 1655T/G (F508C), 1716G/A (E528E), 2377C/T (L749L), 405+46G/T, 3499+37G/A and 4374+13A/G were not found in the control group. Variation 3419T/G in exon 17b is a novel sequence change identified in only one subject from the general population, that changes a non-polar (leucine) for a positively charged (arginine) amino acid residue (L1096R) in a highly conserved cytoplasmic loop of the CFTR protein.
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Discussion |
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Several missense variations identified in this study (R31C, R75Q, F508C, G576A, R668C, or E528E) have previously been described as potentially disease-causing mutations in attenuated CF-related phenotypes such as bronchiectasis (Bombieri et al., 1998), non-CBAVD obstructive azoospermia (Kanavakis et al., 1998
) or CBAVD (Meschede et al., 1993
; Chillon et al., 1995
; Dork et al., 1997
). However, we considered them as neutral variants on the basis of several criteria. First, they are still classified as polymorphisms in the CFGAC. Second, some variants can been found on chromosomes carrying `true' CFTR mutations (for instance, R668C on CBAVD alleles carrying D443Y). Third, some variants can be found in the normal allele of healthy parents of CF children (for instance F508C associated in trans with
F508 (Desgeorges et al., 1994
). Fourth, even when they have been demonstrated to reduce CFTR activity in vitro, their involvement in disease remains controversial. For instance, quantitative and qualitative studies in nasal epithelial cells revealed that E528E, which involves the last nucleotide of exon 10 (1716G/A), results in exon 10 skipping (Cuppens et al., 1998b
). The resulting transcripts without exon 10 fail to mature to fully glycosylated proteins and therefore do not contribute to apical chloride transport activity. However, a role in disease is unlikely as the rate of exon skipping induced by E528E is only 10% (Cuppens et al., 1998b
).
Analysis of three polymorphic loci with frequent alleles in the general population showed: (i) an increase in the proportion of both the TG11-T7 haplotype and the 1540G allele; and (ii) a 1.7-fold increase of homozygotes for haplotype TG11-T7-1540G in men with OAT compared with the controls. Our data confirm those reported for Canadian populations (Zielenski et al., 1998), in which a 2.8-fold increase of homozygotes for TG11-T7-1540G was found compared with the controls, in a cohort of infertile men with oligozoospermia. These observations are interesting in consideration of recent findings on the involvement of specific alleles at the three loci in the modulation of CFTR expression. It was also demonstrated (Cuppens et al., 1998a
) that, in addition to the known effects of the (T)n locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci, (TG)n and 1540A/G. Longer (TG)n and/or shorter (T)n repeats are less favourable for the efficiency of exon 9 splicing, leading to higher proportions of CFTR transcripts lacking exon 9 and non-functional resulting proteins. This explains why, on a TG12 background, a CFTR gene carrying the 5T allele will be fully penetrant as a CBAVD mutation, whereas shorter (TG)n backgrounds will not be deleterious (Cuppens et al., 1998a
). The 1540A/G locus in exon 10 is the most polymorphic two-allele locus of the CFTR gene and, in contrast with intron 8 (TG)n and (T)n, is polymorphic at the amino acid level, a G allele encoding valine instead of methionine at codon 470 (M470V). By in-vitro studies, it was shown that the V470 allele yielded a lower functional CFTR protein rate than the M470 allele, independently of the intron 8 genotypes (Cuppens et al., 1998a
). This finding may explain our previous observation of strong linkage disequilibrium between the 5T and the V470 alleles in the CBAVD population, but not in the normal population (de Meeus et al., 1998
). Thus, although a particular allele may not by itself have deleterious consequences, the combination of specific alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. It was postulated (Cuppens et al., 1998a
) that such `polyvariant mutant' genes might be involved in the partial penetrance of CFTR gene mutations (such as the 5T allele), might be responsible for variations in the phenotype of CFTR mutations, and could explain why apparently normal CFTR genes cause disease.
CFTR expression in testis has been determined to occur in round spermatids in rodents, and to begin in the human testis at puberty, suggesting that the CFTR gene could be involved in later steps of the spermatogenesis process (Trezise et al., 1993). Recently, a significant reduction in mature spermatids per tubule was observed in CBAVD patients carrying the 5T allele, but not in CBAVD patients carrying one or two CFTR mutations in the absence of the 5T allele (Larriba et al., 1998
). This observation suggested that CFTR dysfunctions due to mutations may not be commonly implicated in the efficiency of spermatogenesis in CBAVD patients, whereas the severe reduction in normal CFTR mRNA transcripts in the testis resulting from a gene carrying the 5T allele could alter spermatogenesis in CBAVD.
Our data show that neither the 5T allele nor other CFTR mutations are likely to play an important role in the cause of OAT. The factors associated with reduced sperm quality, including other genetic or chromosomal defects (for review, see Meschede and Horst, 1997) remain to be elucidated. Is homozygosity for haplotype TG11-T7-V470 a sufficient condition to alter CFTR function in spermatogenesis or to contribute to OAT in combination with mutations in other genes? In nasal epithelial cells, it has been shown that, on a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, compared with the (TG)10 allele, and a V470 protein has a 1.7-fold less efficient chloride channel activity than M470 (Cuppens et al., 1998a). The rates of CFTR functional transcripts and proteins derived from haplotype TG11-T7-V470 in sperm cells are unknown.
In conclusion, the results of an extensive analysis of CFTR coding/flanking sequences do not support the involvement of CF or CBAVD mutations in a sample of 56 infertile men with non-CBAVD-reduced sperm quality. If confirmed by further analysis in other populations, these findings will have consequences with regard to the genetic counselling provided to infertile patients treated by in-vitro fertilization. Analysis of haplotype TG11-T7-V470 in a larger cohort will be necessary to substantiate the hypothesis of a putative link between a particular combination of CFTR polymorphisms and male infertility.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 9, 1999; accepted on September 7, 1999.