Hum. Reprod. Advance Access originally published online on February 28, 2007
Human Reproduction 2007 22(5):1285-1291; doi:10.1093/humrep/dem024
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Detection of cystic fibrosis transmembrane conductance regulator (CFTR) gene rearrangements enriches the mutation spectrum in congenital bilateral absence of the vas deferens and impacts on genetic counselling
1 Service de Biochimie et Génétique Moléculaire, AP-HP et INSERM U841, IMRB, eq 21, Hôpital Henri-Mondor, Créteil, France 2 Service de Biologie de la Reproduction et du développement 3 Service d'Urologie, AP-HP, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France
4 To whom correspondence should be addressed at: Service de Biochimie et Génétique Moléculaire, AP-HP et INSERM U654, Hôpital Henri-Mondor, 94010 Créteil, France. Tel.: +33 1 49 81 28 57; Fax: +33 1 49 81 28 42; E-mail: emmanuelle.girodon{at}creteil.inserm.fr
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Abstract |
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BACKGROUND: Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene have been widely detected in infertile men with congenital bilateral absence of the vas deferens (CBAVD). Despite extensive analysis of the CFTR gene using varied screening methods, a number of cases remain unsolved and could be attributable to the presence of large gene rearrangements, as recently shown for CF patients.
METHODS: We carried out a complete CFTR gene study in a group of 222 CBAVD patients with strict diagnosis criteria and without renal anomaly, and searched for rearrangements using a semi-quantitative assay in a subgroup of 61 patients.
RESULTS: The overall mutation detection rate was 87.8%, and 82% of patients carried two mutations. Ten out of the 99 different mutations accounted for 74.6% of identified alleles. Four large rearrangements were found in patients who already carried a mild mutation: two known partial deletions (exons 17a to 18 and 22 to 23), a complete deletion and a new partial duplication (exons 11 to 13). The rearrangements accounted for 7% of the previously unknown alleles and 1% of all identified alleles.
CONCLUSIONS: Screening for rearrangements should be part of comprehensive CFTR gene studies in CBAVD patients and may have impacts on genetic counselling for the patients and their families.
Key words: congenital bilateral absence of the vas deferens/cystic fibrosis transmembrane conductance regulator mutations/deletion/duplication/gene rearrangement
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Introduction |
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Cystic fibrosis (CF) (MIM#219700) is one of the most common autosomal recessive diseases in Caucasians (Welsh et al., 2001
). It affects about one neonate in 2500 and approximately one individual in 25 is a heterozygote, with marked regional variations (Estivill et al., 1997; Bobadilla et al., 2002) (www.genet.sickkids.on.ca/cftr). The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR or ABCC7) gene (MIM*602421). Up to 1500 CFTR sequence variations have been described so far, and these are also involved in a broad spectrum of phenotypes, including male infertility by congenital bilateral absence of the vas deferens (CBAVD) (Dumur et al., 1990; Chillon et al., 1995a; Costes et al., 1995; Bienvenu et al., 1997; Dörk et al., 1997), disseminated bronchiectasis (Pignatti et al., 1995; Girodon et al., 1997) and chronic pancreatitis (Cohn et al., 1998; Sharer et al., 1998). Since the first identification of CFTR mutations in CBAVD in the 1990s (Dumur et al., 1990; Anguiano et al., 1992), extensive studies have shown that 45–80% of CBAVD patients carry two CFTR mutations, mostly in compound heterozygosity, these mutations accounting for 60–86% of disease alleles (Chillon et al., 1995a; Costes et al., 1995; Dörk et al., 1997; Casals et al., 2000; Claustres et al., 2000; Grangeia et al., 2004). All mutation types can be encountered, with effects ranging from mild to severe. The most frequent are the main CF-associated defect, F508del (21–40% of alleles in CBAVD patients, depending on studies), and two mild defects, the T(5) variant of the polypyrimidin tract of the intron 8 (IVS8) acceptor splice site (19–37%) and R117H (3–14%). Unlike genotypes found in CF patients, the majority of those identified in CBAVD patients combine either a severe CF mutation and a mild mutation or two mutations with a mild or variable effect (Claustres et al., 2000; Stuhrmann and Dörk, 2000). Nonetheless, in 20–55% of patients, only one or no CFTR mutation could be found. Unidentified CFTR mutations may lie in introns or in regulatory regions, which are not routinely investigated, or may correspond to gene rearrangements such as large deletions at the heterozygous state which escape detection using current PCR-based techniques. Recent sudies on CF patients have shown that 14–23% of the alleles that remain unknown after extensive analysis of the CFTR gene using scanning techniques consist of large deletions, insertions and duplications (Audrézet et al., 2004; Niel et al., 2004; Bombieri et al., 2005; Ferec et al., 2006; Hantash et al., 2006b). Only one study has reported on screening for rearrangements in CBAVD patients: of 48 patients with one or no identified CFTR mutation, one, heterozygous for the IVS8(T)5 variant, was found to carry a deletion that removed exons 22 to 24 (Hantash et al., 2006a).
Investigation for the presence of such large rearrangements in 61 CBAVD patients using a semi-quantitative fluorescent PCR assay developed in our laboratory (Niel et al., 2004) led us to identify four rearrangements. Here we discuss these findings together with the results of comprehensive CFTR gene studies in a group of 222 CBAVD patients.
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Materials and methods |
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Patients
A group of 222 unrelated infertile patients with isolated CBAVD and from diverse ethnic and geographic origins were investigated. They were recruited between 1992 and 2006 from different French clinics. Preliminary data have already been reported for a number of them (Costes et al., 1995
; Claustres et al., 2000). Diagnosis of CBAVD was assessed by the clinicians on the following criteria: (i) absence of palpable vas deferens at physical examination; (ii) azoospermia with low seminal fluid volume (<2.0 ml); (iii) typical biochemical changes: pH < 7.2, absence or decreased level of fructose and alpha1–4 glucosidase (markers of seminal vesicles and epididymal secretions, respectively) and (iv) anomalies at transrectal ultrasound: absence of the vas deferens and of the distal part of the epididymis, globus major and different degrees of hypoplasia of the seminal vesicles. These anomalies were also confirmed at surgical sperm retrieval whenever patients and their partners requested assisted reproduction. Patients with renal ultrasound anomalies were not included, as a number of studies failed to show a significantly increased frequency of CFTR mutations in patients with CAVD and renal ultrasound anomalies, which may thus represent a distinct clinical entity (Stuhrmann and Dörk, 2000). Plasma FSH, LH and testosterone levels were normal in all subjects. No other CF symptom was evidenced in the patients from questioning or clinical examination at referral. However, follow-up data were not always available and occurrence of late CF symptoms cannot be ruled out. Sweat chloride tests were not performed systematically. According to French legislation and the recommendations of the local ethics committee, written consent to the genetic study were obtained from all patients.
CFTR gene analysis
Genomic DNAs were extracted from whole blood samples collected on EDTA using varied protocols, mostly a phenol chloroform reference protocol or a commercial kit (Nucleon, BACC3, Amersham Biosciences, Saclay, France). DNA concentration and quality were determined for each sample.
Screening for point mutations
CFTR gene analyses included: (i) screening for frequent mutations by means of diverse commercial assays; (ii) scanning of the 27 exons and their boundaries using denaturing gradient gel electrophoresis (DGGE) (Fanen et al., 1992; Costes et al., 1993) or denaturing high-performance liquid phase chromatography (Le Marechal et al., 2001), followed by sequencing to characterize the variants and (iii) screening for the intronic splicing 1811 + 1.6 kbA > G mutation (Chillon et al., 1995b). The (TG)m(T)n haplotype of the IVS8 acceptor splice site was analysed by combination of DGGE and DNA sequencing (Costes et al., 1995).
Mutation names were those reported to the international consortium mutation database (www.genet.sickkids.on.ca/cftr). For nucleotide changes, the A of the ATG translation start codon was numbered as + 133, in accordance with the current CFTR gene numbering based on cDNA sequence (GenBank NM_000492 [GenBank] .2).
Screening for large CFTR rearrangements
We used a semi-quantitative fluorescent multiplex PCR (QFM-PCR) assay recently developed in our laboratory, which enabled detection of rearrangements in 20% of the previously unidentified alleles in CF patients (Niel et al., 2004). Briefly, the assay is based on comparison of fluorescent patterns of multiplex PCR fragments obtained from patients and control samples, the amplification being stopped in the exponential phase. This procedure allows the detection of heterozygous deletions (2-fold reduction in fluorescence intensity) and heterozygous duplications (1.5-fold increase).
All abnormal patterns were confirmed by at least one other QFM-PCR experiment, preferably using different sets of primers. When a pattern was compatible with an already described deletion, identification was performed using specific primers [available upon request and adapted from Lerer et al. (1999) and Audrézet et al. (2004)].
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Results |
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First screening for point mutations in the group of 222 CBAVD patients led to the identification of 95 different mutations, scattered over the whole gene, of all types and with effects ranging from mild to severe. Apart from the IVS8(T)5 variant, sequence variations which are reported as neutral because of segregation analysis in CF families or their frequency in the general population (Bombieri et al., 2000
) (such as 356G > A (R75Q), 1540A > G (M470V) or 1716G > A) were not considered as disease-causing when found in isolation. CFTR mutations were detected in 387 out of 444 alleles (87.2%), most of them being previously described in patients with CF of varying severity, CBAVD or other CFTR diseases: 45% of identified alleles consisted of severe CF mutations (e.g. F508del, W1282X, 2183AA > G); 13.8% of mild or variable CF mutations (e.g. L206W, 3272-26A > G, R117H, D1152H); 36.7% of mild CFTR defects which are currently not considered CF-causing (e.g. IVS8(T)5, Q1352H, the complex alleles [D443Y;G576A;R668C] and [R74W;D1270N]) and 4.5% of rare missense mutations whose effect is difficult to predict (e.g. A959V, G1069R, V1153E). Twenty-nine mutations were found more than once and 10 were found at a frequency above 1% among the identified mutations (Table I).
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Among the 112 IVS8(T)5 identified alleles, all in the heterozygous state, 16 (14.3%) were (TG)13(T)5, 82 (73.2%) were (TG)12(T)5 and 14 (12.5%) were (TG)11(T)5. In five out of the 14 patients carrying the (TG)11(T)5 allele, V562I was also identified, probably in cis as documented in two of these cases and in other patients who do not have CBAVD (Girodon et al., unpublished data).
With a view to segregation analysis, family studies were performed, especially when rare missense mutations were detected. In several instances, however, patients were reluctant to disclose their medical status to their relatives and opposed our request to perform genetic analysis in their family. Compound heterozygous genotypes were suggested, based upon literature data and linkage of F508del with the neutral IVS8(TG)10(T)9 variant.
According to the results of the first screening, the patients fell into three groups: 179 (80.6%) had two CFTR defects, including the IVS8(T)5 variant, 29 (13.1%) had one detectable CFTR mutation and in the 14 remaining patients (6.3%), no CFTR defect was detected.
Sixty-one patients were then investigated for the presence of large rearrangements by QFM-PCR (Table II): (i) the 14 patients with no identified mutation (28 unidentified alleles); (ii) the 29 patients with one identified mutation, of whom 16 carried a mild or variable mutation, 10 carried a severe CF mutation and three carried a rare missense mutation whose effect was difficult to predict (29 unidentified alleles) and (iii) 15 patients with two mutations of mild or unknown effect and three who were apparently homozygous for a mild mutation (0–18 unidentified alleles). Three heterozygous deletions (2-fold reduction in fluorescence intensity) and one duplication (1.5-fold increase) were detected in four patients who already carried a mild mutation (Tables II and III). No rearrangement was found in the patients with no identified point mutation. Patient #1, heterozygous for (TG)12(T)5, exhibited a deletion of exons 17a to 18; by the use of specific primers, the presence of the known 3120 + 1.6 kbdel8.6 kb (or CFTRdele17a_18) (Lerer et al., 1999) was confirmed. Patient #2, heterozygous for the mild V938G missense mutation, had a deletion of exons 22 and 23, for which sequencing analysis showed the same breakpoints as those previously described (Audrézet et al., 2004). Patient #3 had already been referred to in Niel et al. (2004): he was apparently homozygous for R117H with IVS8(T)7 in cis and was found to carry a complete deletion of the CFTR gene, inherited from his mother. Patient #4, heterozygous for (TG)12(T)5, exhibited a duplication pattern involving exons 11 to 13. The presence of the duplication was confirmed upon four independent QFM-PCR experiments. The use of different sets of primers targeted on normal and duplicated exons showed that the duplication may not affect the whole exon 13 (Figure 1).
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Rearrangements, found in 6.9% of 58 unknown alleles (57 alleles from the first two groups of patients and one allele from the patient who was apparently homozygous for R117H), thus accounted for 0.9% of CBAVD alleles and 1% of the identified alleles. The QFM-PCR assay was sensitive enough to detect a number of microdeletions/insertions within exons, which modified fragment size, such as F508del, 2221dupA, as well as variants at the IVS8(TG)m(T)n polymorphic site. Overall, the complete screening has enabled the identification of 87.8% of CBAVD alleles.
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Discussion |
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Screening for large CFTR rearrangements enriches the mutational spectrum in CBAVD
Four large CFTR gene rearrangements were identified in four of 61 CBAVD patients investigated (Tables II and III). We have included all the patients with one or no detected mutation after complete screening for point mutations (57 unidentified alleles), as well as patients with two putative allelic mutations, because their effect may not be deleterious enough to account for the disease or because homozygous status could not be confirmed by family analysis (0–18 unidentified alleles). The four rearrangements accounted for nearly 7% of the previously unidentified alleles. This proportion is lower than that found in populations of CF patients, where 14–23% of unidentified alleles turned out to be large rearrangements (Audrézet et al., 2004
; Niel et al., 2004; Bombieri et al., 2005; Ferec et al., 2006; Hantash et al., 2006b). This can be partly explained by the fact that patients with isolated CBAVD usually carry mutations of a wide range of effects but not two severe mutations. Therefore, CFTR rearrangements, that had been considered null mutations, are expected to be present at most in one allele in CBAVD patients, in trans with a mild mutation, as was observed in the four cases of the present study. In other respects, the proportion of 7% is much higher than that found by Hantash in a recent study of 48 CBAVD patients (Hantash et al., 2006a): a deletion, removing exons 22 to 24, was found in one patient, which accounted for 1.4% of unidentified alleles. This discrepancy could be attributed to a difference in patients' origin or to less stringent inclusion criteria.
Among the four rearrangements identified in our study, two had already been described: CFTRdele17a_18 (Lerer et al., 1999) and CFTRdele22_23 (Audrézet et al., 2004). CFTRdele17a_18 is one of the first reported CFTR gene deletions and was found to account for 13% of CF chromosomes in Israeli Arab patients (Laufer-Cahana et al., 1999). It was subsequently identified in other groups of CF patients (Niel et al., 2004; Hantash et al., 2006b). In keeping with these data, we found it in a patient from Syria. CFTRdele22_23, initially reported in a French patient (Audrézet et al., 2004), was also further identified in other CF patients originating from Southern Europe (Audrézet M.P., personal communication). We found it in a patient of Italian descent. The availability of multiplex semi-quantitative PCR assays applied to the CFTR gene (Audrézet et al., 2004; Niel et al., 2004; Chevalier-Porst et al., 2005; Hantash et al., 2006b) has resulted in an increasing description of large CFTR rearrangements (www.genet.sickkids.on.ca/cftr). Like point mutations, some appear quite frequent, especially in particular populations, as is the case for CFTRdele2,3(21 kb) in Slavic patients (Dörk et al., 2000), CFTRdele17a_18 and CFTRdele3_10,14b_16 in Arab patients (Lerer et al., 1999; Niel et al., 2006) or CFTRdele17a_17b in Eastern France (Niel et al., 2004). This observation further underlines the need to take into account patients' origin as regards genetic counselling.
A complete CFTR gene deletion was found in one patient who had previously been referred to in Niel et al. (2004) and who was apparently homozygous for R117H. The patient was further investigated for the presence of mild CF symptoms but none was found. Characterization of the breakpoints has shown that the deletion actually extends over 3 Mb of chromosome 7 and removes other genes (Niel et al., manuscript in preparation). This finding highlights the importance of looking for rearrangements in apparently homozygous patients, especially when the genotype may not explain the disease phenotype.
The duplication involving exons 11 to 13 was found in a patient from Morocco, a country where very few data are available on CF and related diseases. Although duplications are more difficult to detect than deletions, because they result in 1.5-fold increase in fluorescence intensity, when compared with 2-fold reduction in case of a deletion, the duplication pattern was clear from the different experiments performed at separate times. The use of distinct pairs of primers for the large exon 13 allowed to show that the 3'breakpoint of the duplication is actually located next to the end of exon 13. Although the position and orientation of the duplication have not been determined yet, we postulate that the rearrangement is located inside the CFTR gene and interferes with the transcription or translation process, thus resulting in a null mutation.
Comprehensive mutation and genotype spectrum in CBAVD
Overall, with comprehensive CFTR gene study, 390 out of 444 alleles were identified, which corresponds to a mutation detection rate of 87.8%. This rate is somewhat higher than those described elsewhere for large CBAVD groups from various European origins (Chillon et al., 1995a; Dörk et al., 1997; Casals et al., 2000; Claustres et al., 2000), probably because only CBAVD patients with documented semen biochemical markers and transrectal but no renal ultrasound anomaly were selected in our series. This is also in keeping with the low proportion of patients who carry no CFTR mutation. Of the 222 patients, 82.0% had two CFTR mutations, 11.7% had one identified CFTR mutation and only 6.3% had no detected mutation.
A total of 99 different molecular anomalies were found, which have been described in patients with a broad range of phenotypes, from classical CF to CBAVD. Most of them were found only once. Among the most frequent defects, 10 were observed at a frequency above 1% and their cumulated frequency reached 74.6% among the identified mutations (Table I). This mutation spectrum is similar to those already described in other European populations (Chillon et al., 1995a; Dörk et al., 1997; Claustres et al., 2000).
Beside F508del, the IVS8(T)5 variant was the second most frequent mutation encountered in the present study. It is considered as a mild CFTR defect whose disease penetrance increases with the adjacent (TG)m length (Cuppens et al., 1998; Groman et al., 2004). (TG)11(T)5 penetrance reaching only 11% (Groman et al., 2004), we systematically investigated the whole CFTR gene to look for other cis mutations in the 14 patients carrying this variant and we found V562I in five of them. Three of the five carried a mutation on the other chromosome: L997F, S977F and W1282X. Although V562I has been considered a severe CF mutation, a series of arguments question its severe deleterious effect: its presence in trans of the severe W1282X mutation, the case of a V562I homozygous CF patient who carried in cis the frameshift 2347delG (Girodon et al., unpublished data), and the fact that residue V562 is not conserved in other proteins containing the ATP-binding cassette motif. We suggest reconsidering V562I as a mild and CBAVD-associated mutation.
CBAVD genotypes were varied, the most frequent combined F508del either with the IVS8(T)5 variant (28.0% of 222) or with R117H (6.3%). They were also heterogeneous in their expected effect, as previously documented (Lissens et al., 1996; Claustres et al., 2000; Stuhrmann and Dörk, 2000). No patient carried two severe CF mutations. Some genotypes had already been described in patients with moderate or late CF, such as those combining F508del with L206W, D1152H, 3272-26A > G or 2789 + 5G > A. These patients might develop other symptoms related to a mild form of CF, and thus need long-term follow-up. On the other hand, these mild, possibly variable CF mutations were found in our CBAVD population in combination with milder, non CF-causing defects, such as the IVS8(T)5 variant. Moreover, genotypes combining two mild alleles were found, such as [R117H]+[(TG)13(T)5], [(TG)11(T)5;V562I]+[L997F] or homozygosity for [R74W;D1270N]. Patients carrying such genotypes were thus screened for rearrangements but all tested negative. In these cases, however, as well as in those with only one or no detectable CFTR mutation, we cannot rule out the presence of mutations undetected with the methods used or because they lie inside introns or regulatory regions. In other respects, the proportion of cases without a detectable mutation after extensive gene screening by classical techniques and QFM-PCR (14 of 222) is higher than expected using calculation of genotype frequencies from allele frequencies according to Hardy–Weinberg equilibrium, and further strengthens the hypothesis that a number of these cases are not related to CFTR mutations (Mercier et al., 1995; Rave-Harel et al., 1995). Other factors might be responsible for CBAVD in these 14 patients and in others with one CFTR mutation as well or, even, in patients with two mild mutations, potentially in a multifactorial context.
Revised CFTR molecular strategy and implications for genetic counselling
The results presented here highlight the importance of carrying out a complete CFTR gene analysis, including screening for rearrangements in CBAVD patients with strict diagnosis criteria, as regards diagnostic and genetic counselling matters. Given the varied CBAVD genotype combinations and severities, molecular characterization of both CFTR alleles is of great concern for couples who request assisted reproduction. Strategy for identification of CFTR defects would be a multistep process, implying collaboration networks between routine and specialized laboratories where scanning tools and screening for rearrangements are available. The strategy would combine (Table IV): (i) the search for frequent mutations using commercial kits; (ii) complementary screening for the other six frequent CBAVD defects; (iii) extensive analysis of the 27 exons to search for other point mutations and (iv) screening for rearrangements by QFM-PCR. The first two steps, which may be available in routine CF laboratories, would have enabled detection of almost 80% of the 390 identified alleles in our CBAVD population. In other respects, screening for large deletions that are frequent in particular populations could also be performed in routine laboratories using specific and simple tools, as for the Slavic CFTRdele2,3 (Dörk et al., 2000) or CFTRdele17a_18 (Lerer et al., 1999).
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Confirmation of compound heterozygous status in CBAVD patients by studying the parents could be crucial and the parents should therefore be referred for genetic counselling. Moreover, whenever assisted reproduction is requested, CBAVD patients' partners should be screened for at least frequent CF mutations, and their geographical or ethnic origin taken into account. In cases where a CF mutation is identified in the partner, the couples have a high risk of having a CF child, depending on the patient's genotype, and may request prenatal or preimplantation diagnosis. As an illustration, patient #1's partner, a Bosnian Muslim woman, was found to carry 2184insA, a CF mutation that is not included in commercial kit panels. The couple therefore ran a 25% risk of having a CF child but only obtained this information 10 years after the first referral because the large duplication was identified recently and only carriage for IVS8(T)5 was known for the patient. CBAVD patients should also be informed that their relatives may be CF carriers and could benefit from genetic counselling. Again, knowledge of the actual mutation status greatly facilitates cascade screening in relatives.
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Acknowledgements |
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The authors are indebted to the clinicians who referred the patients: Dr M. Albert, Poissy; Dr E. Amar, Neuilly; Dr F. Fellmann, Besançon; Dr J. Flori, Strasbourg; Dr E. Gautier, Neuilly; Dr E. Ginglinger, Mulhouse; Dr F. Girard-Lemaire, Strasbourg; Dr V. Izard, Le Kremlin-Bicêtre; Pr P. Jonveaux, Nancy; Dr J. Lespinasse, Chambéry; Dr M.O. Peter, Mulhouse; Dr B. Simon-Bouy, Versailles; Dr J.C. Soufir, Le Kremlin-Bicêtre; Pr A. Toutain, Tours. Annick Lefloch, Brigitte Boissier, Rachel Medina and Fabienne Rossi are thanked for valuable technical assistance. David Kerridge is acknowledged for re-reading the manuscript.
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References |
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Submitted on November 17, 2006; resubmitted on January 9, 2007; accepted on January 16, 2007.
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