Mutations of the CFTR gene in Turkish patients with congenital bilateral absence of the vas deferens

Didem Dayangaç1,3,4, Hayat Erdem1, Engin Yilmaz1, Ahmet Sahin2, Christof Sohn3, Meral Özgüç1 and Thilo Dörk3,4

1 Department of Medical Biology and 2 Department of Urology, Faculty of Medicine, Hacettepe University, Sihhiye, 06100 Ankara, Turkey and 3 Clinics of Obstetrics and Gynecology, Medical School Hannover, Podbielskistrasse 380, D-30659 Hannover, Germany

4 To whom correspondence should be addressed: e-mail: didayan{at}hacettepe.edu.tr or doerk.thilo{at}mh-hannover.de


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) can cause congenital bilateral absence of the vas deferens (CBAVD) as a primarily genital form of cystic fibrosis. The spectrum and frequency of CFTR mutations in Turkish males with CBAVD is largely unknown. METHODS: We investigated 51 Turkish males who had been diagnosed with CBAVD at the Hacettepe University, Ankara, for the presence of CFTR gene mutations by direct sequencing of the coding region and exon/intron boundaries. RESULTS: We identified 27 different mutations on 72.5% of the investigated alleles. Two-thirds of the patients harboured CFTR gene mutations on both chromosomes. Two predominant mutations, IVS8-5T and D1152H, accounted for more than one-third of the alleles. Five mutations are described for the first time. With one exception, all identified patients harboured at least one mutation of the missense or splicing type. Presently available mutation panels would have uncovered only 7–12% of CFTR alleles in this population cohort. CONCLUSIONS: Although cystic fibrosis is relatively rare in Turkey, CFTR mutations are responsible for the majority of CBAVD in Turkish males. Because of a specific mutation profile, a population-specific panel should be recommended for targeted populations such as CBAVD in Turkey or elsewhere.

Key words: congenital absence of vas deferens/cystic fibrosis/genotype–phenotype correlation/male infertility/splicing mutation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia and is responsible for 1–2% of male infertility (Mak and Jarvi 1996Go). CBAVD is an autosomal recessive condition that has been classified as a primarily genital form of cystic fibrosis (Anguiano et al., 1992; Oates and Amos 1994Go). Cystic fibrosis (CF), in its classic form, is characterised by chronic pulmonary disease, pancreatic insufficiency, intestinal obstruction, male infertility and elevated sweat chloride (Welsh et al., 2001Go). However, its clinical expression can vary widely and partly depends on the nature of the underlying mutation genotype (Zielenski and Tsui, 1995Go). The gene responsible for CF encodes the Cystic Fibrosis Transmembrane conductance Regulator (CFTR, syn. ABCC7), that is a cAMP regulated chloride channel found in the apical membrane of epithelial cells (Welsh et al., 2001Go). More than 1000 CFTR gene mutations have been reported so far (http://www.genet.sickkids.on.ca) and distinct mutation genotypes have been reported in patients with full-blown CF and with isolated CBAVD (Anguiano et al., 1992Go; Chillón et al., 1995Go; Costes et al., 1995Go; Zielenski et al., 1995Go; Dumur et al., 1996Go; Dörk et al., 1997Go; Casals et al., 2000Go; Claustres et al., 2000Go).

Little is known about the spectrum and frequency of CFTR gene mutations in Turkey. Classic cystic fibrosis is less common in the Turkish population than in Central Europe, with an estimated incidence of about 1:10 000 newborns, but other entities such as CBAVD have not been investigated yet. Two recent studies have provided evidence for an extensive allelic heterogeneity in Turkish patients with classic CF (Onay et al., 2001Go; Kilinç et al., 2002Go). We here report the results of a detailed investigation of CFTR mutation genotypes in 51 Turkish CBAVD patients. We have identified by direct sequencing a high frequency of different CFTR gene alterations and a mutational spectrum that is distinct from other populations.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A total of 51 CBAVD patients had been referred to our laboratory at the Department of Medical Biology, Hacettepe University, Ankara, Turkey, during the years 1996–2000. All patients originated from Anatolia. These patients were diagnosed before as having obstructive azoospermia at different urology departments in Ankara. The diagnosis of CBAVD was based upon clinical examination with impalpable vas deferens and normal or nearly normal testicular volume. Transrectal ultrasonography showed seminal vesicle abnormalities such as hypoplasia and aplasia. Testicular biopsies demonstrated normal or hypospermatogenetic activity. Renal agenesis was tested in 13 patients, two were found with unilateral renal agenesis and one patient had chronic renal insufficiency and agenesis of both kidneys. All patients were between 28 and 45 years old. All males presented with no discernible lung disease or presentation of disease in other organs. Consanguinity of the parents was recorded in 8 of 32 patients for whom data were available. None of the patients had a family history of cystic fibrosis, and only patients who were apparently unrelated were included in this study.

Mutation analysis
Genomic DNA was extracted from peripheral blood leukocytes by routine procedures. All 51 samples were first screened for the {Delta}F508 and 1677delTA mutations in exon 10 with PCR followed by polyacrylamide gel electrophoresis. We next screened for six further CFTR gene mutations of the coding region and flanking intron sequences by previously described restriction-enzyme based methods: G85E, D110H, R347H, 2789+5G->A, D1152H, N1303K (Dörk et al., 1994aGo, 1997). One deep intronic mutation 3849+10kBC->T was tested by restriction enzyme analysis (Highsmith et al., 1994Go) and four large genomic deletions CFTRdele2(5kB), CFTRdele2(ins186), CFTRdele2,3(21kB) and 3120+1kBdel8.6kB were screened using specific primer pairs (Lerer et al., 1999Go; Dörk et al., 2000aGo,b). Missense polymorphism M470V in exon 10 was typed by HphI restriction enzyme analysis and the exon 9 and its flanking intron regions were sequenced in all samples to determine the status of polyvariant alleles that modulate the splicing and maturation of CFTR (Cuppens et al., 1998Go). The 33 samples with no or only one clearly pathogenic mutation identified after this initial screening were then subjected to direct sequencing of the entire coding sequence and all intron/exon boundaries of the CFTR gene using dye terminator chemistry and capillary electrophoresis on an ABI 310 genetic analyser (Applied Biosystems). We used published primer pairs for PCR and sequencing of most of the 27 exons (Zielenski et al., 1991Go) except for the following primers:

5'-GGTCTTTGGCATTAGGAGCTTG-3' and 5'-TCAGTTGCA AGTAGATGTGGC-3' (exon 1, annealing 59°C); 5'-GGAA GATGAAATTGTGTGTACCTTG-3' and CCACTACCATAATG CTTGGGAG-3' (exon 14b, annealing 58°C); 5'-CTACCCCAT GGTTGAAAAGCTG-3' and 5'-GCAGGAACTATCACATGTGAG-3' (exon 23, annealing 58°C); 5'-CTGACCTGCCTTCTGTCCCAG-3' and 5'-CTGAGGCAGAGGTAACTGTTCC-3' (exon 24, annealing 61°C). Finally, the 5'-UTR and minimum promoter region were amplified and sequenced using the primer pair 5'-GTCCTC CAGCGTTGCCAACTGG-3' and 5'-CAACGCTGGCCTTTTCC AGAGG-3' (annealing at 62°C).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 27 mutations was identified on 74 of the 102 alleles, as listed in Table I. Two mutations were found to be very common in Turkish CBAVD patients: first, the IVS8-5T allele was observed, with TG12 or TG13 haplotypes, on 20 chromosomes thus confirming the association of this splice site variant with CBAVD in Turkish patients. Secondly, the D1152H mutation in exon 18 was uncovered on 15 chromosomes, thereby revealing an unexpected high frequency of this missense substitution in Turkish CBAVD patients. Screening for the IVS8-5T and D1152H mutations together led to the identification of more than one-third of alleles. The D1152H mutation was found in the homozygous state in five patients and the IVS8-5T allele was found homozygous in four patients (Table II).


View this table:
[in this window]
[in a new window]
 
Table I. CFTR gene mutations identified in 51 CBAVD patients
 

View this table:
[in this window]
[in a new window]
 
Table II. CFTR genotypes in 51 patients with congenital bilateral absence of the vas deferens
 
Missense and splice site alterations were also predominant among the other 25 identified mutations whereas truncating mutations and the {Delta}F508 allele played a minor role (Table I). There was one patient compound heterozygous for two truncating mutations (1677delTA/E831X), but all other patients with completely resolved mutation genotype carried a missense or splicing mutation on at least one allele. Several mutations were already known as ‘mild’ alleles in cystic fibrosis, e.g. D110H, R347H or 2789+5G->A, and have been described previously in studies of patients with CBAVD. A few other substitutions, e.g. R74W, 2751-15C->G, L997F, are not classic cystic fibrosis mutations but we cannot exclude the possibility that they may contribute to a CBAVD phenotype, and the L997F substitution was reported to be associated with mild forms of cystic fibrosis such as pancreatitis (Gomez Lira et al., 2000Go). An unusual variant of the polyT tract in intron 8 was also identified, i.e. the IVS8-6T allele (Figure 1A), and was considered as a candidate mutation for CBAVD in one patient who also was heterozygous for the CFTRdele2(ins186) deletion with no other mutation detected.




View larger version (65K):
[in this window]
[in a new window]
 
Figure 1. Identification by direct sequencing of splicing mutations targeting the polypyrimidine tract. (A) Allelic heterogeneity of the polythymidine tract in intron 8. Three genotypes are shown for comparison. Top: IVS8-5T/IVS8-7T, middle: IVS8-6T/IVS8-7T, bottom: IVS8-7T/IVS8-7T. The sequence of the reverse strand is shown. (B) Mutations of the polypyrimidine tract in intron 15. Two new splice site mutations are shown in comparison to the wildtype sequence. Top: homozygous wildtype, middle: homozygous 3041-15T->G, bottom: homozygous 3041-13del7. The sequence of the forward strand is shown. An asterisk marks the position of the 3041-15T->G substitution. An insertion above the 3041-13del7 sequence indicates the deleted seven nucleotides. This figure can be viewed in colour online as supplementary data.

 
Five mutations are described here for the first time: 1767del6, 3041-13del7, 3041-15T->G, I853F and G1130A. The first three were found in the homozygous state. The 1767del6 mutation results in a two-amino-acid deletion within the first nucleotide binding fold of CFTR adjacent to the conserved ABC signature termed ‘Walker C’ motif. The deletion 3041-13del7 and the substitution 3041-15T->G both disrupt the polypyrimidine tract of the intron 15 acceptor splice site and thus may result in aberrant splicing (Figure 1B). The missense mutations I853F and G1130A lead to subtle amino acid alterations in less conserved portions of the CFTR protein. Although we cannot prove that these five new mutations are pathogenic, no other sequence alteration was identified through direct sequencing in these patients.

In summary, this study led to the identification of 27 CFTR gene mutations and presumably pathogenic alterations on 72.5% of chromosomes from 51 Turkish CBAVD patients. Two mutant alleles were identified in 34 patients (66.7%). One mutant allele was identified in six patients (11.7%), of whom two carried clearly pathogenic mutations ({Delta}F508 and R347H, respectively) and four harboured unclassified variants. No mutations were found in 11 patients (21.6%), including the three patients with known renal malformations.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutation analysis of the CFTR gene has become a standard procedure for couples seeking genetic testing before assisted reproduction if the male infertility is caused by obstructive azoospermia. Because lung disease and other CF-like symptoms may develop later in life in those young adults who are mutation carriers, careful work-up and counselling is mandatory. However, while the spectrum and frequency distribution of CFTR gene mutations in most patients of European descent has been the subject of extensive research, information is scarce about other populations, even those at the European–Asian border, and thus the efficacy of the present diagnostic tools for genetic testing in Turkish couples is largely unknown. Two recent reports have demonstrated a marked heterogeneity of CFTR mutations in Turkish patients with classic cystic fibrosis. In the first study of 98 families, Onay et al., (2001Go) have described 28 different CFTR mutations, including the IVS8-5T allele, on 105 of 168 CF chromosomes (62.5%) after scanning the whole coding region using a multiplex heteroduplex method. In the second study, Kilinç et al., (2002Go) investigated 83 Turkish CF patients and reported 36 different mutations, excluding the IVS8-5T allele, that accounted for 125 of the 166 CF chromosomes (75%). The most common mutation {Delta}F508 was present on about one in four alleles in both reports, this is a significantly lower frequency than in CF patients from Central European populations. The relatively low incidence of CF and the low frequency of prominent CF mutations could lead one to suggest that CFTR gene mutations might be rare in Turkey, but such an interpretation may not necessarily be correct since there are other CFTR-related diseases such as CBAVD which are caused by a different mutational spectrum than CF (Dörk et al., 1997Go; Claustres et al., 2000Go). To elucidate this issue further, we have performed a sequencing analysis of the CFTR gene in 51 Turkish patients with CBAVD.

The results of this study reflect the high allelic heterogeneity of CFTR gene mutations, although two mutations, IVS8-5T and D1152H, were found to be very common in Turkish CBAVD patients. First, the IVS8-5T allele was observed on 19.6% of all chromosomes. This polythymidine tract deletion is known as a splicing mutation with reduced penetrance (Chillón et al., 1995Go; Costes et al., 1995; Zielenski et al., 1995Go) and is found at an allele frequency of only 3–5% in the general Turkish population (Onay et al., 2001Go; Kilinç et al., 2002Go). Consistent with previous reports (Costes et al., 1995Go; Dörk et al., 1997Go; Cuppens et al., 1998Go), we find that the IVS8-5T allele in CBAVD patients is exclusively associated in cis with long IVS8-(TG)12–13 tracts which have been shown to increase the extent of exon 9 skipping induced by the 5T variant (Cuppens et al., 1998; Niksic et al., 1999; Buratti et al., 2001Go). It has previously been found that IVS8-5T is a recurrent mutation on different ethnic backgrounds, thus our study corroborates the suggestion that it may contribute substantially to the etiology of CBAVD worldwide (Dörk et al., 1997Go; Lissens et al., 1999Go). Secondly, the D1152H missense substitution was uncovered on 14.7% of all chromosomes. This finding was unexpected because this mutation has not been detected in Turkish CF patients before (Onay et al., 2001Go; Kilinç et al., 2002Go). It has, however, been previously observed in CBAVD patients of diverse ethnicities at low frequencies (Dörk et al., 1997Go; Kerem et al., 1997Go; Claustres et al., 2000Go) and was initially reported as a mild cystic fibrosis mutation. In vitro studies have shown that the D1152H substitution does not interfere with the maturation of the CFTR protein but strongly reduces its cAMP-activated chloride conductance (Vankeerberghen et al., 1998Go) which may provide the basis for a mild expression of disease. Our results implicate D1152H as a common missense mutation in Turkey which appears to be specifically associated with CBAVD, perhaps comparable to the role of the R117H missense substitution in Central Europe (Gervais et al., 1993Go; Dörk et al., 1997Go). All D1152H alleles appeared to be linked with the same haplotype comprising the IVS8-7T(TG)11 and Val470 alleles (Table II). Screening for the IVS8-5T and D1152H mutations together led to the identification of more than one-third of alleles in Turkish CBAVD males.

Most of the less frequent mutations were also of the missense or splicing type and, beyond the IVS8-5T allele, four other putative splicing mutations have been identified that also target a polypyrimidine tract. The IVS8-6T allele constitutes a new member of the growing family of polyT variants in intron 8 and may have clinical implications. Although the 6T allele has first been observed in an apparently healthy female carrier seeking assisted reproduction (Dörk et al., 2001Go), it could nevertheless be a CBAVD causing mutation. With the four alleles that have previously been identified in intron 8 of the CFTR gene (T9, T7, T5 and T3), the efficiency of the splice acceptor site usage consistently decreases with shorter polythymidine tracts which results in a lower than normal level of full-length CFTR mRNA (Chu et al., 1993Go; Mak et al., 1997Go; Teng et al., 1997Go; Larriba et al., 1998Go; Buratti et al., 2001Go). The 6T allele can be predicted to affect splicing to a degree between the 5T and 7T alleles and thus it could represent a low-penetrance mutation, at the border between health and mild disease. The other polypyrimidine tract variants 2752-15C->G, 3041-15T->G, and 3041-13del7 all reduce the maximum entropy score of the respective acceptor splice sites (Yeo and Burge 2004;Go http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq_acc.html), but the quantitative impact of these mutations on the accuracy and efficiency of splicing remains to be determined.

The observation that almost every Turkish CBAVD patient with identified mutation genotype harboured a missense or splicing mutation on at least one allele is in line with previous reports of such a distinction between mutation genotypes in CF and CBAVD (Dörk et al., 1997Go; Claustres et al., 2000Go). The first exception is one patient in our study who is a compound heterozygote for the two truncating mutations 1677delTA and E831X. Although early reports have suggested that CF patients with two null alleles may have milder lung disease compared with {Delta}F508 homozygotes (Gasparini et al., 1992Go; Wine, 1992Go), such patients are generally pancreatic insufficient and previous studies of patients with isolated CBAVD have not revealed any patient carrying two truncating mutations, thus far (Dörk et al., 1997Go; Claustres et al., 2000Go). It may thus be the particular genotype of our single case that confers an unusually mild phenotype. Exon 14a is the subject of alternative splicing (Hull et al., 1994Go; Bienvenu et al., 1996Go) and the mutation E831X at the first base of this exon may increase its in-frame skipping. Such a by-pass of a premature termination codon has been shown to ameliorate the disease phenotype in other clinical conditions (Morisaki et al., 1993Go; Ginjaar et al., 2000Go; Su et al., 2000Go).

Our study detected CFTR gene mutations on both alleles in two-thirds of the Turkish CBAVD males. This is comparable with thorough studies of European CBAVD patients as well as with the detection rate obtained for Turkish CF patients. Eleven patients, including three with confirmed renal agenesis, remained with no mutation identified after sequencing all exons. The reasons for the incomplete detection could not be fully established but may be related to some heterogeneity in the etiology of CBAVD (Rave-Harel et al., 1995Go; McCallum et al., 2001Go). There is evidence that the prevalence of CFTR mutations is significantly reduced in patients who have CBAVD accompanied by renal agenesis (Augarten et al., 1994Go; Schlegel et al., 1996Go; Dörk et al., 1997Go; Casals et al., 2000Go; McCallum et al., 2001Go). It is also possible that some unknown mutations outside of the analysed regions account for a proportion of unidentified alleles in our study and in Turkish CF patients (Onay et al., 2001Go; Kilinç et al., 2002Go). Despite these limitations, we believe that direct sequencing is the most sensitive of the presently available methods for a whole CFTR mutation genotyping in Turkish CBAVD patients. Any of today’s commercially distributed kits would miss the majority of the mutant alleles in our patient cohort. For instance, the analysis of the 31 most common cystic fibrosis mutations found within the white population would have detected three mutations, accounting for 8 of the 102 alleles in the Turkish CBAVD patients (8%, compared with 31% in Canadian CBAVD males; Mak et al., 1999Go). The ACMG 25 mutation panel that includes core mutations recommended for general population CF carrier screening, would also have detected three mutations on 7 of the 102 alleles in our cohort (7%; Grody et al., 2001Go). An extension of this panel to 100 CF mutations together with mass spectrometry analysis would have detected 7 mutations on 12 of the 102 alleles (12%; Wang et al., 2002Go).

In conclusion, none of the currently used routine mutation panels is well-suited for Turkish couples seeking genetic testing before assisted reproduction. It would be desirable to have more population-specific mutation panels which could help to overcome the underdiagnosis of cystic fibrosis and other CFTR-related diseases in those countries where the mutational distribution does not match that of Central Europe. In this regard, a population-specific mutation panel including the D1152H mutation and the IVS8-5T allele should be highly recommended for Turkish CBAVD patients. Our data add to a growing body of evidence that CFTR mutations can have a substantial clinical impact also in populations where cystic fibrosis in its classic form may be infrequent. The spectrum of mutations and genotypes that we have presented here, including the identification of a common missense mutation, should be useful to refine the diagnosis of CBAVD or other CFTR-related diseases in patients of Turkish descent.


    Acknowledgements
 
We cordially thank the CBAVD males for taking part in this study and providing blood samples. Part of this work was supported by a travel grant from the Cystic Fibrosis European Network.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anguiano A, Oates RD, Amos JA, Dean M, Gerrard B, Stewart C, Maher TA, White MB and Milunsky A (1992) Congenital bilateral absence of the vas deferens: a primarily genital form of cystic fibrosis. J Am Med Assoc 267, 1794–1797.[Abstract]

Audrezet MP, Mercier B, Guillermit H, Quere I, Verlingue C, Rault G and Ferec C (1993) Identification of 12 novel mutations in the CFTR gene. Hum Mol Genet 2, 51–54.[Abstract]

Augarten A, Yahav Y, Kerem BS, Halle D, Laufer J, Szeinberg A, Dor J, Mashiach S, Gazit E and Madgar I (1994) Congenital bilateral absence of the vas deferens in the absence of cystic fibrosis. Lancet 344, 1473–1474.[CrossRef][ISI][Medline]

Beaudet AL and Tsui L-C (1993) A suggested nomenclature for designating mutations. Hum Mutat 2, 245–248.[ISI][Medline]

Bienvenu T, Beldjord C, Chelly J, Fonknechten N, Hubert D, Dusser D and Kaplan JC (1996) Analysis of alternative splicing patterns in the cystic fibrosis transmembrane conductance regulator gene using mRNA derived from lymphoblastoid cells of cystic fibrosis patients. Eur J Hum Genet 4, 127–134.[Medline]

Buratti E, Dörk T, Zuccato E, Pagani F, Romano M and Baralle F (2001) Nuclear factor TDP-43 and SR proteins binding on either side of CFTR exon 9 promote in vitro and in vivo exon skipping. EMBO J 20, 1774–1784.[Abstract/Free Full Text]

Casals T, Bassas L, Egozcue S, Ramos MD, Giménez J, Sigura A, Garcia F, Carrera M, Larriba S, Sarquella J and Estivill X (2000) Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens. Hum Reprod 15, 1476–1483.[Abstract/Free Full Text]

Chillón M, Casals T, Mercier B et al. (1995) Mutations in the cystic fibrosis gene in patients with congenital absence of vas deferens. N Engl J Med 332, 1475–1480.[Abstract/Free Full Text]

Chu C-S, Trapnell BC, Curristin S, Cutting GR and Crystal RG (1993) Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nat Genet 3, 151–156.[ISI][Medline]

Claustres M, Guittard C, Bozon D et al. (2000) Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France. Hum Mutat 16, 143–156.[ISI][Medline]

Costes B, Girodon E, Ghanem N, Flori E, Jardin A, Soufir JC and Goossens M (1995) Frequent occurrence of the CFTR intron 8 (TG)n5T allele in men with congenital bilateral absence of the vas deferens. Eur J Hum Genet 3, 285–293.[ISI][Medline]

Cremonesi L, Ferrari M, Belloni E, Magnani C, Seia M, Ronchetto P, Rady M, Russo MP, Romeo G and Devoto M (1992) Four new mutations of the CFTR gene (541delC, R347H, R352Q, E585X) detected by DGGE analysis in Italian CF patients, associated with different clinical phenotypes. Hum Mutat 1, 314–319.[Medline]

Cuppens H, Lin W, Jaspers M et al. (1998) Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation. J Clin Invest 101, 487–496.[Abstract/Free Full Text]

Dean M, White MB, Amos J, Gerrard B, Stewart C, Khaw KT and Leppert M (1990) Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients. Cell 61, 863–70.[ISI][Medline]

Dörk T, Neumann T, Wulbrand U et al. (1992) Intra- and extragenic marker haplotypes of CFTR mutations in cystic fibrosis families. Hum Genet 88, 417–425.[ISI][Medline]

Dörk T, Mekus F, Schmidt K et al. (1994a) Detection of more than 50 different CFTR mutations in a large group of German cystic fibrosis patients. Hum Genet 94, 533–542.[ISI][Medline]

Dörk T, Fislage R, Neumann T, Wulf B and Tümmler B (1994b) Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations. Hum Genet 93, 67–73.[ISI][Medline]

Dörk T, Dworniczak B, Aulehla-Scholz C et al. (1997) Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens. Hum Genet 100, 365–377.[CrossRef][ISI][Medline]

Dörk T, Macek M Jr, Mekus F et al. (2000a) Characterization of a novel 21-kb deletion, CFTRdele2,3(21kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central and East Europe. Hum Genet 106, 259–268.[CrossRef][ISI][Medline]

Dörk T, Mekus F, Seydewitz HH, Özgüç M, Onay T, Ferec C, Rawashdeh M, Aznarez I and Zielenski J (2000b) An unusual deletion/insertion mutation of the CFTR gene in German, Turkish and Jordanian cystic fibrosis patients. Eur J Hum Genet 8 (Suppl. 1), 148.

Dörk T, Gläser D, Stuhrmann M, Buratti E, Pagani F and Baralle FE (2001) Novel, rare splice site variants of IVS8 in the cystic fibrosis gene. Eur J Hum Genet 9 (Suppl. 1), 405.

Dumur V, Gervais R, Rigot J-M, Delomel-Vinner E, Decaestecker B, Lafitte J-J and Roussel P (1996) Congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis transmembrane regulator: correlation between genotype and phenotype. Hum Genet 97, 7–10.[CrossRef][ISI][Medline]

Fanen P, Ghanem N, Vidaud M, Besmond C, Martin J, Costes B, Plassa F and Goossens M (1992) Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions. Genomics 13, 770–776.[ISI][Medline]

Feldmann D, Rochemaure J, Plouvier E, Magnier C, Chauve C and Aymard P (1995) Mild course of cystic fibrosis in an adult with the D1152H mutation. Clin Chem 41, 1675.[ISI][Medline]

Gasparini P, Borgo G, Mastella G, Bonizzato A, Dognini M and Pignatti PF (1992) Nine cystic fibrosis patients homozygous for the CFTR nonsense mutation R1162X have mild or moderate lung disease. J Med Genet 29, 558–562.[Abstract]

Gervais R, Dumur V, Rigot J-M, Lafitte J-J, Roussel P, Claustres M and Demaille J (1993) High frequency of the R117H cystic fibrosis mutation in patients with congenital absence of the vas deferens. N Engl J Med 328, 446–447.[Free Full Text]

Ginjaar IB, Kneppers ALJ, Meulen J-DM, Anderson LVB, Bremmer-Bout M, van Deutekom JCT, Weegenaar J, den Dunnen JT and Bakker E (2000) Dystrophin nonsense mutation induces different levels of exon 29 skipping and leads to variable phenotypes within one BMD family. Eur J Hum Genet 8, 793–796.[CrossRef][ISI][Medline]

Gomez-Lira M, Bertazzo MG, Marzari MG, Bombieri C, Belpinati F, Castellani C, Cavallini GC, Mastella G and Pignatti PF (2000) High frequency of cystic fibrosis transmembrane conductance regulator mutation L997F in patients with recurrent idiopathic pancreatitis and in newborns with hypertrypsinemia. Am J Hum Genet 66, 2013–2014.[CrossRef][Medline]

Grody WW, Cutting GR, Klinger KW, Richards CS, Watson MS and Desnick RJ (2001) Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 3, 149–154.[Medline]

Highsmith WE Jr, Burch LH, Zhou Z et al. (1994) A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations. N Engl J Med 331, 974–980.[Abstract/Free Full Text]

Highsmith WE Jr, Burch LH, Zhou Z, Olsen JC, Strong TV, Smith T, Friedman KJ, Silverman LM, Boucher RC, Collins FS and Knowles MR (1997) Identification of a splice site mutation (2789+5G>A) associated with small amounts of normal CFTR mRNA and mild cystic fibrosis. Hum Mutat 9, 332–338.[CrossRef][ISI][Medline]

Hull J, Shackleton S and Harris A (1994) Analysis of mutations and alternative splicing patterns in the CFTR gene using mRNA derived from nasal epithelial cells. Hum Mol Genet 3, 1141–1146.[Abstract]

Ivaschenko TE, White MB, Dean M and Baranov VS (1991) A deletion of two nucleotides in exon 10 of the CFTR gene in a Soviet family with cystic fibrosis causing early infant death. Genomics 10, 298–299.[Medline]

Kerem B, Chiba-Falek O and Kerem E (1997) Cystic fibrosis in Jews: frequency and mutation distribution. Genet Test 1, 35–9.[Medline]

Kilinç MO, Ninis VN, Dagli E, Demirkol M, Ozkinay F, Arikan Z, Cogulu O, Huner G, Karakoc F and Tolun A (2002) Highest heterogeneity for cystic fibrosis: 36 mutations account for 75% of all CF chromosomes in Turkish patients. Am J Med Genet 113, 250–257.[CrossRef][Medline]

Larriba S, Bassas L, Gimenez J, Ramos MD, Segura A, Nunes V, Estivill X and Casals T (1998) Testicular CFTR splice variants in patients with congenital absence of the vas deferens. Hum Mol Genet 7, 1739–1744.[Abstract/Free Full Text]

Lerer I, Laufer-Cahana A, Rivlin JR, Augarten A and Abeliovich D (1999) A large deletion mutation in the CFTR gene (3120+1Kbdel8.6Kb): a founder mutation in the Palestinian Arabs. Hum Mutat 13, 337.[Medline]

Lissens W, Mahmoud KZ, El-Gindi E, Abdel-Sattar A, Seneca S, Van Steirteghem A and Liebaers I (1999) Molecular analysis of the cystic fibrosis gene reveals a high frequency of the intron 8 splice variant 5T in Egyptian males with congenital bilateral absence of the vas deferens. Mol Hum Reprod 5, 10–13.[Abstract/Free Full Text]

Macek M Jr, Mackova A, Hamosh A, Hilman BC, Selden RF, Lucotte G, Friedman KJ, Knowles MR, Rosenstein BJ and Cutting GR (1997) Identification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%. Am J Hum Genet 60, 1122–1127.[Medline]

Mak V and Jarvi K (1996) The genetic basis of male infertility. J Urol 156, 1245–1257.[CrossRef][ISI][Medline]

Mak V, Jarvi KA, Zielenski J, Durie P and Tsui LC (1997) Higher proportion of intact exon 9 CFTR mRNA in nasal epithelium compared with vas deferens. Hum Mol Genet 6, 2099–2107.[Abstract/Free Full Text]

Mak V, Zielenski J, Tsui L-C, Durie P, Zini A, Martin S, Longley TB and Jarvi KA (1999) Proportion of cystic fibrosis gene mutations not detected by routine testing in men with obstructive azoospermia. J Am Med Assoc 281, 2217–2224.[Abstract/Free Full Text]

McCallum TJ, Milunsky JM, Munarriz R, Carson R, Sadeghi-Nejad H and Oates RD (2001) Unilateral renal agenesis associated with congenital bilateral absence of the vas deferens: phenotypic findings and genetic considerations. Hum Reprod 16, 282–288.[Abstract/Free Full Text]

Morisaki H, Morisaki T, Newby LK and Holmes EW (1993) Alternative splicing: a mechanism for phenotypic rescue of a common inherited defect. J Clin Invest 91, 2275–2280.[ISI][Medline]

Niksic M, Romano M, Buratti E, Pagani F and Baralle FE (1999) Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9. Hum Mol Genet 8, 2339–2349.[Abstract/Free Full Text]

Oates RD and Amos JA (1994) The genetic basis of congenital bilateral absence of the vas deferens and cystic fibrosis. J Androl 15, 1–8.[Free Full Text]

Onay T, Zielenski J, Topaloglu O, Gokgoz N, Kayserili H, Apak MY, Camcioglu Y, Cokugras H, Akcakaya N, Tsui L-C and Kirdar B (2001) Cystic fibrosis mutations and associated haplotypes in Turkish cystic fibrosis patients. Hum Biol 73, 191–203.[Medline]

Rave-Harel N, Madgar I and Goshen R (1995) CFTR haplotype analysis reveals genetic heterogeneity in the etiology of congenital bilateral aplasia of the vas deferens. Am J Hum Genet 56, 1359–1366.[ISI][Medline]

Schlegel PN, Shin D and Goldstein M (1996) Urogenital anomalies in men with congenital absence of the vas deferens. J Urol 155, 1644–1648.[CrossRef][ISI][Medline]

Su L-K, Barnes CJ, Yao W, Qi Y, Lynch PM and Steinbach G (2000) Inactivation of germline mutant APC alleles by attenuated somatic mutations: a molecular genetic mechanism for attenuated familial adenomatous polyposis. Am J Hum Genet 67, 582–590.[CrossRef][ISI][Medline]

Teng H, Jorissen M, van Poppel H, Legius E, Cassiman J-J and Cuppens H (1997) Increased proportion of exon 9 alternatively spliced CFTR transcripts in vas deferens compared with nasal epithelial cells. Hum Mol Genet 6, 85–90.[Abstract/Free Full Text]

Vankeerberghen A, Wei L, Teng H, Jaspers M, Cassiman J-J, Nilius B and Cuppens H (1998) Characterization of mutations located in exon 18 of the CFTR gene. FEBS Lett 437, 1–4.[CrossRef][ISI][Medline]

Wang Z, Milunsky J, Yamin M, Maher T, Oates R and Milunsky A (2002) Analysis by mass spectrometry of 100 cystic fibrosis gene mutations in 92 patients with congenital bilateral absence of the vas deferens. Hum Reprod 17, 2066–2072.[Abstract/Free Full Text]

Welsh MJ, Ramsey BW, Accurso F and Cutting GR (2001) Cystic fibrosis. In Scriver C, Vogelstein B, Beaudet AL, Childs B, Kinzler KW, Sly WS, Valle D (eds) The metabolic and molecular bases of inherited disease (8th edn). McGrawHill, New York, pp. 5121–5188.

Wine J (1992) No CFTR: are CF symptoms milder? Nat Genet 1, 10.[Medline]

Yeo G and Burge CB (2004) Maximum Entropy Modeling of Short Sequence Motifs with Applications to RNA Splicing Signals. J Comp Biol, in press.

Zielenski J, Patrizio P, Corey M, Handelin B, Markiewicz D, Asch R and Tsui L-C (1995) CFTR gene variant for patients with congenital absence of the vas deferens. Am J Hum Genet 57, 958–960.[ISI][Medline]

Zielenski J, Rozmahel R, Bozon D, Kerem B, Grzelczak Z, Riordan JR, Rommens J and Tsui LC (1991) Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Genomics 10, 214–228.[ISI][Medline]

Zielenski J and Tsui L-C (1995) Cystic fibrosis: genotypic and phenotypic variations. Annu Rev Genet 29, 777–807.[CrossRef][ISI][Medline]

Submitted on October 13, 2003; accepted on February 24, 2004.