Analysis by mass spectrometry of 100 cystic fibrosis gene mutations in 92 patients with congenital bilateral absence of the vas deferens

Zhenyuan Wang1, Jeff Milunsky1, Moshe Yamin1, Thomas Maher1, Robert Oates2 and Aubrey Milunsky1,3

1 Center for Human Genetics and the Departments of Pediatrics and 2 Urology, Boston University School of Medicine and Boston Medical Center, Boston, MA, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Limited mutation analysis for congenital bilateral absence of the vas deferens (CBAVD) has revealed only a minority of men in whom two distinct mutations were detected. We aimed to determine whether a more extensive mutation analysis would be of benefit in genetic counselling and prenatal diagnosis. METHODS: We studied a cohort of 92 men with CBAVD using mass spectrometry and primer oligonucleotide base extension to analyse an approximately hierarchical set of the most common 100 CF mutations. RESULTS: Analysis of 100 CF mutations identified 33/92 (35.9%) patients with two mutations and 29/92 (31.5%) with one mutation, compound heterozygosity accounting for 94% (31/33) of those with two mutations. This panel detected 12.0% more CBAVD men with at least one mutation and identified a second mutation in >50% of those considered to be heterozygotes under the two routine 25 mutation panel analyses. CONCLUSION: Compound heterozygosity of severe/mild mutations accounted for the vast majority of the CBAVD patients with two mutations, and underscores the value of a more extensive CF mutation panel for men with CBAVD. The CF100 panel enables higher carrier detection rates especially for men with CBAVD, their partners, partners of known CF carriers, and those with ‘mild’ CF with rarer mutations.

Key words: congenital bilateral absence of the vas deferens/cystic fibrosis/genetic testing/mass spectrometry/mutation detection


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Congenital bilateral absence of the vas deferens (CBAVD, MIM #277180) is present in 1–2% of the infertile but otherwise healthy male population and accounts for >=6% of cases of obstructive azoospermia (Holsclaw et al., 1971Go). Bilateral vasal agenesis is also present in ~97% of male patients with cystic fibrosis (CF) (Welsh et al., 2001Go). These men with CF are azoospermic due to anomalies in Wolffian duct-derived structures, with the body and tail of the epididymis, vas deferens, seminal vesicles, and ejaculatory ducts being atrophic or absent (Kaplan et al., 1968Go; Taussig et al., 1972Go; Welsh et al., 2001Go).

In 1971, Holsclaw et al. showed that bilateral vassal agenesis was present in almost all male CF patients (Holsclaw et al., 1971Go). Our studies (Anguiano et al., 1992Go) and those of others (Dumur et al., 1990Go; Osborne et al., 1993Go; Culard et al., 1994Go) showed that otherwise healthy men with CBAVD frequently had cystic fibrosis transmembrane regulator (CFTR) gene mutations. This observation led to our suggestion that CBAVD was a primarily genital form of CF (Anguiano et al., 1992Go). Some CFTR gene mutations associated with CBAVD are uncommon in CF patients. In CBAVD, compound heterozygosity is common, there being mostly one severe and one mild mutation or two mild mutations (Chillon et al., 1995Go; Costes et al., 1995Go; Zielenski et al., 1995Go; Dork et al., 1997Go; Kanavakis et al., 1998Go). The 5-thymidine variant of the polythymidine tract (IVS8-5T) in the splice acceptor site of intron 8 is one of the mild mutations that were discovered to be present with high frequency in CBAVD patients (Chillon et al., 1995Go; Costes et al., 1995Go; Zielenski et al., 1995Go; Bienvenu et al., 1997Go). This variant has been demonstrated to cause a high level of exon 9 skipping, leading to a non-functional CFTR protein (Chu et al., 1993Go; Bienvenu et al., 1997Go). The variable level of expression of this 5T allele explains the incomplete penetrance of this allele, resulting in CBAVD and a mild form of CF (Mak et al., 1997Go; Rave-Harel et al., 1997Go; Cuppens et al., 1998Go).

Routine, but limited, mutation analysis for CBAVD men has revealed only a minority in which two distinct mutations were detected. Given the frequency of CF mutations, especially in the Caucasian population (1 in 25), and the common request by CBAVD men to sire their own offspring by using surgical sperm aspiration in conjunction with ICSI, we sought to determine whether a more extensive mutation analysis might prove to be of benefit for genetic counselling and risk estimation, as well as for subsequent prenatal diagnosis. We studied a cohort of men with clinically diagnosed CBAVD using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry to analyse an approximately hierarchical set of the most common 100 CF mutations.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
The 92 patient samples in this study were sent to the Center for Human Genetics for our routine CF mutation panel analysis. None was known to have symptoms or signs of CF. Diagnosis of CBAVD was made clinically by urologists and most had renal ultrasound studies. These patients were studied consecutively except when the DNA supply was exhausted.

Methods
25 mutation panel (CF25):
Genomic DNA was extracted from peripheral blood from each patient. The polymerase chain reaction (PCR) was performed on genomic DNA by the standard method. The PCR-amplified products from each patient were digested with specific restriction enzymes prior to gel electrophoresis. The mutations in the 25 mutation panel were: {Delta}F508, G542X, N1303K, G551D, W1282X, 1717–1G->A, R553X, 621+1G->T, R1162X, 2183AA->G, R117H, {Delta}I507, R560T, 3849+10kbC->T, S549N, S549I, S549R, R1283M, R1283K, R553G, R560K, R117L, 1774delCT, 1811+1G->C, and 4006–61del14.

ACMG 25 mutation panel (ACMG25):
The following mutations are the recommended core mutations for general population CF carrier screening by American College of Medical Genetics (ACMG) (Grody, et al 2001Go): {Delta}F508, G542X, N1303K, G551D, W1282X, 1717–1G->A, R553X, 621+1G->T, R1162X, R117H, {Delta}I507, 1898+1G->A, G85E, R347P, A455E, R560T, R334W, 3849+10kbC->T, 3659delC, 1078delT, 2789+5G->A, 711+1G->T, 2184delA, 3120+1G->A and I148T. These mutations are included in the CF100 mutation panel below.

100 mutation panel (CF100).
DNA extraction and PCR amplification were carried out the same way as in the routine CF25 mutation panel, except that the reverse primer had a universal sequence tail, and a third primer of the same universal sequence was also added to the reaction. The third primer was also 5' end-labelled with biotin which attaches to the streptavidin coated magnetic beads. The exons of the CFTR gene amplified were 3, 4, 5, 6a, 7, 9, 10, 11, 12, 13, 14b, 15, 16, 17b, 19, 20, 21 and their immediate flanking intronic sequences as well as intron 19. PCR primers were selected to cover whole exons, except for 13 and 17b, where only partial exons were amplified. Three or more exons were co-amplified in a multiplex PCR, followed by a multiplex primer extension of the oligonucleotides at the mutation sites. The extended diagnostic products were then measured by MALDI-TOF mass spectrometry. The CF100 panel includes the most common mutations and the 5T allele as well as other ‘less common’ mutations (Table IGo) (Cystic Fibrosis Genetic Analysis Consortium, 1994Go; Zielenski and Tsui, 1995Go; Estivill et al., 1997Go). This assay was validated in blinded analyses of all the mutations in the CF25 panel plus the 5T, 100% concordance being achieved in repeated assays.


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Table I. The 100 most common cystic fibrosis mutations listed by exon
 
Probe/Mass/Array.
Primer oligonucleotide base extension (PROBE) was carried out according to the Sequenom protocol (Braun et al., 1997Go; Haff and Smirnov, 1997Go; Higgins et al., 1997Go; Little et al., 1997Go) Magnetic beads were first added to the amplified DNA of specific regions of the CFTR gene. Amplified DNA was then exposed to primers that target the mutation sites. By extending the primer into the mutation site, the distinction between normal and mutated sequences is translated into a change in the primer’s mass. The extended DNA primer was isolated, with low femtomole quantities transferred to the SpectroCHlP, a derivatized silicon microarray. The array contained 96 individually addressable positions that provide an optimized launching pad for MALDI-TOF analysis.

MALDI-TOF.
This is a technology that enables the mass of large biomolecules, like DNA, to be measured directly (Hillenkamp et al., 1991Go; Little et al., 1997Go). DNA is combined with a small organic compound (the matrix) that is able to absorb energy from a laser beam. The DNA and matrix solution is dried onto the surface of a Sequenom SpectroCHIP microarray to produce a crystalline dispersion. Once the sample is introduced into the mass spectrometer and air is removed, a laser pulse causes a spontaneous volatilization and ionization of matrix and associated DNA fragments. These gas-phase ions are accelerated through a voltage potential and their TOF is recorded. The TOF results are then converted into mass values that are, in turn, translated into diagnostic results. As an example, a MALDI-TOF mass spectrum provides the diagnostic results in Figure 1Go.



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Figure 1. Matrix-assisted laser desorption ionization–time of flight mass spectra for multiplex primer oligonucleotide base extension reactions of mutations {Delta}F508, Q493X and R1066C. (A) A healthy individual with only normal alleles at all three loci; (B) a homozygous patient with {Delta}F508; (C) a heterozygous patient with {Delta}F508.

 
Mutation controls.
Lacking a full set of native control DNA, we constructed DNA fragments of multiple CF mutations to be tested. Using normal human DNA as a template, we produced two PCR products separately, that partially overlapped in sequence, together constituting the desired control fragments and the mutations that were introduced as part of the PCR primer. The two products were denatured and re-annealed together forming two possible heteroduplexes. The heteroduplex with recessed 3' end was able to extend by PCR to produce the fragment that is the sum of the two overlapping PCR products and the mutations in them. The extended heteroduplex then served as template in a subsequent PCR to generate a continuous supply as a positive control.

Data analysis.
CBAVD patients’ samples were subjected to our new CF100 mutation panel and assay results compared with our earlier routine CF25 mutation panels, as well as the recently announced ACMG25 mutation panel as if it were tested separately. Results were also analysed including the 5T allele for the CF25 and ACMG25 panels (Tables II and IIIGoGo).


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Table II. CFTR mutations in 92 men with congenital bilateral absence of vas deferens
 

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Table III. CFTR genotypes in 92 men with congenital bilateral absence of vas deferens
 

    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CFTR mutations in CBAVD patients
Six and eight different mutations (Table IIGo) respectively were detected in the original cohort of 92 men using the CF25 panel and ACMG25 panel. Analysis of the 5T allele of the poly-T tract of introns 8 added a further 33 mutant alleles. The CF100 panel analysis yielded a further seven different mutations compared with our routine CF25 panel, and five more mutations against the ACMG25 panel. A total of 14 different mutations were identified in 62/92 (67.4%) men studied (Table II and IIIGoGo) by analysis of the 100 mutations in CF100. Excluding 5T, CF100 mutation analysis would still identify one additional mutant allele for every seven mutant alleles found under the ACMG25 panel analysis. The {Delta}F508 mutation was found most frequently and accounted for 41% of mutant alleles. The 5T allele was the next most common mutation and represented 35% of mutant alleles. After the 5T allele, the relative frequent mutations with two to four alleles were: R117H (four alleles), W1282X (four alleles), G551D (three alleles), L206W (three alleles) and D1270 (two alleles). The remaining seven mutations were only present in a single patient (Table IIGo).

CFTR genotypes in CBAVD patients
In CF25 mutation analysis, 49/92 (53.3%) of the patients had at least one mutation identified, including three (3.3%) with two mutations and 46 (50.0%) with one mutation (Table IIIGo). The ACMG25 panel, if performed, would have identified 50/92 (54.3%) patients with at least one mutation, including a new heterozygote and a second mutation on a previously considered heterozygote by CF25 analysis. Further analysis of the 5T allele yielded an increase in the total number of patients with at least one mutation by 20.4% (from 49 to 59 patients) for the CF25 + 5T and 20.0% (from 50 to 60 patients) for the ACMG25 + 5T. However, the number of the patients detected with two mutant alleles was 6.8- and 8.6-fold greater than with the ACMG25 and CF25 panels without the 5T allele. CF100 analysis resulted in even greater increases of 26.5% (from 49 to 62 patients) and 24.0% (from 50 to 62 patients), when compared with the CF25 and ACMG25 panels respectively, in the total number of patients with at least one mutation. The number of the patients detected with two mutant alleles increased 8- and 11-fold respectively compared with the ACMG25 and CF25 panels. Here, our routine CF25 and the ACMG25 panels would miss 4/92 (4.3%) patients with two mutations and 8–9/92 (8.7–9.8%) patients with one mutation. In addition, 25–26/33 (76–79%) compound heterozygotes (two mutations) would be incorrectly identified as heterozygotes (Table IIIGo).

Through CF100 mutation analysis, 33/92 (35.9%) of the CBAVD patients were found to have two mutations and 29/92 (31.5%) had one mutation, and in 30/92 (32.6%) no mutation was detected. Compound heterozygosity accounts for 94% (31/33) of the patients with two mutations. Two homozygotes for 5T and D1270N were also detected. The majority of the compound heterozygotes had genotypes of one severe and one mild mutation (Table IIIGo). The most common was the genotype of {Delta}F508 and 5T (16 patients). Two relatively frequent compound heterozygotes were F508/R117H (3/33) and W1282X/5T (4/33). Unilateral renal agenesis was noted in 4/92 patients, one of whom had the {Delta}F508 mutation.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our CF100 mutation analysis led to identification of an additional 4/92 (4.3%) of previously unrecognized homozygotes and compound heterozygotes, and discovery of an additional 8–9/92 (8.7–9.8%) heterozygous CBAVD patients (Table IIIGo). In addition, this panel also identified a second mutation in >50% of the 46 heterozygotes under the CF25 and ACMG25 panels. Mak et al. (1999) used extensive screening of all 27 CFTR exons followed by gene sequencing in a study of CBAVD patients. Their efforts identified an additional 46% of mutations over their panel of 31 mutations using their costly approach. Our studies and those of others (Mak et al., 1999Go) indicate that CF100 mutation panel analysis could detect ~82% of the mutations that are potentially identifiable under extensive screening and sequencing protocols. Gene scanning using multiplex heteroduplex and single strand confirmation polymorphism analysis is likely to be more insensitive and yield a lower detection rate than direct mutation analysis by mass spectrometry. Furthermore, it is interesting to note that the five additional mutations over the ACMG25 panel were previously reported in CF and three of them are relatively frequent in CBAVD. However, the impact of CF100 mutation analysis on general population carrier screening would still need further studies.

The distribution of mutation genotypes in CBAVD was clearly different from that seen in the CF population. Of our 33 patients with two mutations, 30/33 (90.9%) of the patients were compound heterozygotes of severe/mild and 3/33 (9.1%) had mild/mild mutations. In CF, the common mutation genotypes are homozygotes or compound heterozygotes with severe mutations such as {Delta}F508 and others (i.e. nonsense or frameshift mutations). Compound heterozygotes and homozygotes of severe/severe mutations were not found in our cohort of CBAVD men. These results are consistent with those reported previously (Chillon et al., 1995Go; Mercier et al., 1995Go; Dork et al., 1997Go; Mak et al., 1999Go; Claustres et al., 2000Go), reflecting the general observation of compound heterozygosity with severe/mild or mild/mild mutations in CBAVD patients. In a large French CBAVD study (Claustres et al., 2000Go), the authors found that in CF, 87.77% of patients carried two severe CF mutations, whereas only 11.33% had a severe mutation in trans with a mild mutation. In contrast, none of their CBAVD patients had two severe mutations, all having severe/mild or mild/mild mutation combinations. The high frequency of mild or very mild CFTR mutations in patients with CBAVD led us and others to the hypothesis that the vas deferens is one of the tissues most susceptible to the effect of changes in CFTR activity and that CBAVD is a primarily genital form of CF (Anguiano et al., 1992Go; Oates and Amos, 1993Go; Mercier et al., 1995Go). The one unilateral renal agenesis patient with CBAVD and a {Delta}F508 mutation may be a chance finding for a phenotype due to a different aetiology and pathogenesis (McCallum et al., 2001Go).

The compound heterozygote {Delta}F508/R117H, previously reported to occur commonly in CBAVD patients (Dork et al., 1997Go), was also a frequent genotype (3/33) in this study. It was noted that R117H occurred on two chromosomal backgrounds, one carrying a 5T allele in CF patients and the other carrying a 7T allele in CBAVD patients, when the other chromosome carries a severe mutation such as {Delta}F508 (Kiesewetter et al., 1993Go). A strong linkage between {Delta}F508 and the 9T allele in cis on a chromosome has been reported in several studies (Chu et al., 1993Go; Kiesewetter et al., 1993Go; Cuppens et al., 1994Go). It is interesting that, in this study, 7T/9T was the only haplotype found in all three CBAVD patients with {Delta}F508/R1I7H. Although it was not confirmed, our results indicate that {Delta}F508 and R117H were on chromosome backgrounds of 9T and 7T respectively, because at least one 9T allele was seen in each one of the 41 patients having a {Delta}F508 mutation, a 7T in each of the four patients with R117H mutation. Therefore, our results tend to lend further support to the concept of {Delta}F508 mutation originally arising on a single chromosome background, possibly with 9T. The association of R117H in cis with a 5T allele results in CF when patients carry in trans {Delta}F508 or one of the severe mutations, and probably represents a general theme that a splicing mutation such as 5T, in cis with a mild mutation, can aggravate that mild mutation. This observation could have clinical implications in genetic counselling of patients with mild mutations, especially for those compound heterozygotes with severe/mild mutations.

L206W is another mild mutation reported with relatively high frequency in both CF and CBAVD patients (Chillon et al., 1995Go; Mak et al., 1999Go; Claustres et al., 2000Go) and which we noted in three compound heterozygotes, two with {Delta}F508 and one with 5T. It appears that L206W is associated in cis with the 9T allele since both {Delta}F508/L206W patients have a homozygous 9T background, while 5T/L206W exists in a 5T/9T background.

The 5T allele is the second most frequent mutation (33/95) in this study, after the {Delta}F508 which had a frequency of 39/95. The frequent 5T allele was previously proposed to be a partially penetrant and ‘leaky’ splicing mutation (Chillon et al., 1995Go; Costes et al., 1995Go; Zielenski et al., 1995Go). Homozygotes for ‘5T’ in our one CBAVD patient and in others add weight to the argument that the 5T allele is a disease-causing mutation resulting in mild CF or CBAVD (Cuppens et al., 1998Go) and probably the most common one of the CFTR gene, since it has a 5% allele frequency in most investigated populations (Kiesewetter et al., 1993Go; Chillon et al., 1995Go). Partial penetrance could be a feature not only of the 5T allele but also of other missense mutations or other variants (polymorphisms).

CFTR gene mutation analysis is recommended for the female partners of men with CBAVD planning surgical sperm aspiration coupled with ICSI (Johnson, 1998Go; Lawler and Gearhart, 1998Go). Negative results for the female partner by testing for only the major CF mutations still leave residual risk of having affected offspring. Therefore, the most extensive and least expensive test that contains both the major and ‘less common’/mild mutations is strongly recommended. Our CF100 mutation analysis helps diminish this risk. One of the residual difficulties in counselling the family with CBAVD is the lack of a clear genotype–phenotype correlation, which precludes prediction of a specific phenotype.

In summary, we have developed an accurate assay for 100 CF mutations including not only the mutations found in classical CF but also mild/‘less common’ mutations that have been found in both CF and CBAVD patients. We have further demonstrated the utility of mass spectrometry (MALDI-TOF) as a diagnostic tool with its inherent qualities of high accuracy and ease for automation. When compared with the two routine, but limited, CF mutation panels, our CF100 panel detected 4.3% more homozygotes and compound heterozygotes and 8.0% more heterozygotes. It also identified a second mutation in >50% of those considered heterozygotes under CF25 and ACMG25 analyses. Excluding IVS8-5T, CF100 mutation analysis would identify one newly recognized mutation for every seven carriers found under the ACMG25 panel analysis. This panel provides significantly greater opportunities for mutation detection especially for men with CBAVD, their partners, the partners of known CF carriers, and for those with ‘mild’ CF with rarer mutations.


    Notes
 
3 To whom correspondence should be addressed at: Center for Human Genetics, Boston University School of Medicine, 715 Albany Street, W-408, Boston, MA 02118, USA. E-mail: amilunsk{at}bu.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anguiano, A., Oates, R.D., Amos, J.A., Dean, M., Gerrard, B., Stewart, C., Maher, T.A., White, M.B. 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]

Bienvenu, T., Adjiman, M., Thiounn, N., Jeanpierre, M., Hubert, D., Lepercoq, J., Francoual, C., Wolf, J., Izard, V., Jouannet, P. et al. (1997) Molecular diagnosis of congenital bilateral absence of the vas deferens: analyses of the CFTR gene in 64 French patients. Annales de Génétique, 40, 5–9.[ISI][Medline]

Braun, A., Little, D.P. and Koster, H. (1997) Detecting CFTR gene mutations using primer oligo base extension and mass spectrometry. Clin. Chem., 43, 1151–1158.[Abstract/Free Full Text]

Chillon, M., Casals, T., Mercier, B., Bassas, L., Lissens, W., Silber, S., Romey, M.C. Ruiz-Romero, J., Verlingue, C., Claustres, M. et al. (1995) Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. New Engl. J. Med., 332, 1475–1480.[Abstract/Free Full Text]

Chu, C.S., Trapnell, B.C., Curristin, S., Cutting, G.R. and Crystal, R.G. (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., Chevalier, F., Verlingue, C., Ferec, C., Girodon, E., Cazeneuve, C., Bienvenu T., Lalau, G. et al. (2000) Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France. Hum. Mut., 16, 143–156.[ISI][Medline]

Costes, B., Girodon, E., Ghanem, N., Flori, E., Jardin, A., Soufir, J.C. and Goossens, M. (1995) Frequent occurrence of the CFTR intron 8 (TG)n 5T allele in men with congenital bilateral absence of the vas deferens. Eur. J. Hum. Genet., 3, 285–293.[ISI][Medline]

Culard, J.F., Desgeorges, M., Costa, P., Laussel, M., Razakatzara, G., Navratil, H., Demaille, J. and Claustres, M. (1994) Analysis of the whole CFTR coding regions and splice junctions in azoospermic men with congenital bilateral aplasia of epididymis or vas deferens. Hum. Genet., 93, 467–470.[ISI][Medline]

Cuppens, H., Teng, H., Raeymaekers, P., De Boeck, C. and Cassiman, J.J. (1994) CFTR haplotype backgrounds on normal and mutant CFTR genes. Hum. Mol. Genet., 3, 607–614.[Abstract]

Cuppens, H., Lin, W., Jaspers, M., Costes, B., Teng, H., Vankeerberghen, A., Jorissen, M., Droogmans, G., Reynaert, I., Goossens, M. et al. (1998) Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (TG)n locus explains the partial penetrance of the T5 polymorphism as a disease mutation. J. Clin. Invest., 101, 487–489.[Abstract/Free Full Text]

Cystic Fibrosis Genetic Analysis Consortium (1994) Population variation of common cystic fibrosis mutations. Hum. Mut., 4, 167–177.[ISI][Medline]

Dork, T., Dworniczak, B., Aulehla-Scholz, C., Wieczorek, D., Bohm, I., Mayerova, A., Seydewitz, H.H., Nieschlag, E., Meschede, D., Horst, J., Pander, H.J. et al. (1997) Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens. Hum. Genet., 100, 365–377.[ISI][Medline]

Dumur, V., Gervais, R., Rigot, J.M., Lafitte, J.J., Manouvrier, S., Biserte, J., Mazeman, E. and Roussel, P. (1990) Abnormal distribution of CF {Delta}F508 allele in azoospermic men with congenital aplasia of epididymis and vas deferens. Lancet, 336, 512.[ISI][Medline]

Estivill, X., Bancells, C., Ramos, C. and the Biomed CF Mutation Analysis Consortium (1997) Geographic distribution and regional origin of 272 cystic fibrosis mutations in European populations. Hum. Mut., 10, 135–154.[ISI][Medline]

Grody, W.W., Cutting, G.R., Klinger, K.W., Richards, C.S., Watson, M.S. and Desnick, R.J. (2001) Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet. Med., 3, 149–154.[Medline]

Haff, L.A. and Smirnov, I.P. (1997) Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF MS. Genome Res., 7, 378–388.[Abstract/Free Full Text]

Higgins, G.S., Little, D.P. and Koster, H. (1997) Competitive oligonucleotide single base extension combined with mass spectrometric detection for mutation screening. BioTechniques, 23, 710–714.[ISI][Medline]

Hillenkamp, F., Karas, M., Beavis, R.C. and Chait, B.T. (1991) Introduction of MALDI principles and review of early applications. Anal. Chem., 63, 1193A–1202A.[Medline]

Holsclaw, D.S., Perlmutter, A.D., Jockin, H. and Shwachman, H. (1971) Genital abnormalities in male patients with cystic fibrosis. J. Urol., 106, 568–574.[ISI][Medline]

Johnson, M.D. (1998) Genetic risks of intracytoplasmic sperm injection in the treatment of male infertility: recommendations for genetic counseling and screening. Fertil. Steril., 70, 397–411.[ISI][Medline]

Kanavakis, E.,. Tzetis, M.,. Antoniadi, T., Pistofidis, G., Milligos, S. and Kattamis, C. (1998) Cystic fibrosis mutation screening in CBAVD patients and men with obstructive azoospermia or severe oligozoospermia. Mol. Hum. Reprod., 4, 333–337.[Abstract]

Kaplan, E., Shwachman, H., Perlmutter, A.D., Rule, A., Khaw, K.T. and Holsclaw, D.S. (1968) Reproductive failure in males with cystic fibrosis. New Engl. J. Med., 279, 65–69.[ISI][Medline]

Kiesewetter, S., Macek, M. Jr., Davis, C., Curristin, S.M., Chu, C.S., Graham, C., Shrimpton, A.E., Cashman, S.M., Tsui, L.C. and Mickle, J. (1993) A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat. Genet., 5, 274–278.[ISI][Medline]

Lawler, A.M. and Gearhart, J.D. (1998) Genetic counseling for patients who will be undergoing treatment with assisted reproductive technology. Fertil. Steril., 70, 412–413.[ISI][Medline]

Little, D.P., Braun, A., O’Donnell, M. and Koster, H. (1997) Mass spectrometry from miniaturized arrays for full comparative DNA analysis. Nat. Med., 3, 1413–1416.[ISI][Medline]

Mak, V., Jarvi, K.A., Zielenski, J., Durie, P.and Tsui, L.C. (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, T.B. and Jarvi, K.A. (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, T.J., Milunsky, J.M., Munarriz, R., Harris, D.H., Maher, T.A. and Oates, R.D. (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]

Mercier, B., Verlingue, C., Lissens, W., Silber, S.J., Novelli, G., Bonduelle, M., Audrezet, M.P. and Ferec, C. (1995) Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? Analyses of the CFTR gene in 67 patients. Am. J. Hum. Genet., 56, 272–277.[ISI][Medline]

Oates, R.D. and Amos, J.A. (1993) Congenital bilateral absence of the vas deferens and cystic fibrosis. World J. Urol., 11, 82–88.[ISI][Medline]

Osborne, L.R., Lynch, M., Middleton, P.G., Alton, E.W., Geddes, D.M., Pryor, J.P., Hodson, M.E. and Santis, G.K. (1993) Nasal epithelial ion transport and genetic analysis of infertile men with congenital bilateral absence of the vas deferens. Hum. Mol. Genet., 2, 1605–1609.[Abstract]

Rave-Harel, N., Kerem, E., Nissim-Rafinia, M., Madjar, I., Goshen, R., Augarten, A., Rahat, A., Hurwitz., A., Darvasi A. and Kerem, B. (1997) The molecular basis of partial penetrance of splicing mutations in cystic fibrosis. Am. J. Hum. Genet., 60, 87–94.[ISI][Medline]

Taussig, L.M., Lobeck, C.C., di Sant’Agnese, P.A., Ackerman, D.R. and Kattwinkel, J. (1972) Fertility in males with cystic fibrosis. New Engl. J. Med., 287, 586–589.[ISI][Medline]

Welsh, M.J., Ramsey, B.W., Accurso, F. and Cutting, G.R. (2001) In Scriver, C.R., Beaudet, A., Sly, W.S. and Valle, D. (eds), The Metabolic & Molecular Bases of Inherited Disease, 8th edn. McGraw-Hill, New York, pp. 5121–5188.

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

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

Submitted on January 10, 2002; accepted on April 8, 2002.