Cystic fibrosis transmembrane conductance regulator gene screening and clinical correlation in Taiwanese males with congenital bilateral absence of the vas deferens

C.C. Wu1,2, H.M. Hsieh-Li1, Y.M. Lin3 and H.S. Chiang1,4,5

1 Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, 2 Department of Urology, Taipei Medical University Hospital, Taipei, 3 Department of Urology, National Cheng Kung University Hospital, Tainan and 4 College of Medicine, Fu Jen Catholic University, Taipei, Taiwan

5 To whom correspondence should be addressed at: College of Medicine, Fu Jen Catholic University, 510 Chung Cheng Road, Hsinchuang, Taipei Hsien 24205, Taiwan. e-mail: hansun{at}tmu.edu.tw


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: In Taiwan, an area with a very low incidence of cystic fibrosis (CF), we first screened for the most common mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and looked for clinical correlations in 27 patients with clinically diagnosed congenital bilateral absence of the vas deferens (CBAVD). METHODS AND RESULTS: The clinical results showed that none of the 27 patients had CF symptoms. We did not detect any definite renal anomaly ultrasonographically. Mutation analysis was carried out on these 27 cases and 46 normal fertile males as controls. No mutations of {Delta}F508 or R117H were identified in any of the samples analysed. In the screening of IVS8-poly T, five of the 27 CBAVD patients showed the homozygous genotype for 5T/5T, 14 showed the heterozygous genotype for 5T/7T and eight showed the homozygous genotype for 7T/7T. The frequency of 5T alleles was 44.4%, which was significantly higher than in the 46 normal fertile males, for which there was a 5T frequency of 5.4%. CONCLUSIONS: The absence of major mutations of CFTR genes could be related to the much lower CF incidence in Taiwan. Further investigations into differences in the mutation spectrum of other CFTR genes are needed for a better understanding of the development of Taiwanese-Oriental CBAVD.

Key words: congenital bilateral absence of the vas deferens/cystic fibrosis/cystic fibrosis transmembrane conductance regulator/gene screening/male infertility


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Congenital bilateral absence of the vas deference (CBAVD) is the cause of 1.5% of male infertility and up to 25% of obstructive azoospermia (Patrizio et al., 1993Go; Chillon et al., 1995Go). It is a common finding in males with cystic fibrosis (CF) (de la Taille et al., 1998Go), and some, if not all, otherwise healthy men with CBAVD reflect a newly recognized, primarily genital phenotype of CF after genetic screening (Anguiano et al., 1992Go). CF, which is the most common autosomal recessive disorder in Caucasians, has an estimated frequency of one in 2500, and one in 25 are carriers of the disease (Casals et al., 2000Go). The CF defective gene has a 250-kb sequence of DNA located in the long arm of chromosome 7 (Mickle et al., 1995Go). The gene, CF transmembrane conductance regulator (CFTR), regulates the transmembrane transport of (most likely chloride) ions of epithelial cells. A high incidence of mutation of the CFTR gene has recently been reported in males with CBAVD (McCallum et al., 2001Go). Moreover, patients with congenital unilateral absence of the vas deferens (CUAVD), and patients with unilateral renal agenesis (URA) and CBAVD (URA/CBAVD), have been shown to have a lower frequency of CFTR mutations; therefore, the two conditions have different aetiologies of CBAVD (Imaizumi, 1995Go; Dork et al., 1997Go).

CBAVD patients are common in our practice among male infertility patients with obstructive azoospermia (Chiang et al., 1986Go). However, the CF incidence is low in Taiwan and in other Oriental countries: 3.12 per million live births in the Japanese population (Imaizumi, 1995Go), and less than one in 90 000 live births among Orientals (Wright and Morton, 1968Go). To date, only six cases of CF have been reported in Taiwan (J.Y.Wang et al., 1987Go; M.C.Wang et al., 1993Go; Wu et al., 2000Go).

This study is the first survey to screen for the most common CFTR mutations of CBAVD patients in Taiwan.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and clinical evaluations
From January 1, 1994 to December 31, 2002, 27 infertile males were diagnosed as having CBAVD at the Taipei Medical University Hospital and National Cheng Kung University Hospital, Tainan, Taiwan. The diagnosis was based on physical examination of the scrotal content showing the absence of a palpable vas deferens on both sides, with normal-sized testes (long axis >2 cm) and variable epididymal shape. In total, 16 cases were confirmed by surgical exploration for microscopic epididymal sperm aspiration (MESA) (14 cases) or testicular sperm extraction (TESE) (two cases) for subsequent ICSI.

In all patients, a detailed history was taken and a clinical examination performed for the identification of CF symptoms and signs. In addition to routine semen analysis, special examinations for semen pH and fructose were determined to confirm the CBAVD diagnosis. The patient received transrectal ultrasonography to be checked for the morphology and size of the seminal vesicles, prostate and ejaculatory ducts. To detect any renal anomaly, renal ultrasonography was carried out to evaluate the existence and outline of both kidneys. We also carried out hormone assays, chromosome examination and screened for microdeletion of the Y chromosome to detemine any possible testicular azoospermia.

DNA analysis
We isolated genomic DNA from peripheral blood lymphocytes and amplified it with a PCR-based assay to evaluate the {Delta}F508 and R117H mutations. PCR was performed with 10 pmol of each primer in a total volume of 50 µl. Ten microliters of the DNA sample served as a template for amplification through 30 cycles of denaturation (1 min at 95°C), primer annealing (1 min at 55°C) and DNA extension (2 min at 72°C) in an automated PCR cycler (GeneAmp PCR System 9700; Applied Biosystems, Foster City, CA, USA). The ends were ‘polished’ by a final 10-min incubation at 70°C. We performed amplifications with a single set of gene-specific sense and antisense oligonucleotide primers according to a previous study (Ferrie et al., 1992Go). We identified amplified PCR products with electrophoresis of 5–10 µl sample aliquots on a 1.5% agarose gel stained with 0.5 µg/ml ethidium bromide. The sample products were visualized by ultraviolet transillumination, and the gels were photographed.

Cloning and sequencing of the poly T alleles in intron 8 of CFTR
We separated PCR products of the poly T alleles in intron 8 (IVS8-poly T) of the CFTR gene with electrophoresis on a 2% regular agarose gel. For cloning, PCR products were gel-purified with a GFX gel purification kit (Amersham Biosciences, San Francisco, CA, USA). We cloned purified PCR product inserts into a pGEM-T vector (Promega, Madison, WI, USA). DNA minipreparations of individual clones, obtained by alkaline lysis (Sambrook and Russell, 2001Go), were assayed for appropriately sized inserts and characterized by PCR, as confirmed using T7 and SP6 primers. The plasmid DNA of the clones was sequenced and analysed using an ABI PRISMTM Dye-Terminator automated cycle-sequencing system (Applied Biosystems).

Screening by an INNO-LiPA CFTR17+Tn kit
For further confirmation, we used a commercial kit (INNO-LiPA CFTR17+Tn; Innogenetics, Ghent, Belgium) that allowed the detection of 17 mutations (including 394delTT, G85E, E60X, 621+1G->T, R117H, 711+5G->A, 1078delT, R347P, R334W, A455E, 2143delT, 2183AA->G, 2184delA, 2789+5G->A, R1162X, 3659delC and 3849+10kbC->T) associated with IVS8-Tn polymorphisms (5T/7T/9T) in the CFTR gene to analyse our 27 patients and 46 normal, fertile control males.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
We collected 27 subjects who were infertile, with a diagnosis of CBAVD. Clinically, none of them had CF symptoms such as chronic dysfunction of the pancreas or lungs, or an electrolyte imbalance. Table I lists their clinical variables. Of 27 subjects, 17 subjects received renosonographic examination and 19 received transrectal examination.


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Table I. The clinical variables of 27 patients
 
Mutation analysis was performed on all 27 patients and 46 normal, fertile controls. We did not identify any mutations of {Delta}F508 or R117H in any of the samples analysed (Table I). On screening of the poly T alleles in intron 8, five of the 27 CBAVD patients were shown to be homozygous for the 5T/5T genotype, 14 were heterozygous for the 5T/7T genotype and eight were homozygous for the 7T/7T genotype. The frequency of 5T alleles was 44.4%, which was significantly higher than that of the 46 normal fertile males, with a 5T frequency of 5.4% (the numbers of patients shown to have the 5T/5T, 5T/7T and 7T/7T genotypes were one, three and 42, respectively).


    Discussion
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 Materials and methods
 Results
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CBAVD is thought to be the most frequent atypical CF genital form (Stuhrmann and Dork, 2000Go). Of course, the clinical phenotype of some CBAVD males is evidently associated with other symptoms regularly seen in CF (Colin et al., 1996Go). None of our 27 cases was found to have CF symptoms by a detailed questionnaire and related clinical examinations. In the transrectal ultrasound study, as well as in the operative findings of surgical exploration, local anomalies of the seminal vesicles and epididymides of our 27 cases with CBAVD were found to be quite variable (Table I). The findings of our study are similar to those published previously (Goldstein and Schlossberg, 1988Go). However, the results of the routine screening renosonogram in our study revealed no cases of renal anomalies, including URA or an ectopic kidney in the pelvic cavity. This finding does not agree with previous reports, in which the frequency of renal anomalies in CBAVD patients was reported to be 17–21% (de la Taille et al., 1998Go).

Since the discovery of the CFTR gene in 1989, investigators have documented more than 800 mutations (Stuhrmann and Dork, 2000Go). Dumur et al. (1990)Go were the first to report an increased frequency of the major CFTR mutation {Delta}F508 in azoospermic CBAVD men. Subsequent publications reported the occurrence of the {Delta}F508 gene mutation in Caucasian CBAVD patients to be 12.0–26.9% in several large series (Table II) (Chillon et al., 1995Go; Costes et al., 1995Go; Jarvi et al., 1995Go; Dork et al., 1997Go). Another CF mutation, R117H, was found to occur at a high frequency in CBAVD patients (Gervais et al., 1993Go). Molecular analysis of these two most-frequent CFTR mutations involved in CBAVD was performed in our 27 cases, and none of them was found to carry a single allele mutation of either {Delta}F508 or R117H. The extremely low frequency of {Delta}F508 and R117H mutations in CBAVD patients reported here might be correlated to the rarity of CF occurrence in Taiwan.


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Table II. Series review of {Delta}F508, R117H and IVS8-poly T frequencies in patients with CBAVD
 
Besides examing major CFTR genes, we also analysed our 27 patients for the presence of IVS8-5T. This splice variant, which is localized on the branch acceptor site of exon 9, and alternative variants, consist of 7T or 9T nucleotides. An intron 8 splice variant, 5T (IVS8-5T), is frequently observed in CBAVD patients (Kiesewetter et al., 1993Go). IVS8-5T itself may be the most common atypical CF mutation worldwide (Chillon et al., 1995Go). It is present on ~5% of CFTR alleles in normal Caucasian populations, and on 12.27% of CFTR alleles from Caucasians with CBAVD (Chu et al., 1993Go). In a recent report on the molecular analysis of CFTR dysfunction in CBAVD males from Egypt (Lissens et al., 1999Go), a country with a presumed low CF frequency, only one {Delta}F508 carrier was identified, but an exceptional number, as high as 14 out of 32 alleles (43.7%), were also found to have the IVS8-5T variant. This is 8- to 9-fold higher than the proportion in the general Caucasian population. The results of our study also showed a higher frequency of the IVS8-5T variant in CBAVD patients from an Oriental country with an even lower CF incidence. In an analysis of 27 Taiwanese CBAVD patients, none of whom had any renal anomaly, the molecular analysis of 18 CFTR gene mutations revealed no {Delta}F508 carriers; however, 24 of 54 alleles (44.4%) were identified as having the IVS8-5T variant. Although there is no population study yet available, in our 46 normal, fertile Taiwanese control males, the frequency of the IVS8-5T variant was five of 92 alleles (5.4%) (Table II). Therefore, a significantly higher frequency of IVS8-5T in Taiwanese males with CBAVD, compared with normal controls, could be postulated from our study.

In the present study, we found a low frequency of major CFTR mutations ({Delta}F508 and R117H) in Taiwanese CBAVD patients. This finding may be due to the rare occurrence of CF in Taiwan. Our data, which showed a high frequency of IVS8-5T in Taiwanese CBAVD patients (44.4%), are similar to those reported for CBAVD patients in Egypt (43.7%), which is also a non-Caucasian country with a low incidence of CF. Therefore, more comprehensive screening of the CFTR gene mutation may provide further evidence to explore the racial differences in this genetic disorder.


    Acknowledgements
 
The authors thank Ping-Ching Hsu, Patrick Lee and Jen-Feng Liang for their expert technical assistance. This work was supported by grants from Taipei Medical University (TMC89-Y05-A111 and TMU90-Y05-A143).


    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. JAMA 267,1794–1797.[Abstract]

Casals T, Bassas L, Egozcue S, Ramos MD, Gimenez J, Segura A, Garcia F, Carrera M, Larriba S, Sarquella J et al. (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]

Chiang HS, Huang SH, Chuang SM and Chiang WH (1986) Analysis of 110 patients with total azoospermia. Formos J Surg 19,152–162.

Chillon M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, Romey MC, 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. N Engl J Med 332,1475–1480.[Abstract/Free Full Text]

Chu CS, 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]

Colin AA, Sawyer SM, Mickle JE, Oates RD, Milunsky A and Amos JA (1996) Pulmonary function and clinical observations in men with congenital bilateral absence of the vas deferens. Chest 110,440–445.[Abstract/Free Full Text]

Costes B, Girodon E, Ghanem N, Flori E, Jardin A, Soufir JC 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]

delaTaille A, Rigot JM, Mahe P, Vankemmel O, Gervais R, Dumur V, Lemaitre L and Mazeman E (1998) Correlation between genito-urinary anomalies, semen analysis and CFTR genotype in patients with congenital bilateral absence of the vas deferens. Br J Urol 81,614–619.[ISI][Medline]

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

Dumur V, Lafitte JJ, Gervais R, Debaecker D, Kesteloot M, Lalau G and Roussel P (1990) Abnormal distribution of cystic fibrosis delta F508 allele in adults with chronic bronchial hypersecretion. Lancet 335,1340.

Ferrie RM, Schwarz MJ, Robertson NH, Vaudin S, Super M, Malone G and Little S (1992) Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene. Am J Hum Genet 51,251–262.[ISI][Medline]

Gervais R, Dumur V, Rigot JM, Lafitte JJ, 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]

Goldstein M and Schlossberg S (1988) Men with congenital absence of the vas deferens often have seminal vesicles. J Urol 140,85–86.[ISI][Medline]

Imaizumi Y (1995) Incidence and mortality rates of cystic fibrosis in Japan, 1969–1992. Am J Med Genet 58,161–168.[ISI][Medline]

Jarvi K, Zielenski J, Wilschanski M, Durie P, Buckspan M, Tullis E, Markiewicz D and Tsui LC (1995) Cystic fibrosis transmembrane conductance regulator and obstructive azoospermia. Lancet 345,1578.[CrossRef][ISI][Medline]

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

McCallum TJ, Milunsky JM, Munarriz R, Carson R, Sadeghi-Nejad H and Oates R (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]

Mickle J, Milunsky A, Amos JA and Oates RD (1995) Congenital unilateral absence of the vas deferens: a heterogeneous disorder with two distinct subpopulations based upon aetiology and mutational status of the cystic fibrosis gene. Hum Reprod 10,1728–1735.[Abstract]

Patrizio P, Asch RH, Handelin B and Silber SJ (1993) Aetiology of congenital absence of vas deferens: genetic study of three generations. Hum Reprod 8,215–220.[Abstract]

Sambrook J and Russell DW (2001) Molecular Cloning: A Laboratory Manual. 3rd edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

Stuhrmann M and Dork T (2000) CFTR gene mutations and male infertility. Andrologia 32,71–83.[CrossRef][ISI][Medline]

Wang JY, Hsieh KH, Chang MH, Chen SH and Lue HC (1987) Cystic fibrosis: report of a Chinese case with suggestive family history. J Formos Med Assoc 86,897–901.

Wang MC, Shu SG, Chang SM, Ho WL and Chi CS (1993) Cystic fibrosis in two Chinese infants in Taiwan. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi 34,314–321.[Medline]

Wright SW and Morton NE (1968) Genetic studies on cystic fibrosis in Hawaii. Am J Hum Genet 20,157–169.[ISI][Medline]

Wu CL, Shu SG, Zielenski J, Chiang CD and Tsui LC (2000) Novel cystic fibrosis mutation (2215insG) in two adolescent Taiwanese siblings. J Formos Med Assoc 99,564–567.[ISI][Medline]

Submitted on October 21, 2002; resubmitted on August 11, 2003; accepted on October 15, 2003.