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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: congenital bilateral absence of the vas deferens/cystic fibrosis/cystic fibrosis transmembrane conductance regulator/gene screening/male infertility
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CBAVD patients are common in our practice among male infertility patients with obstructive azoospermia (Chiang et al., 1986). However, the CF incidence is low in Taiwan and in other Oriental countries: 3.12 per million live births in the Japanese population (Imaizumi, 1995
), and less than one in 90 000 live births among Orientals (Wright and Morton, 1968
). To date, only six cases of CF have been reported in Taiwan (J.Y.Wang et al., 1987
; M.C.Wang et al., 1993
; Wu et al., 2000
).
This study is the first survey to screen for the most common CFTR mutations of CBAVD patients in Taiwan.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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., 1992
). We identified amplified PCR products with electrophoresis of 510 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, 2001), 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+1GT, 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since the discovery of the CFTR gene in 1989, investigators have documented more than 800 mutations (Stuhrmann and Dork, 2000). Dumur et al. (1990)
were the first to report an increased frequency of the major CFTR mutation
F508 in azoospermic CBAVD men. Subsequent publications reported the occurrence of the
F508 gene mutation in Caucasian CBAVD patients to be 12.026.9% in several large series (Table II) (Chillon et al., 1995
; Costes et al., 1995
; Jarvi et al., 1995
; Dork et al., 1997
). Another CF mutation, R117H, was found to occur at a high frequency in CBAVD patients (Gervais et al., 1993
). 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
F508 or R117H. The extremely low frequency of
F508 and R117H mutations in CBAVD patients reported here might be correlated to the rarity of CF occurrence in Taiwan.
|
In the present study, we found a low frequency of major CFTR mutations (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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 14761483.
Chiang HS, Huang SH, Chuang SM and Chiang WH (1986) Analysis of 110 patients with total azoospermia. Formos J Surg 19,152162.
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 332,14751480.
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 3,151156.[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 110,440445.
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 3,285293.[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 81,614619.[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 100,365377.[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 51,251262.[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 328,446447.
Goldstein M and Schlossberg S (1988) Men with congenital absence of the vas deferens often have seminal vesicles. J Urol 140,8586.[ISI][Medline]
Imaizumi Y (1995) Incidence and mortality rates of cystic fibrosis in Japan, 19691992. Am J Med Genet 58,161168.[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 5,274278.[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 5,1013.
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 16,282288.
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 10,17281735.[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 8,215220.[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 32,7183.[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 86,897901.
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 34,314321.[Medline]
Wright SW and Morton NE (1968) Genetic studies on cystic fibrosis in Hawaii. Am J Hum Genet 20,157169.[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 99,564567.[ISI][Medline]
Submitted on October 21, 2002; resubmitted on August 11, 2003; accepted on October 15, 2003.