1 Graduate Institute of Medical Sciences, Taipei Medical University, 2 Department of Urology, Taipei Medical University Hospital, 4 College of Medicine, Fu Jen Catholic University, Taipei, Taiwan and 3 Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC, USA 5 These authors contributed equally to this work 6 Present address: Akdeniz University, School of Medicine, Department of Medical Biology-Genetics, Antalya, Turkey
7 To whom correspondence should be addressed at: College of Medicine, Fu Jen Catholic University, 510, Chung-Cheng Road, Hsin-Chuang, Taipei Hsien, 24205 Taiwan. Email: hansun{at}tmu.edu.tw
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Abstract |
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Key words: CBAVD/CFTR/IVS8-5T/male infertility/Taiwanese CF
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Introduction |
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Although CF is one of the most common autosomal recessive diseases in Caucasians, it is very rare in Asian populations (Welsh et al., 2001; Wong et al., 2003
). Little is known about the mutation spectrum and frequency of CFTR gene mutations in Asian populations. A recent survey on a small number of the Asian CFTR mutations revealed mostly private mutations that have never been reported in Caucasian CF patients (Wong et al., 2003
). Screening of the CFTR gene for 17 common Caucasian mutations, including the polymorphic polythymidine tract in intron 8 (IVS8 poly T), detected only the presence of the IVS8-5T mutation in Taiwanese CBAVD patients (Wu et al., 2004
). The frequency of the IVS8-5T allele was found to be significantly higher in CBAVD patients than in normal controls (Wu et al., 2004
).
The IVS8-5T of the CFTR gene is found in 510% of individuals in the general population (Groman et al., 2004). When found in trans with a severe CFTR mutation, IVS8-5T can result in male infertility, non-classic CF or a normal phenotype (Chillon et al., 1995
; Zielenski et al., 1995
). The incomplete penetrance is due to the number of TG repeats adjacent to 5T (Groman et al., 2004
). A recent study found that those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit a disease phenotype (Groman et al., 2004
). In order to understand the molecular aetiology of CF and CBAVD and to determine the CFTR gene mutations in the Taiwanese population, we analysed the whole CFTR gene in 36 infertile males with CBAVD using the newly developed temporal temperature gradient gel electrophoresis (TTGE) (Wong et al., 2001
, 2003
; Wong and Alper, 2004
).
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Materials and methods |
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We performed clinical examination for CF symptoms on every patient. However, no classic CF symptoms were identified in any of the patients. Every patient provided detailed clinical and family history. In addition to routine semen analysis, special examination for semen pH and fructose content was carried out to confirm CBAVD diagnosis. Eighteen patients received transrectal ultrasonography for the evaluation of morphology and size of the seminal vesicles, prostate and ejaculatory ducts. To detect any renal anomaly, we carried out renal ultrasonography to assess the existence and outline of both kidneys, and also hormonal assays and chromosomal analyses to rule out testicular azoospermia. Table I lists their clinical variables.
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TTGE (temporal temperature gradient gel electrophoresis) mutational analysis
Patients' DNA was analysed by TTGE for unknown mutations in the exons and intronexon junctions of the entire CFTR gene (Wong et al., 2001, 2003
; Wong and Alper, 2004
). The primer sequences used for the amplification of the 27 coding exons and their flanking intronexon junctions, as well as PCR and TTGE conditions have been described in detail previously (Wong et al., 2001
). The size of the PCR product varies from 260 bp for exon 23 to 862 bp for exon 13 (Wong et al., 2001
). Briefly, 5 µl of denatured and reannealed PCR products were loaded onto a polyacrylamide gel containing 6 mol/l urea. The electrophoresis was carried out at 130 V at constant temperature increments of
12°C/h over a range of temperatures suitable for each exon (Wong et al., 2001
). The temperature range of TTGE for each PCR fragment was determined empirically with the aid of computer simulation (MacMelt, Bio-Rad Laboratories) (Wong et al., 2001
; Wong and Alper, 2004
). The gels were stained in 2 µg/ml ethidium bromide for 5 min and imaged with a digital charged-coupled device (CCD) gel documentation system. TTGE analysis reveals homozygous change as a bandshift and heterozygous change as multiple bands (Wong et al., 2001
; Wong and Alper, 2004
).
The DNA fragments that showed abnormal banding patterns on TTGE analysis were sequenced using the Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) and analysed on an ABI Prism 377 DNA Sequencer (Applied Biosystems) according to the manufacturer's protocols. The sequencing data were analysed using ABI DNA sequencing analysis software (version 3.0) and compared with the GenBank sequence by using Mac VectorTM (version 7.0). The mRNA (GenBank NM_000492.2) sequence of the CFTR gene is used as the reference sequence. DNA mutation numbering is based on the cDNA sequence that uses the A of the ATG translation initiation start site as nucleotide +1. The traditional nomenclature is also included using nucleotide position 133 as the translational start site. Mutation nomenclature follows journal and Human Genome Variation Society (HGVS) guidelines. Exon 9 and its 5' upstream intron 8 region that contains the polymorphic polythymidine tract and polymorphic TG dinucleotide repeats were sequenced to determine the length of IVS8 poly(T) and TG repeats.
Genotyping of M470V polymorphism by allele-specific oligonucleotide (ASO) dot blot hybridization
The p.M470V polymorphism was assayed using PCR/ASO dot blot analysis. Briefly, exon 10 of the CFTR gene containing the p.M470V (c.1408A>G) polymorphic site was amplified with forward primer 5'gcagagtacctgaaacagga3' and reverse primer 5'cattcacagtagcttaccca3' located in the flanking intron regions of exon 10. A 2 µl aliquot of PCR products was dotted onto positively charged nylon zeta membrane. Two blots were prepared, one for normal probe M470 and the other one for mutant probe 470V (DeMarchi et al., 1994; Wong and Senadheera, 1997
). Hybridization and wash were carried out according to published procedures (DeMarchi et al., 1994
; Wong and Senadheera, 1997
).
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Results |
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5T allele and its adjacent polymorphic TG repeats
Since the disease penetrance of 5T is affected by its adjacent polymorphic TG repeats, the number of TG dinucleotide repeats in intron 8 of each patient was determined by direct sequencing. It was found that in all patients carrying 5T, the mutant allele was associated with either 12 or 13 TG repeats (Table I). In 51 7T alleles, 24 were associated with 11TG, 26 were with 12TG and one 7T allele was linked with 13TG (Table II). In 23 normal fertile males, only one 5T allele was identified, which was linked with 11TG; of 45 7T alleles from these normal fertile males, 19 were associated with 11TG, 25 with 12TG and one with 13TG (data not shown). None of our patients or the normal fertile males had a 9T allele.
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Discussion |
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Previous studies have suggested strong linkage disequilibrium between 5T and p.M470V in Caucasian CBAVD patients but not in the normal population (de Meeus et al., 1998b). Although our sample size was small, the data in our study showed that M470 alleles were associated with 7T (M/V=24/20) whereas 470V alleles were associated with 5T (M/V=6/8) (Table II). This could be due to an ethnicity difference that includes the absence of the 9T allele and
F508 in the Taiwanese population. Furthermore, in Caucasian populations, 5T11TG was always found with M470,
95% of 5T12TG was found with V470, and 5T13TG was found to occur only with M470 (Groman et al., 2004
). Our data also showed that TG repeat number, rather than M470V status, is the major determinant of penetrance for 5T. However, further studies of a larger sample size will be required in order to confirm this observation.
In conclusion, our studies of the CFTR mutations in Taiwanese CBAVD patients showed that the number of mutations was limited, that the most common mutation IVS8-5T accounted for 81% of the mutations identified, and that most mutant alleles (64%) remained unknown. Those observations are consistent with the finding that the CF incidence is rare in the Taiwanese population. Based on the finding of this study, we suggest that either the mutations in the CFTR gene are yet to be identified, or other novel pathological mechanisms are responsible for Taiwanese CBAVD. Despite the low detection rate, the information is important to facilitate our understanding of CF pathogenesis in the Taiwanese population. Comprehensive analysis of the CFTR gene in its entirety for both the infertile male and his partner is essential for those who are considered for IVF (Danziger et al., 2004; Wong et al., 2004
).
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Acknowledgements |
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References |
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Submitted on January 3, 2005; resubmitted on April 8, 2005; accepted on April 19, 2005.
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