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Ramin Radpour studied Human Genetics in the University of Social Welfare & Rehabilitation Sciences, Tehran, Iran (2004). He took up a post in the department of Reproductive Genetics, Royan Institute, Tehran where he contributed to a number of projects, resulting in several publications on infertility and genetics. In 2007 he joined the Laboratory for Prenatal Medicine and Gynecologic Oncology, University Hospital of Basel, Switzerland to undertake a PhD. His current research interests are mainly focused on the molecular mechanisms of male and female infertility, epigenetics of gynaecological cancers and non-invasive prenatal medicine.

Dr Ramin Radpour

Ramin Radpour1,2,5, Elahe Taherzadeh-Fard3, Hamid Gourabi2, Sahar Aslani4, Ahmad Vosough Dizaj2, Ali Aslani41Laboratory for Prenatal Medicine and Gynecologic Oncology, Department of Medicine, University of Basel, Switzerland; 2Department of Reproductive Genetics, Reproductive Biomedicine Research Centre of Royan Institute, Tehran, Iran; 3Department of Human Genetics, Ruhr-University, Bochum, Germany; 4Department of Urology, Biomedical Research Centre of Medical Sciences University, Tehran, Iran5Correspondence: e-mail: radpourr@uhbs.ch

Abstract

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia, and caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A novel TG13T2 allele was identified in a CBAVD patient with no clinical cystic fibrosis phenotype, normal pancreatic function, normal sweat chloride concentrations and no Y chromosome microdeletions. This case was studied for CFTR mutations, IVS8-poly(T), and M470V exon 10 mis-sense polymorphism. One novel allele was detected in the (TG)m(T)n loci that had not been reported previously. This patient carried a [TG11T9; R117H; p.Met470Val] haplotype on the other chromosome. Since the TG13T2 allele was a compound heterozygote with R117H mutation, it was difficult to judge the severity of this allele. To better understand the complex regulation of exon 9 splicing, the levels of correctly spliced CFTR transcripts in CFTR-expressing epithelial cells derived from vas deferens and epididymis were analysed. These data emphasize the role of the T2 allele in CBAVD, and identify the T2 allele as a severe CBAVD disease-causing mutation. According to the data, the longer (TG)m polymorphic tract increases the proportion of transcripts with exon 9 deletion (9–), but only when activated by the short T allele.

Keywords: CBAVD, CFTR, male infertility, obstructive azoospermia, vas deferens

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia. Nearly 75% of men with CBAVD have at least one detectable common CFTR mutation (Claustres et al., 2000; Radpour et al., 2008a). Over 1500 mutations have been described in the Cystic Fibrosis Mutation Database (2007). These mutations are clustered in six different classes, including defective CFTR biosynthesis, defective protein processing, alteration in CFTR regulation, disruption of the pore activity, alteration of CFTR localization and genesis of unstable CFTR (Zielenski and Tsui, 1995; Radpour et al., 2008b).

The poly(T) tract Tn, near a poly-TG loci [(TG)m], in the branch/acceptor splicing site of intron 8, exists in three variants with five, seven or nine thymidines (the T5, T7 and T9 alleles respectively)

(Kiesewetter et al., 1993). The T7 and T9 alleles generate a predominantly normal transcript, whereas the T5 variant generates two transcripts, one normal with exon 9 intact, and the other with an in-frame deletion of exon 9 (Chu et al., 1993). The T5 allele, the most common mutation in CBAVD after p.F508del, is considered a mild mutation with incomplete penetrance (Zielenski et al., 1991).

The degree of exon skipping in the CFTR gene is inversely related to the length of the Tn tract. The transcripts derived from genes that carry five thymidines (T5) at this locus have the highest levels of exon 9 skipping, whereas those with seven or nine thymidines (T7 and T9 respectively) exhibit progressively lower levels of skipping (Chu et al., 1993). Besides the Tn

Case report

Novel cause of hereditary obstructive azoospermia: a T2 allele in the CFTR gene

Introduction

Case report - CBAVD and T2 allele in the CFTR gene R Radpour et al.

locus, additional cis-acting elements have been described that influence CFTR exon 9 alternative splicing (Niksic et al., 1999; Pagani et al., 2000; Hefferon et al., 2002). In particular, another polymorphism consisting of a series of 10–13 (TG)m, is located upstream of the T(n) repeat. It has been reported that the T5 CFTR genes carried by most CBAVD patients have a high number of (TG)m repeats, whereas T5 genes carried by healthy fathers of individuals with cystic fibrosis (carrying a cystic fibrosis mutation on the other gene) have a lower number of (TG)m repeats (Cuppens et al., 1998; Groman et al., 2004). These observations suggest that the (TG)m tract can further modulate exon 9 skipping, and may account for the partial penetrance of the T5 allele (Radpour et al., 2008b).

The present study reports the clinical features and mutational data of one CBAVD patient, who carried a novel T2 allele in (TG)m(T)n loci of the CFTR gene, as a new cause of hereditary obstructive azoospermia.

Materials and methods

Samples

The study was approved by the local institutional review board. A blood sample was collected from one patient with azoospermia and CBAVD without any renal or urinary tract malformations. A complete medical history and a physical examination were performed. This patient was excluded for androgen receptor mutations and Y chromosome microdeletions, and had normal karyotype (data not shown). The diagnosis of CBAVD was initially suggested by impalpable scrotal vas on physical examination and trans-abdominal/rectal ultrasonography. This diagnosis was confirmed by cyto-biochemical characteristics: azoospermia with low semen volume (<1.5 ml), decrease in fructose (vesicular marker), carnitine (epididymal marker) concentrations, pH 6.7 and normal FSH concentrations evaluated according to World Health Organization (1999) criteria. He had no pulmonary or gastrointestinal manifestations of cystic fibrosis, and sweat chloride values of 82 and 86 mmol/l. The genomic DNA was prepared from peripheral blood lymphocytes using the Roche high pure polymerase chain reaction (PCR) template preparation kit (Roche Diagnostics GmbH, Germany).

CFTR mutation scanning and genomic analysis of (TG)m(T)n loci

All 27 exons of the CFTR gene were analysed by previously reported methods (Radpour et al., 2006, 2007). Mis-sense polymorphism M470V in exon 10 was typed by HphI restriction enzyme analysis. To evaluate the incidence of the poly-T allele of intron 8, amplification and sequencing of the polypyrimidine tract in front of exon 9 was performed according to a previously published method (Radpour et al., 2007) (Figure 1). Sequencing of PCR products was carried out by VBC Genomics (VBC Genomics Bioscience Research, Austria), using 50 ng (2 µl) of PCR product and 4 pmol/l (1 µl) of non-fluorescent primer (forward and reverse separately), 4 µl of BigDye Terminator ready reaction kit (Perkin Elmer, USA) and 3 µl of double-distilled water to adjust the volume to 10 µl. Sequencing results were compared with the sequence of the wild-type CFTR gene published in the Cystic Fibrosis Mutation Database (2007).

Analysis of the rate of exon-9 skipping in the patient carrying the T2 allele

To check the rate of exon 9 skipping in CFTR mRNA, total RNA was isolated from cultured CFTR-expressing epithelial cells derived from vas deferens, and epididymis after biopsy, using RNA extraction PrepMate kit (Bioneer, Korea). The concentration and purity of the RNA samples were determined by measuring the absorbance in a spectrophotometer at 260 nm (A260) and 280 nm (A280). The calculated A260/A280 ratio of pure RNA was between 1.8 and 2.0. The integrity of the RNA samples was further confirmed by electrophoresis on 1% agarose gels. cDNA was synthesized by First Strand cDNA Synthesis kit (Fermentas, USA) using poly-T primer. Exon 9 amplification was performed using specific primers for exon 9 (Radpour et al., 2007). The reaction mixtures were preheated at 95°C for 10 min before thermal cycling. The PCR program was 35 cycles of 30 s at 94°C, 30 s at 55°C and 45 s at 72°C. The amount of RNA in each sample was standardized by PCR amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH was amplified for 25 cycles at an annealing temperature of 55°C using primers published previously (Zielenski et al., 1991; Radpour et al., 2006). PCR products were resolved on 2% agarose gel containing 10 µg/ml ethidium bromide.

Results

Clinical findings of the CBAVD case with a novel mutation were as follows: sperm count of zero with semen pH 6.7, and ejaculate volume 0.6 ml. This patient did not have clinical manifestations (pulmonary or gastrointestinal), or a family history suggestive of CF. Sweat chloride concentration was normal. Absence of the pelvic part of the vas deferens and the seminal vesicles was detected by transabdominal/rectal ultrasonography (Figure 2).

Mutation screening

After complete analysis of the CFTR gene, The T2 allele at the c.1209–6Tn (IVS8–6Tn) polymorphic locus in intron 8 of the CFTR gene was identified in trans of a T9 allele in association with TG13 and TG11 alleles ([TG11T9] + [TG13T2]). Whole gene screening for base substitutions and exon rearrangements identified the R117H mutation and M470V polymorphism. The T2 allele was found to be associated with haplotype [TG13T2; p.Met470], while haplotype [TG11T9; p.Val470; R117H] was present on the other allele (Table 1).

Analysis of rate of exon-9 skipping in the patient carrying the T2 allele

To further explore the role of the T2 allele in the alternative splicing of CFTR exon 9 and its implication in the pathophysiology of CBAVD, the rate of exon 9 skipping in CFTR mRNA was assessed. In the mRNA extracted from epididymal cells and vas deferen cells, the exon 9 sequence could not be detected, which means that the IVS8–2T allele could induce the extent of exon 9 skipping, especially when associated with long IVS8-(TG)13 (Figure 3).328

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Case report - CBAVD and T2 allele in the CFTR gene R Radpour et al.

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Figure 1. Poly-T Alleles in intron 8 of the CFTR gene. A new IVS8–2T allele which was heterozygous with 9T allele in other chromosomes was compated with homozygote form of IVS8–5T, 7T and 9T (bp = base pairs).

Figure 2. Trans-abdominal ultrasonography in patient with R117H and IVS8–2T mutations. Absence of the vas deferens and seminal vesicles are indicated by black arrows.

Table 1. Genotype of CFTR gene mutations in one CBAVD case with novel mutation. Mutation type IVS8–(TG)mTn M470V

R117H TG11T9/TG13T2 M/V

Mutation genotypes were designated following the recommended nomenclature (Beaudet and Tsui, 1993). The IVS8-(TG)mTn haplotypes were named by the number of upstream TG, followed by the number of consecutive thymidines (Costes et al., 1995; Dork et al., 1997). The M470V alleles were designated M for nucleotide A at position 1540 and V for nucleotide G at that position.

Figure 3. DNA variants in intron 8 of the Iranian CFTR gene: the alleles at (TG)m modulate the partial penetrance of the T5 allele. Sequences at the 5' and 3' splice site in intron 8 (IVS8) share most of the canonical features of splicing site, including the 5'-GT donor site, 3'-AG acceptor site and pyrimidine-rich region at the splice acceptor site. (A) Seven different haplotypes of (TG)m(T)n have been found in the Iranian population. TG12T7 was the most common combination (Radpour et al., 2007). (B) The novel (TG)13(T)2 allele as a newly reported cause of obstructive azoospermia in CBAVD patients.

Mutation nomenclature

Nucleotide numbering was done based on the CFTR cDNA sequence (GenBank NM_000492.2) with the A of the ATG translation initiation codon at position 133. Current mutation nomenclature recommendations (den Dunnen and Antonarakis, 2000) suggest numbering the A of the ATG translation initiation codon as +1. The numbering of the reported novel allele is as follows: c.1209–6Tn (recommended nomenclature) for the polymorphic locus in intron 8, and IVS8–6T(n) or c.1342–6Tn (traditional nomenclature).

Case report - CBAVD and T2 allele in the CFTR gene R Radpour et al.

Bioinformatics analysis of exon 9 skipping in CFTR protein

To better understand the pathogenicity of exon 9 skipping by T5 or T2 alleles in CFTR protein, a complete bioinformatics analysis of CFTR protein was performed, including exons

8–10, and CFTR protein with deletion of exon 9 (Figure 4). Data showed that after deletion of exon 9, hydrophobicity, antigenicity and surface probability of CFTR would decrease, but protein charge in isoelectric pH would greatly increase. Also by this deletion, the protein would lose three important beta-sheet domains. This means that exon 9 has a critical role

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Figure 4. Bioinformatics analysis of exon 9 skipping in CFTR protein. (A) Partial sequence of CFTR protein contains exons 8, 9 and 10. Black horizontal arrows indicate beta-sheet and grey arrows indicate alpha-helix domains. (B−F) compared hydrophobicity, antigenicity, protein charge, surface probability and secondary structure domains in normal CFTR protein (+exon 9) and protein with exon 9 skipping (−exon 9). Black arrows indicate important differences between normal and defective protein.

in protein folding and conformation. As a result of this in-frame deletion, the CFTR protein would lose the ability to create transmembrane channels. The pathogenicity of T5 or T2 alleles that exacerbate exon 9 skipping is related to these changes.

Discussion

The studied CBAVD patient had no urinary tract malformations or renal agenesis. This is probably explained by the fact that

defects in the genital ducts, due to CFTR dysfunction, occur

after the splitting of the Wolffian duct into its reproductive and ureteral parts at 7 weeks of gestation.

Since the TG13T2 allele was a compound heterozygote with R117H, it was difficult to judge the severity of this allele and its role in the CBAVD phenotype. To better understand the complex regulation of exon 9 splicing, the levels of correctly spliced CFTR transcripts in cultured CFTR-expressing epididymal cells and vas deferens cells were analysed. These data emphasize the role of the T2 allele in CBAVD, and identify the T2 allele as a severe CBAVD disease-causing mutation. This novel allele has not been found in other chromosomes of the normal Iranian population and in a sample of 224 chromosomes from local classical CBAVD patients (Radpour et al., 2007). It has also not been reported in the Cystic Fibrosis Database (2007).

Previously one group in France were able to detect a novel T3 allele (TG12T3) in a CBAVD patient who carried a [TG11T7; p.Phe508Cys; p.Met470Val] haplotype on the other chromosome (Disset et al., 2005). They also showed that the T3 allele may facilitate exon 9 skipping in CBAVD patients. Comparing previous studies about IVS8–5T and the data from T3 and T2 alleles supports the critical role of IVS8-(T)n in the accuracy of CFTR mRNA splicing and exon 9 skipping. Recently, after performing a comprehensive study in Iranian CBAVD patients, it was shown that the TG repeat number is a predictor of benign versus pathogenic T5 alleles (Radpour et al., 2007). Using a minigene assay designed to include all the cis-acting elements reported so far in the vicinity of exon 9 and variable combinations at the (TG)m(T)n polymorphic loci, it was determined that the residual amount of normally spliced CFTR transcripts in the presence of a short Tn repeat does not depend on the general splicing efficiency (Radpour et al., 2007).

Previous studies have suggested that when IVS8–5T is combined with additional activity-reducing variations, such as the higher number of IVS8 (TG)m, it shows higher disease penetrance (Cuppens et al., 1998). The longer (TG)m tract increases the proportion of exon 9– transcripts, but only when activated by the short Tn allele, with TG13T5 > TG12T5. Finding the new TG13T2 completely confirmed this hypothesis (Figure 3). This new allele shows that CFTR transcripts lacking exon 9 lose 21% of the amino terminal end of the NBF1 region, and produce a protein that is misfolded and non-functional (Figure 4).

In summary, cystic fibrosis is rare in Iran and little is known about the spectrum of CFTR mutations in the general Iranian population. This study has found a novel TG13T2 allele in the (TG)m(T)n loci that is considered as a severe CBAVD disease-causing mutation.

Acknowledgements

The authors are indebted to the patient for his co-operation. They thank Dr Mohamad Ali Sedighi Gilani and Dr Solmaz Samandar for their excellent assistance and support, Dr Monika Girodon for her help and Regan Geissmann for proofreading the text.

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Case report - CBAVD and T2 allele in the CFTR gene R Radpour et al.

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Case report - CBAVD and T2 allele in the CFTR gene R Radpour et al.

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Declaration: The authors report no financial or commercial conflicts of interest.

Received 31 March 2008; refereed 14 July 2008; accepted 20 October 2008.

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