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Mol. Hum. Reprod. Advance Access originally published online on September 14, 2006
Molecular Human Reproduction 2006 12(11):717-721; doi:10.1093/molehr/gal077
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Two novel missense and one novel nonsense CFTR mutations in Iranian males with congenital bilateral absence of the vas deferens

Ramin Radpour1,5, Hamid Gourabi1, Mohamad A.Sadighi Gilani2, Ahmad Vosough Dizaj2, Mina Rezaee3 and Sepideh Mollamohamadi4

1Department of Reproductive Genetics, 2Department of Male Infertility, Reproductive Biomedicine Research Center of Royan Institute, 3Genetic Research Center of Social Welfare and Rehabilitation Sciences University and 4Department of Stem Cell, Reproductive Biomedicine Research Center of Royan Institute, Tehran, Iran

5 To whom correspondence should be addressed at: Department of Reproductive Genetics, Reproductive Biomedicine Research Center of Royan Institute, PO Box 19395-4644, Tehran, Iran. E-mail: rradpour{at}royaninstitute.org


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 cystic fibrosis (CF) transmembrane conductance regulator (CFTR) mutation. To study the involvement of CFTR mutations in the Iranian population with presumed low CF frequency, we analysed 112 Iranian CBAVD males. Three Iranian CBAVD males with no clinical CF phenotype indicated by a normal karyotype, normal pancreatic function and sweat chloride concentration and no Y chromosome microdeletions were studied for CFTR mutations, IVS8-5T mutations and M470V exon 10 missense polymorphism. The entire coding sequence of each gene was analysed using a combination of the denaturing gradient-gel electrophoresis or by single-strand conformation analysis and direct DNA sequencing. Also, 52 fertile males were tested as controls to rule out polymorphism. This approach allowed us to detect one novel nonsense mutation (K536X) in the nucleotide-binding domain 1 (NBD1) region and two novel missense mutations (Y122H and T338A) in the M2 and M6 regions of CFTR gene in our studied population, which were not reported previously. Also, the conservation of changed nucleotide and amino acid in mutated regions was analysed by aligning with nine different species. K536X nonsense mutation (transversion) was found in the first NBD (NBF1), which plays an important regulatory role in CFTR function. It was, therefore, considered as a severe allele responsible for elevated sweat chloride levels and obstructive azoospermia. Because Y122H and T338A mutations were compound heterozygote with the IVS8-5T, it is difficult to judge the severity of these mutations and their role in the CBAVD phenotype.

Key words: CBAVD/CFTR/male infertility/new mutation


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia and accounts for ~1.5% of male infertility (Jequier et al., 1985Go). CBAVD is caused by mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, which contains 27 exons encompassing ~180kb of DNA on chromosome 7q31.2. The CFTR gene encodes a 1480-amino acid protein that acts as a cyclic adenosine monophosphate (cAMP)-regulated chloride channel in the apical membrane of epithelial cells. CFTR protein is composed of two repeated parts; each contains a transmembrane domain (TMD1 or TMD2); each part includes six transmembrane helices (M1–M6 and M7–M12) and two nucleotide-binding domains (NBDs) (NBD1 and NBD2) (Riordan et al., 1989Go). The two parts are linked by a cytoplasmic hydrophilic regulatory (R) domain, which is required for protein kinase A sensitivity of channel gating (Riordan et al., 1989Go; Cheng et al., 1991Go).

Over 1000 mutations have been described in the Cystic Fibrosis Mutation Consortium (Cystic fibrosis mutation database, 2006Go)—mutations 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, 1995Go). The most common mutations found in isolated CBAVD phenotypes are F508del, R117H and the T5 allele (IVS8-T5) (Chillon et al., 1995Go; Jarvi et al., 1995Go). 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 5, 7 or 9 thymidines (the T5, T7 and T9 alleles, respectively) (Kiesewetter et al., 1993Go). 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 (Chillon et al., 1995Go). The translated product of the CFTR transcript lacking exon 9 is apparently devoid of cAMP-activated chloride conductance. The T5 allele, the most common mutation in CBAVD after F508del, is considered as a mild mutation with an incomplete penetrance (Zielenski et al., 1991Go). In this study, we report the clinical features and mutational data of three CBAVD patients who had novel CFTR mutations.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Samples
Blood samples were collected from 112 patients with azoospermia and CBAVD referred to the Reproductive Biomedicine Research Center of Royan Institute, Iran. These patients were excluded for androgen receptor mutations and Y chromosome microdeletions, and all of them had normal karyotype (data not shown). The diagnosis of CBAVD patients were initially suggested by impalpable scrotal vas on physical examination and trans-abdominal/rectal ultrasonography, subsequently confirmed by Cyto-biochemical characteristics: azoospermia with low semen volume (<1.5ml), decrease of fructose (vesicular marker), carnitine (epididymal marker) concentrations, pH (range of 6–6.7) and normal FSH concentrations according to World Health Organization (1999)Go criteria.

CFTR mutation scanning
DNA samples of three cases were analysed by previously reported methods (Radpour et al., 2006Go). All 27 exons of CFTR were amplified by PCR using the published primer pairs for sequencing (Zielenski et al., 1991Go) and were studied by denaturing gradient-gel electrophoresis (Culard et al., 1994Go) or by single-strand conformation analysis (Liechti-Gallati et al., 1999Go). Missense polymorphism M470V in exon 10 was typed by HphI restriction enzyme analysis. To evaluate the incidence of the 5T allele of intron 8 in CBAVD and normal population, we performed amplification and sequencing of the polypyrimidine tract in front of exon 9 using primers 9i-5 and 9i-3 (Zielenski et al., 1991Go; Chillon et al., 1995Go). Nested PCR was performed to amplify the polypyrimidine sequence with previous reported primers RF9 and RR9 (Radpour et al., 2006Go). The nested PCR conditions were as follows: denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 74°C for 40 s, for 32 cycles. The reaction mixture contained 5µl of PCR buffer, 200µM each of dNTPs, 20pmol of each primer and 1.0U of DNA polymerase (AmpliTaq GoldTM; PE Applied Biosystems, Foster City, CA, USA) in a final volume of 50µl, containing 1µl of the first PCR product. Nested PCR products finally were digested with XmnI enzyme and visualized on 12% non-denaturing polyacrylamide gel.

Sequencing of PCR products was carried out by VBC-Genomics (VBC-Genomics Bioscience Research) using 50ng (2 µl) of PCR product and 4pM (1µl) of non-fluorescent primer (forward and reverse separately), 4µl of BigDye Terminator ready reaction kit (Perkin Elmer) and 3µl of double-distilled water to adjust the volume to 10µl. Sequencing results were compared with the sequence of wild-type CFTR gene published on Cystic Fibrosis Mutation Database (http://www.genet.sickkids.on.ca/cftr/).

Protein Truncation Test analysis
To confirm the novel nonsense mutation found in one of the patients, we performed Protein Truncation Test (PTT) analysis. Double-stranded DNA (1.497kb) containing the partial coding region of CFTR gene was obtained from genomic DNA by PCR using forward primer that contained the T7 promoter sequence, the Kozac consensus sequence, and the ATG-initiation codon (GGAATTC-TAATACGACTCACTATAGGG-AACAG-CCACC-ATG-AG CTCAGCCTTCTTCTTCTC) and the reverse primer (CAGTTCAGTCAAGTTTGCC). The ATG-initiation codon was in frame and upstream of the natural translation initiation. PCR was performed in a 20-µl reaction mixture containing 50ng of genomic DNA, 10 picomoles of each primer, MgCl2-containing reaction buffer, 250µM dNTPs and 1.0U of polymerase (AmpliTaq GoldTM). Samples were amplified in 35 cycles of 30 s each at 94°C for denaturing, 30 s at 57°C for annealing and 60 s at 72°C for extension. An in vitro translation reaction was performed using a commercial system (TNT T7 Quick Coupled Transcription/Translation System; Promega, Madison, WI, USA). A 25-µl reaction mixture containing 300ng of PCR products, 20µl of TNT Quick Master Mix and 1µl of 35S-methionine was incubated at 30°C for 90 min. A 5-µl aliquot of each reaction mixture was loaded into 18% sodium dodecyl sulphate (SDS)–polyacrylamide gel. Electrophoresis was performed at 30mA for 2 h, and the gel was fixed with acetic acid–methanol–water, dried and visualized using the BAS 1000 system (Fujifilm, Tokyo, Japan) or the autoradiography. When band shifts were observed, nucleotide alterations of the corresponding positions were detected by direct sequence analysis.

Phylogenic study of mutated CFTR nucleotides in different species
CFTR sequence alignment was done to find the conservation of novel mutations in exons 4, 7 and 11. In this study, the cDNA of Homo sapiens (Humans, gi | 6995995) was aligned with another eight species: Macaca mulatta (rhesus monkey, gi | 7413642), Bos taurus (cattle, gi | 3134303), Ovis aries (sheep, gi | 5752639), Sus scrofa (pig, gi| 4645225), Canis familiaris (dog, gi | 5487316), Oryctolagus cuniculus (rabbit, gi| 1100984), Mus musculus (house mouse, gi | 1414118) and Rattus norvegicus (Norway rat, gi | 6264689). cDNA sequences were analysed with DNASIS MAX software version 2.6.6 (Hitachi Software Engineering Co.) and aligned with GeneDoc software version 2.6.002.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
After all exons of CFTR were amplified and sequenced in 112 Persian CBAVD males, we found three novel mutations: a nonsense mutation in exon 11 and two missense mutations in exon 4 and exon 7 (Table I).


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  		Table I. The cystic fibrosis transmembrane conductance regulator (CFTR) allele frequency of three congenital bilateral absence of the vas deferens cases with new mutations

 

Case report
Clinical findings of CBAVD cases with new mutations were as follows: sperm count of zero with semen pH range of 6–6.7 and ejaculate volume 0.2–0.7 ml. None had clinical manifestations (pulmonary or gastrointestinal) or family history suggestive of CF. Sweat chloride levels were normal for these patients. Absence of the pelvic part of the vas deferens and the seminal vesicles was detected by trans-abdominal/rectal ultrasonography. The patient with nonsense mutation also had right kidney agenesis (Figure 1).


Figure 1
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  		Figure 1. (A) Trans-abdominal/rectal ultrasonography in Patient no. 103 with one K536X mutation showed absence of the vas deferens and seminal vesicles (white arrows) (left) with right kidney agenesis (right). (B) Trans-abdominal ultrasonography in Patient no. 100 with Y122H and IVS8-5T mutations. (C) Trans-abdominal ultrasonography in Patient no. 49 with T338A and IVS8-5T mutations. Absence of the vas deferens and seminal vesicles (black arrows).

 

Mutation screening
First mutation was a transversion mutation of 1738A->T (numbering of nucleotide position was based on GenBank accession number NM_00049.2) which causes an amino acid change of lysine to ocher stop codon (TAA) at position 537 of the CFTR polypeptide chain in exon 11 (Figure 2). Sequencing from the opposite direction of the product from another PCR was performed to confirm this mutation. The second mutation was a transition mutation of 496T->C in exon 4 (Figure 2) which causes amino acid change of tyrosine to histidine at position 122 of the CFTR polypeptide. The third mutation was a transition mutation of 1144A->G in exon 7 (Figure 2) which causes an amino acid change of threonine to alanine at position 338 of CFTR polypeptide. Two of new mutations were found in compound heterozygote form with IVS8-5T (Table II).


Figure 2
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  		Figure 2. Sequencing results of three founded mutations. (A) A nonsense mutation with transversion nucleotide alteration. (B, C) Two missense mutations with transition nucleotide alteration.

 

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  		Table II. Genotype of cystic fibrosis transmembrane conductance regulator gene mutations in three congenital bilateral absence of the vas deferens cases with new mutations

 

To verify the novel mutations, we sequenced 104 alleles (52 Iranian normal blood donors, age ranged from 21 to 43 years) and found no CFTR mutations.

Mutation nomenclature
Nucleotide numbering is currently based on the CFTR cDNA sequence (GenBank NM_000492 [GenBank] .2) with the A of the ATG translation initiation codon at position 133. Current mutation nomenclature recommendations (http://www.hgvs.org/mutnomen) suggest numbering the A of the ATG translation initiation codon as +1. The numbering of the reported mutations is as follows: c.1738A>T or p.Lys536Stop (K536X), c.496T>C or p.Tyr122His (Y122H) and c.1144A>G or p.Thr338Ala (T338A).

PTT analysis for nonsense mutation
PTT analysis results confirmed that K536X is a nonsense mutation. After in vitro transcription and translation, we could detect two different polypeptide chains: a full-length (wild-type) product in a normal male as control and a defective polypeptide as an abnormal band that confirmed the nonsense mutation in exon 11 (Figure 3).


Figure 3
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  		Figure 3. Mutation screening by Protein Truncation Test (PTT). PTT analysis shows abnormal fragment pattern (M) comparison with the control (Co). Dash represents wild-type, full-length product. Arrow represents abnormal band. Marker and molecular weights of the marker proteins (in kDa) are shown on the right-hand side of the gel.

 

Sequence alignment and phylogenic study
CFTR nucleotide and amino acid sequence alignment of exons 4, 7 and 11 showed that mutated nucleotides (amino acid) c.496 (p.122) in exon 4 and c.1738 (p.536) in exon 11 were not conserved in different species, but nucleotide c.1144 (p.338) was conserved in most of studied groups (Figure 4).


Figure 4
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  		Figure 4. cDNA sequence alignment results of nine different species for exons 4, 7 and 11 find conserved mutated nucleotides in different species.

 

In summary, we report three novel CFTR mutations in Iranian CBAVD patients without manifestations of CF phenotype that were not reported previously. The novel mutations were submitted to the Cystic Fibrosis Mutation Database.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
CFTR gene encodes a large transmembrane protein of 1480-amino acids, an ATP- and cAMP-dependent chloride channel, found in the apical border of epithelial cells lining most exocrine glands. So far, >1000 different mutations have been discovered in CFTR gene (Cystic Fibrosis Mutation Database). Most of them were located in exons 2–5, 7, 9–13, 17b and 19–21 [encoding transmembrane domain (TMD) and nucleotide binding domain (NBD)] (Riordan et al., 1989Go). Mutations in CFTR include deletions, substitutions and insertions that cause frameshifts and missense, nonsense and splicing mutations. We found three novel mutations in exon 4, exon 7 and exon 11, which are hot spots in the CFTR gene.

K536X
An exon 11 nonsense mutation (transversion) was found in the NBF1 domain. This is a common feature of the ATP-binding cassette (ABC) superfamily and indicates a separate role for the two binding domains. The NBFs contain a number of highly conserved motifs predicted to bind and hydrolyse ATP. This 27-year-old man (Patient no. 103) with obstructive azoospermia and normal sweat test had two hypoplastic vas deferens and bilateral aplasia of the epididymal cauda, and he also had right kidney agenesis. This mutation creates a stop codon leading to the deletion of 944 C-terminus amino acids of the CFTR protein including the NBF2, R domain and TMD (7–12). These conserved sites play an important regulatory role in CFTR function (Mennicke et al., 2005Go). Therefore, the K536X considered as a severe allele responsible for obstructive azoospermia.

Y122H
A nucleotide substitution (transition) in exon 4 was compound heterozygote with IVS8-5T in a patient (Patient no. 100) who presented obstructive azoospermia with hypoplastic vas deferens diagnosed on clinical palpation. This 31-year-old man had a normal sweat test. Y122H is located in TMD-M2. TMD domains have been shown to be comprised of typical {alpha}-helical secondary structure. Many of the residues within these regions that form the channel lining residues have a major role in the regulation of pore function. This mutation changed tyrosine with an aromatic side chain to histidine with a basic side chain. Histidine can be uncharged or positively charged depending on its local environment. Indeed, histidine is often found in the active site of proteins or enzymes, where its imidiazol ring can readily switch between these states to catalyse the making and breaking of bonds; so this mutation can change the conformation of normal membrane channel in M2 position of CFTR.

T338A
An exon 7 missense mutation in TMD-M6 was found with 1144A->G in a CBAVD phenotype (Patient no. 49). T338A was compound heterozygote with IVS8-5T. This 29-year-old man had normal sweat test. Amino acid sequence alignment of CFTR exon 11 in different species showed evolutionary conservation of the threonine amino acid residue at this position in most species (Figure 4). By this mutation, threonine with an aliphatic hydroxyl side chain changed to alanine with just an aliphatic side chain. If threonine is conserved, then a change in this amino acid in the transmembrane domain of CFTR may cause defective channel formation in the resulting protein.

Because Y122H and T338A mutations were compound heterozygote with the IVS8-5T, it is difficult to judge the severity of these mutations and their role in the CBAVD phenotype.

These three novel mutations have not been found in 104 chromosomes of normal Iranian population and in a sample of 224 chromosomes from local classical CBAVD patients, and none of them were reported by any other member of the Cystic Fibrosis Genetic Analysis Consortium. Under these circumstances, polymorphisms could be ruled out.

Different criteria are used to suggest that a novel mutation is pathogenic: (i) the mutation changes a highly conserved base or disrupts a conserved base pair, (ii) the mutation is absent in controls, (iii) the mutation has been reported in several pedigrees with similar phenotypes and (iv) there is a correlation between the levels of mutated DNA and severity of symptoms. As discussed in this research, we studied the first two criteria. Our findings of mutations 496T>C and 1144A>G nucleotide substitutions need to be tested for other criteria.

In summary, CF is rare in Iran and little is known about the spectrum of CFTR mutations in the general Iranian population. In this study, we found one novel 1738A>T nonsense mutation (exon 11) and two novel missense mutations 496T>C (exon 4) and 1144A>G (exon 7). To understand more about mutation spectrum and clinical features of CF in Iran, genetic analysis should be used in Iranian patients with typical and atypical CF and CBAVD manifestations.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We are indebted to the CBAVD patients for their co-operation. This research was supported by grants from the Reproductive Biomedicine Research Center of Royan Institute of Iran.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Chillon M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, Romey MC, Ruiz-Romero J, Verlingue C and Claustres M (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]

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Culard JF, 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.[Web of Science][Medline]

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Radpour R, Sadighi Gilani MA, Gourabi H, Vosough Dizaj A and Mollamohamadi S (2006) Molecular analysis of the IVS8-T splice variant 5T and M470V exon 10 missense polymorphism in Iranian males with congenital bilateral absence of vas deferens. Mol Hum Reprod 12,469–473.[Abstract/Free Full Text]

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Submitted on May 22, 2006; resubmitted on June 28, 2006; accepted on August 15, 2006.


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