Molecular Screening of the CFTR Gene in Mexican Patients with Congenital Absence of the Vas Deferens

Abstract

Background: In several populations CFTR mutations, as well as IVS8-Tn CFTR polymorphism, have been associated with congenital bilateral absence of the vas deferens (CBAVD) and idiopathic obstructive azoospermia diseases. However, the involvement of these mutations in infertility of Mexican males has not been elucidated. Aims: We investigated whether CFTR mutations and IVS8-Tn(TG)m polymorphisms are associated with infertility in azoospermic Mexican patients. Methods: Sixteen CBAVD and 33 idiopathic azoospermic cases were included. The CFTR gene was sequenced in all CBAVD cases. In the idiopathic azoospermic patients, the p.F508del, p.G542X, p.N1303K, p.S549N, p.I507del, and p.R117H mutations and those detected in our CBAVD cases were screened. Results: The p.F508del, p.G85E, p.D1152H, and p.W1089X mutations were found in 3 CBAVD patients (18.8%). None of the 9 CFTR mutations screened for in idiopathic azoospermic were found; however, we documented a high frequency of the Gln1463Gln polymorphism in comparison with healthy controls (20% vs 6%; p=0.0029). Conclusions: These data showed that the CFTR mutations but not the IVS8-Tn polymorphism are involved in CBAVD etiology in a Mexican population. Nevertheless, other screening strategies should be used to rule out the implication of CFTR mutations in idiopathic azoospermic disease.

Introduction

More than 1850 mutations (Cystic Fibrosis Mutation Database; www.genet.sickkids.on.ca/cftr/) causing cystic fibrosis (CF) have been characterized in the CF transmembrane conductance regulator gene (CFTR, OMIM 602421). CFTR mutations can result in severe or mild CF, an atypical phenotype, or a nonsymptomatic form (Mak et al., 1999; Sturhmann and Dörk, 2000; Grangeia et al., 2004; Groman et al., 2004). Patients with atypical and nonsymptomatic phenotypes bear a mild mutation in at least 1 allele, which leads to some residual CFTR function (Chillon et al., 1995; Zielenski et al., 1995; Dörk et al., 1997; Kanavaris et al., 1998; Lissens et al., 1999; Wu et al., 2004; Chiang et al., 2009). In 1990, Dumur and colleagues observed a high frequency of heterozygotes for the p.F508del mutation in patients with congenital absence of the vas deferens (CBAVD; OMIM 277180). Subsequently, many studies confirmed an elevated frequency of CFTR mutations in males with CBAVD and other forms of male infertility due to obstructive azoospermia. p.F508del and p.R117H mutations, as well as the 5T variant of the IVS8-Tn (5, 7 and 9) polymorphism, were the most common mutations found in these patients (Chillon et al., 1995; Zielenski et al., 1995; Dörk et al., 1997; Kanavaris et al., 1998; Jézéquel et al., 2000; Dohle et al., 2002; Danzinger et al., 2004). The 5T allele, considered the most common atypical CF mutation, produces high levels of a CFTR transcript that lacks exon 9 by alternative splicing, which encodes a nonfunctional protein (Chu et al., 1993; Teng et al., 1997). In fact, the frequency of this allele is 12-44% in the CBAVD cases, whereas it is found in less than 5% of the healthy population (Chillon et al., 1995; Dörk et al., 1997; Claustres et al., 2000; Radpour et al., 2007). Recently, a (TG)m repeat, located next to the IVS8-Tn polymorphism, has been implicated as a cis element that favors the penetrance of the IVS8-5T, being the haplotype TG12-T5 the most common genotype found in CBAVD patients (Hefferon et al., 2002; Groman et al., 2004). Because assisted reproduction techniques allow patients with obstructive azoospermia, including CBAVD patients, to father children with an increased risk for CF, the aim of this study was to assess the possible involvement of mutations and polymorphisms in the CFTR gene with infertility in Mexican patients who have obstructive azoospermia.

Materials and Methods

Patients

The following were enrolled in the study: 49 males with zero sperm count by obstructive azoospermia (16 patients with CBAVD and 33 with idiopathic azoospermia), 114 Mexican patients with CF and 124 characterized and 104 uncharacterized CFTR alleles (Orozco et al., 2000), and 103 fertile healthy men with at least 1 child as a control group. The respective local ethics and research committees approved this study. All participating individuals or their parents signed an informed consent form.

Patients with obstructive azoospermia were diagnosed at the Andrology Department of the National Institute of Perinatology in Mexico City. Diagnosis was based on a clinical evaluation that included detailed clinical history, physical examination, semen analysis, and serum hormonal profiling. Additional investigations, such as scrotal examination, transrectal ultrasonography, and testicular biopsy, were performed to confirm the diagnosis. Patients having unilateral renal agenesis by renal ultrasonography were not included.

Screening of CFTR mutations

Genomic DNA from azoospermic patients and healthy controls was isolated from peripheral blood lymphocytes by following conventional methods (QIAgen Systems, Inc., Valencia, CA). DNA samples from CF patients with previously characterized mutations were obtained from the DNA Bank at the National Pediatrics Institute in Mexico City (Orozco et al., 2000).

All CBAVD cases were subjected to direct sequencing of the entire coding sequence and all intron/exon boundaries of the CFTR. The 27 exons were amplified as described elsewhere (Zielenski et al., 1991) and sequenced directly with a DNA Sequencing Kit with Big Dye Terminator chemistry on an automated ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA).

The 33 idiopathic azoospermic patients were screened for the most common CFTR Mexican mutations (p.F508del, p.G542X, p.N1303K, p.S549N, p.I507del), the p.R117H mutation, and those found in our CBAVD patients (Friedman et al., 1991; Zielenski et al., 1991).

CFTR IVS8-Tn and -(TG)m polymorphisms analysis

In CBAVD patients and fertile healthy male controls, exon 9 and its exon-intron junction were directly sequenced by using an automated ABI PRISM 3100 DNA sequencer (Applied Biosystems). By using the primers forward 5′-ATGGGCCATGTGCTTTTCAAAC and reverse 5′-AACCGCCAACAACTGTCCTCT (DNASTAR Lasergene), sequencing allowed screening for both IVS8-Tn and (TG)m loci. In all CF patients and idiopathic azoospermic individuals, allelic discrimination of the IVS8-Tn polymorphism was carried out by the allele-specific polymerase chain reaction assay as described by Friedman et al. (1997).

Statistical analysis

The statistical analysis of an association between polymorphisms and obstructive azoospermia was performed by using the chi-square test (Stat-Calc program, Epic Info 2005 V.3.2; Centers of Disease Control and Prevention, Atlanta, GA). Statistical significance was considered at a p value less than 0.05.

Results

Clinical features

We enrolled 49 male patients who were infertile because of obstructive azoospermia. None had symptoms or family history of CF or related diseases. Sixteen of them were diagnosed with CBAVD, and 33 had idiopathic azoospermia. Twenty-two of the latter also displayed genital abnormalities (varicocele, epididymitis, testicular hypertrophy, and hypotrophy and epididymal cyst).

CFTR mutations and polymorphism analysis in CBAVD

Sequencing analysis of the entire coding sequence of the CFTR in 16 CBAVD patients led us to identify the p.F508del, p.G85E, p.W1089X, and p.D1152H mutations in 3 CBAVD cases (18.8%). The p.F508del and p.W1089X mutations were found in a heterozygous state, and the p.G85E and p.D1152H mutations were documented in the same patient (Table 1).

Table 1.

CFTR Genotype in Patients with Congenital Bilateral Absence of the Vas Deferens

Patient	Mutations	(TG)m-(T)n
1	NA	TG11-T7/TG11-T7
2	NA	TG10-7T/TG11-7T
3	NA	TG11-T7/TG11-T7
4	p.G85E/p.D1152H	TG11-T7/TG11-T7
5	NA	TG11-T7/TG11-T9
6	NA	TG12-T7/TG12-T7
7	p.W1089X	TG13-T5/TG12-T7
8	NA	TG10-T7/TG11-T7
9	NA	TG11-T7/TG12-T7
10	NA	TG11-T7/TG12-T7
11	NA	TG11-T7/TG12-T7
12	NA	TG11-T7/TG12-T7
13	p.F508del	TG11-T7/TG12-T9
14	NA	TG11-T7/TG11-T7
15	NA	TG11-T7/TG12-T7
16	NA	TG11-T7/TG11-T7

NA, not available (no mutation identified).

Several previously reported polymorphisms were found either on the coding sequence—c.869+11C>T (1001+11C/T), c.1408A>G (M470V), c.2909-71G>C (3041-71G/C), and c.4389 G/A (Gln1463Gln)—on the noncoding regions—rs67380110 (T/-), c.-966T>G (-834T/G), and c.-869T[8_9] (-790T9/8). Additionally, a new variant, the c.3273 G/C (L1091L), was found in 1 case.

Regarding the IVS8 polythymidine tract analysis, we did not observe significant differences in the distribution of the Tn alleles between CBAVD cases and fertile male controls (p>0.05) (Table 2).

Table 2.

Analysis of the IVS8-Tn CFTR Polymorphism

	IVS8-Tn, n (%)
Population	5T	7T	9T	p value
Controls (n=206)	3 (1.4)	201 (97.6)	2 (0.98)
Cystic fibrosis (n=228)	7 (3.07)	124 (54.4)	97 (42.5)	>0.05
CBAVD (n=32)	1 (3.1)	30 (93.8)	1 (3.1)	>0.05
Idiopathic azoospermia (n=66)	3 (4.5)	58 (86.4)	5 (7.6)	0.0008

CBAVD, congenital bilateral absence of the vas deferens.

Analysis of the (TG)n tract revealed a similar distribution of TG11 and TG12 genotypes in CBAVD patients (90.6%) and fertile controls (93.7%); the TG11-T7 haplotype was the most common combination in both groups (62.5% and 49.5%, respectively). However, we found a compound heterozygous CBAVD patient to have TG12/13-T7/5 haplotypes, the patient who carried the W1089X mutation.

CFTR mutations and IVS8-Tn polymorphism analysis in patients with idiopathic obstructive azoospermia

We screened idiopathic obstructive azoospermia males for the 5 most prevalent CFTR mutations in the Mexican population (Orozco et al., 2000) or the p.R117H mutation and mutations found in our CBAVD patients. None of the 9 screened mutations were detected in the 33 individuals with idiopathic obstructive azoospermia. However, the Gln1463Gln [C.4389 G/A] variation, documented in 1 CBAVD case, was found in 20% of the idiopathic azoospermic group and was detected in 6% of the 206 healthy chromosomes (p=0.0029) by direct sequencing.

Regarding the IVS8-Tn polymorphism, the 7T allele was the most common (86.4%) in the idiopathic azoospermic patients. Interestingly, the 9T allele frequency was significantly higher in idiopathic azoospermic cases than in fertile male controls (7.6 vs. 0.98%; p=0.0008) (Table 2). Analysis of a large sample size is needed to confirm or discard this association finding.

CFTR mutations and IVS8-Tn polymorphism background

To assess the association between CF mutations and IVS8-Tn polymorphisms of the CFTR gene, we included 124 CF chromosomes bearing 30 characterized mutations. The linkage analysis between characterized CFTR mutations and Tn alleles showed that chromosomes bearing the p.F508del, p.G542X, p.N1303K, p.I148T, and c.406→1G>A mutations were localized on a 9T allele, whereas the p.S549N, p.I507del, p.R75X, c.2055_2063del, c.3199_3204del, and c.2183AA>G mutations and those unique mutations, as well as most uncharacterized CF alleles, were located on a 7T allele. The p.W1098C mutation was uniquely on a 5T variant, and the I506T mutation was found on 7T or 9T alleles (Table 3).

Table 3.

CFTR Mutations and IVS8-Tn Polymorphism Background in Mexican Patients with Cystic Fibrosis

IVS8-Tn	Mutations	N (%)
9T	p.F508del	58 (25.44)
	p.G542X	20 (8.78)
	p.N1303K and p.I148T	6 (2.63)
	c.406 - 1G>A	2 (0.87)
	p.I506T	1 (0.44)
	Uncharacterized	10 (4.38)
7T	p.S549N	5 (2.20)
	p.I507del	4 (1.75)
	p.R75X	3 (1.31)
	c.2055_2063del, c.3199_3204del and c.2183AA>G	6 (2.63)
	c.935delA, p.G551D, p.R553X, c.1924_1930del, p.G551S,	1
	c.1078delT, p.Y1092X, p.R117H, c.1716G>A, p.W1204X,
	c.846delT, p.P750L, p.W1069X, p.L558S, p.I506T,
	c.4160_4161insGGGG, c.297 - 1G>A and p.H199Y	18 (7.90)
	Uncharacterized	88 (38.60)
5T	p.W1098C	1 (0.44)
	Uncharacterized	6 (2.63)
Total alleles		228 (100)

N=Number of alleles.

Discussion

Several authors have documented a high frequency of CFTR mutations in males with CBAVD or obstructive azoospermia without CBAVD, suggesting that a broad spectrum of reproductive tract abnormalities could be associated with a defective CFTR function (Chillon et al., 1995; Meschede et al., 1997; Kanavaris et al., 1998; Mak et al., 2000; Rossi et al., 2004). It has been reported that 30-75% of men with CBAVD have at least 1 detectable common CFTR mutation (Uzun et al., 2005; Radpour et al., 2006). The analysis of the entire coding sequence of CFTR in 16 CBAVD Mexican patients, documented 5 different mutations (including the 5T allele) in 3 patients (18.8%). The p.F508del mutation was found in 1 patient, and the p.G85E/p.D1152H and p.W1089X/T5 mutations were found in 2 compound heterozygous CBAVD cases. This mutation rate is one of the lowest in CBAVD patients reported worldwide. We also observed differences in the mutation distribution between our population and those reported in CBAVD patients elsewhere. The frequency of the p.F508del mutation (3.1%) in Mexican cases was similar to that in Algerian and Egyptian populations (2.17% and 3.12%, respectively) but quite different from that in white patients (9.67-26.9%), including Spaniards (21.57%). Although the frequency of the p.D1152H mutation (3.1%) was similar to that reported in Austrians (2.72%) (Chillon et al. 1995; Dörk et al. 1997; Uzun et al., 2005), it was higher than that in Spaniards (0.98%) and Germans (1.41%) and lower than that found in Turks (14.7%) (Dayangaç et al., 2004). The p.G85E mutation, which shows a low frequency throughout the world (1/800 French and 2/122 Iranian cases), was detected in 1 of our cases (3.1%), suggesting that this mutation could be more common in CBAVD Mexican patients (Claustres et al., 2000; Radpour et al., 2007). As far as we know, the p.W1089X mutation had not been reported in CBAVD patients (Table 4). This mutation creates a stop codon and is predicted to produce a truncated protein at amino acid 1089, affecting the CFTR-TM2 and NBD2 domains.

Table 4.

Frequencies of CFTR Mutations and IVS8-T5 in Several Populations of Patients with Congenital Bilateral Absence of the Vas Deferens

		CFTR mutations, n (%)
Population	Alleles, n	p.F508del	p.G85E	p.D1152H	p.W1089X	5T	Alleles with other mutations n (%)	Reference
Mexican	32	1 (3.12)	1 (3.1)	1 (3.1)	1 (3.1)	1 (3.1)	NA	Current study
Spanish	204	44 (21.57)	0	2 (0.98)	0	42 (20.58)	48 (23.52)	Chillon et al., 1995
German	212	57 (26.9)	0	3 (1.41)	0	26 (12.26)	84 (39.62)	Dörk et al., 1997
Greek	28	4 (14.28)	0	0	0	4 (14.28)	10 (35.71)	Kanavaris et al., 1998
Canadian	128	23 (17.96)	0	0	0	24 (18.75)	16 (12.50)	Mak et al., 1999
Dutch	42	8 (19.04)	-	-	-	4 (9.52)	14 (33.33)	Dohle et al., 1999
Egyptian	32	1 (3.12)	0	0	0	14 (43.75)	-	Lissens et al., 1999
French	1600	348 (21.75)	1 (0.06)	1 (0.06)	0	254 (15.88)	355 (22.18)	Claustres et al., 2000
Portuguese	62	6 (9.67)	0	0	0	14 (22.58)	16 (36.36)	Grangeia et al., 2004
Turkish	102	3 (2.84)	0	15 (14.70)	0	20 (19.6)	35 (34.31)	Dayangaç et al., 2004
Austrian	44	6 (13.63)	0	1 (2.72)	0	0	7 (15.90)	Uzun et al., 2005
Iranian	224	17 (7.58)	2 (0.89)	-	-	59 (26.33)	47 (20.98)	Radpour et al., 2007
Algerian	92	2 (2.17)	-	-	-	8 (8.69)	2 (2.17)	Loumi et al., 2008
Taiwanese	54	0 (0.0)	0	-	-	24 (44.4)	-	Wu et al., 2004
Taiwanese	126	0 (0.0)	0	0	0	42 (33.3)	-	Chiang et al., 2009

It is possible that the low rate of CFTR mutations observed in the populations could be due to limitations in the techniques used; nevertheless, we cannot discard the role of other CFTR mutations in this disease.

On the other hand, other genes have been implicated in the etiology of the CBAVD. Recently the single-nucleotide polymorphism rs5335 on the endothelin receptor type A (EDNRA) and copy number variants either in the Pantothenate kinase 2 gene (PANK2) or 3q26.1 locus have been associated with CBAVD in Turkish and Taiwanese populations, respectively (Lee et al., 2009; Havasi et al., 2010).

Although in the 33 patients with idiopathic azoospermia none of the 9 CFTR mutations (p.F508del, p.G542X, p.N1303K, p.S549N, p.I507del, p.R117H. p.G85E, p.D1152H, and p.W1089X) screened were found, we documented a high frequency of the Gln1463Gln (c.4389 G>A) polymorphism in comparison with the healthy control group (20% vs 6%, respectively; p=0.0029). It is notable that this variant was found in 18.3% of Brazilian patients with CF (Vidigal et al., 2008). The role of this synonymous variant in the etiology of azoospermia needs to be evaluated because evidence shows that certain synonymous SNPs involving frequent-to-rare codon substitutions may result in ribosome stalling due to a lower concentration of cognate tRNAs or an alteration of the RNA structure (Tsai et al., 2008). It is also possible that this variant is in linkage disequilibrium with certain mutations implicated in the azoospermia disease; therefore, other screening strategies should be used to complete the entire mutation characterization or CFTR variants implicated in disease etiology. Otherwise, mutations in other genes, such as the androgen receptor gene, have been reported in infertile Iranian male with azoospermia (Mirfakhraie et al., 2010); thus, screening of other candidate genes is needed in Mexican patients.

The 5T allele of the IVS8-Tn polymorphism has been considered the most common atypical CFTR mutation (Chillon et al., 1995; Dörk et al., 1997; Dohle et al., 2002, Dayangaç et al., 2004; Radpour et al., 2007), however, it was found in a low frequency in our population (2.6%), suggesting that this mutation does not play a significant role in CF-related CBAVD or idiopathic azoospermia in Mexicans.

It has been documented that the spectrum of CBAVD/azoospermic-causing mutations varies among ethnic groups, reflecting the complex and different genetic component of populations. So, given the serious clinical consequences of transmitting such mutations, this study supports the needed of accurate CFTR mutation detection in men with obstructive azoospermia and their partners and making tailored CBAVD screening panels appropriate to each population.

Footnotes

Acknowledgments

We would like to thank all the patients and their families. We also thank the Polymorphism Sequencing and Identification Unit (Instituto Nacional de Medicina Genómica, Mexico) for technical support.

Disclosure Statement

The authors confirm that no competing financial interests exist.

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