Increased frequency of CFTR gene mutations identified in Indian infertile men with non-CBAVD obstructive azoospermia and spermatogenic failure

Gene 548 (2014) 43–47

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Increased frequency of CFTR gene mutations identified in Indian infertile men with non-CBAVD obstructive azoospermia and spermatogenic failure Himanshu Sharma a, Ravimohan S. Mavuduru b, Shrawan Kumar Singh b, Rajendra Prasad a,⁎ a b

Department of Biochemistry, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India Department of Urology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India

a r t i c l e

i n f o

Article history: Received 25 April 2014 Received in revised form 18 June 2014 Accepted 5 July 2014 Available online 7 July 2014 Keywords: Male infertility Cystic fibrosis CFTR Assisted reproduction technology Mutations

a b s t r a c t High incidence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with congenital bilateral absence of the vas deferens (CBAVD) and is considered as the genital form of cystic fibrosis (CF). The CFTR gene may also be involved in the etiology of male infertility in cases other than CBAVD. The present study was conducted to identify the spectrum and frequency of CFTR gene mutations in infertile Indian males with non-CBAVD obstructive azoospermia (n = 60) and spermatogenic failure (n = 150). Conspicuously higher frequency of heterozygote F508del mutation was detected in infertile males with non-CBAVD obstructive azoospermia (11.6%) and spermatogenic failure (7.3%). Homozygous IVS(8)-5T allele frequency was also significantly higher in both groups in comparison to those in normal healthy individuals. Two mutations in exon 25 viz., R1358I and K1351R were identified as novel mutations in patients with non-CBAVD obstructive azoospermia. Mutation R1358I was predicted as probably damaging CFTR mutation. This is the first report from the Indian population, emphasizing increased frequency of CFTR gene mutations in male infertility other than CBAVD. Thus, it is suggested that screening of CFTR gene mutations may be required in infertile Indian males with other forms of infertility apart from CBAVD and willing for assisted reproduction technology. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Cystic fibrosis (CF, MIM# 219700) is the most common severe autosomal recessive genetic disorder in Caucasians with an approximate incidence of 1 in 3500 births and the carrier frequency is ~ 1 in 25 individuals (Castellani et al., 2009). Notwithstanding, cystic fibrosis is still thought to be very rare in Indian population (Prasad et al., 2010). Mutations in the gene encoding CFTR are responsible for pathogenesis of CF. More than 1950 CFTR gene sequence variations have been identified in different ethnic populations, as listed in cystic fibrosis genetic analysis consortium database (http://www.genet.sickkids.on.ca/ StatisticsPage.html). Many of these identified mutations are responsible for a wide spectrum of clinical phenotypes including respiratory distress (Girodon et al., 1997; Pignatti et al., 1996), chronic pancreatitis (Cohn et al., 1998; Sharer et al., 1998) and male infertility due to congenital bilateral absence of the vas deferens (CBAVD, MIM# 22180) (Chillon et al., 1995; Oates and Amos, 1994).

Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane regulator gene; CBAVD, congenital bilateral absence of the vas deferens; SSCP, single strand conformational polymorphism; ARMS, amplification refractory mutation system. ⁎ Corresponding author. E-mail address: [email protected] (R. Prasad).

http://dx.doi.org/10.1016/j.gene.2014.07.005 0378-1119/© 2014 Elsevier B.V. All rights reserved.

The genetic link between CFTR mutation and male infertility due to CBAVD is well established. The majority of male CF patients are infertile due to CBAVD (Cuppens and Cassiman, 2004; Yu et al., 2012). However, the exact molecular mechanism by which defective CFTR leads to male infertility remains largely unknown. For the past few years, mutations in CFTR have been implicated in other forms of male infertility besides CBAVD phenotype. The CFTR gene expression has been reported both in humans and rodent testes, germ cells and Sertoli cells, suggesting its possible involvement in spermatogenesis (Boockfor et al., 1998; Gong et al., 2001; Hihnala et al., 2006; Trezise et al., 1993). Notwithstanding, outcome of various studies involving CFTR mutation screening in infertile patients with defective spermatogenesis or obstructive azoospermia with the presence of vas deferens is variable. Though some reports indicate the association of CFTR mutations and defect in sperm production (Dohle et al., 2002; Jakubiczka et al., 1999; Schulz et al., 2006; Stuppia et al., 2005; Tamburino et al., 2008; Tomaiuolo et al., 2011; Van der Ven et al., 1996) while, few other investigations ruled out the possibility of any association (Mak et al., 2000; Pallares-Ruiz et al., 1999; Ravnik-Glavac et al., 2001). Intriguingly, the findings of various investigators intimate that even heterozygous mutations of CFTR gene may also increase the risk of non-CBAVD male infertility (Mocanu et al., 2010; Safinejad et al., 2011; Stuppia et al., 2005; Van der ven et al., 1996).

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H. Sharma et al. / Gene 548 (2014) 43–47

Our recent study as well as few other investigations has provided evidence for an extensive allelic heterogeneity in Indian infertile males with CBAVD (Sachdeva et al., 2011; Sharma et al., 2009a). Recently, we have reported F508del and IVS(8)-5T allele as the most common CFTR gene mutation in Indian CBAVD males, with an allelic frequency of 11% and 25% respectively (Sharma et al., 2009a). Nonetheless, the spectrum and the frequency of CFTR gene mutations in infertility other than CBAVD are unknown in our population. It is uncertain whether screening for CFTR mutations should be recommended for Indian infertile males without CBAVD willing for assisted reproduction technology. Based on these considerations, this is the first study from Indian population where we have appraised the frequency and spectrum of mutations in the CFTR gene in infertile males with impaired spermatogenesis and with obstructive azoospermia without CBAVD. 2. Material and methods 2.1. Ethical approval The study was approved by the institutional ethics committee at the Post Graduate Institute of Medical Education and Research, Chandigarh (Approval No. 877/PG11-1TRG/16814). An information sheet was provided to each patient/family member and written informed consent was obtained. 2.2. Selection of subjects A total of 210 consecutive Indian infertile men attending male health clinic of the Urology Department at Post Graduate Institute of Medical Education and Research, Chandigarh were included in this study. In all cases, the initial evaluation included a physical scrotal examination and a semen analysis including volume, pH, sperm count and motility, performed as per World Health Organization guidelines (World Health Organization, 2010). Complete clinical data concerning infertility was evaluated. Hormonal assays were done using chemiluminescence. Transrectal ultrasonography was conducted for the morphology and size of the seminal vesicles, prostate and ejaculatory ducts. Abdominal ultrasonography was performed in order to evaluate the pelvis and the upper urinary tract. Testicular fine needle aspiration cytology (FNAC) was carried out for studying the patterns of spermatogenesis. After detailed clinical and physical examination by the urologist, all infertile male patients (n = 210) were categorized into two groups, one with spermatogenic failure (n = 150) (oligospermia with sperm count b 10 million/ml and non-obstructive azoospermia) and other with obstructive azoospermia (n = 60) having normal spermatogenesis and palpable vas deferens but obstruction in any other part of the reproductive tract. Sweat chloride analysis was performed only in subjects with obstructive azoospermia (Gibson and Cooke, 1959) considering that probability of defect in both alleles of CFTR is more in this group of patients. Sweat chloride levels N 60 mEq/l were considered as positive, 40–60 mEq/l as borderline and b40 mEq/l as negative. Healthy subjects (n = 100) with sperm count N 20 million/ml and having no sign and symptoms of infertility served as control. 2.3. CFTR gene analysis Genomic DNA was isolated from whole blood following the method of Daly et al. (1996). CFTR gene analysis, for the presence F508del, R117H, N1303K and R553X mutations was performed by single ARMS PCR as described by Ferrie et al. (1992). Other common mutations viz., 621+1GNT, G542X, G551D and W1282X were screened by multiplex ARMS PCR (Ferrie et al., 1992). The T5 variant in the polymorphic region IVS8-5T was analyzed as described previously (Chillon et al., 1995). The presence of novel/rare mutations in the 27 exons and at the exon–intron boundaries of the CFTR gene was screened by single-

strand conformational polymorphism (SSCP) analysis as reported earlier (Sharma et al., 2009b). Normal controls were used in each run to predict interpretation of SSCP pattern as abnormal. The patient samples exhibiting shifts relative to normal samples on SSCP were subjected to automated DNA sequencing with forward and reverse primers, using an ABI Prism Big Dye Terminator Sequencing Ready Reaction Kit (Perkin Elmer, USA) and a DNA sequencer ABI Prism (Model 3100, Perkin Elmer). 2.4. Pathological prediction and computational analysis The output prediction score of all novel mutations was tested using online available software, PolyPhen-2 (http://genetics.bwh.harvard. edu/pph2/). PolyPhen-2 is an automatic tool for prediction of the possible impact of an amino acid substitution on the structure and function of a human protein. This prediction is based on a number of features comprising the sequence, phylogenetic and structural information characterizing the substitution. The results obtained from PolyPhen-2 were validated using the PMut tool of molecular modeling and bioinformatics (mmb) program for pathological prediction (http://mmb2.pcb.ub.es/ pmut/temp/pmut1380564661173/). Structural stability of mutated CFTR protein was also predicted using online available iStable software (http://predictor.nchu.edu.tw/iStable/indexSeq.php). 2.5. Statistical analysis The difference in the genotype was analyzed by χ2 statistics. Fisher exact probability test was implicated for comparison between two groups using online available VassarStats website for statistical computation (www.vassarstats.net). P value b 0.05 is considered as significant. Hormonal profile, age and semen volume in different groups were compared by one way ANOVA followed by post hoc test using the SPSS statistical software package. 3. Results 3.1. Demographics and clinical variables In this study, we have genotyped entire coding region of CFTR gene in 210 infertile patients. These patients were broadly categorized into 2 groups for instances: 60 patients with normal spermatogenesis and palpable vas deferens, but having nil sperm count in ejaculation due to obstruction in the reproductive tract and 150 patients of non-obstructive azoospermia or oligospermia due to defective spermatogenesis. Healthy controls (n = 100) with a sperm count N 20 million/ml and having no sign and symptoms of infertility were also screened for F508del mutation and IVS(8)-5T allele polymorphism. The mean age of the infertile oligospermic male patients at the time of enrollment was 33 ± 5.5 years, which was significantly higher in comparison to the mean age of the patients with non-obstructive azoospermia and obstructive azoospermia (Table 1). Mean semen volume in obstructive azoospermic patients (1.26 ± 0.5) was significantly less in comparison to normal range (2–4 ml). However, all males with obstructive azoospermia had sweat chloride value b 60 mEq/l but the mean sweat chloride value for these patients was 39.81 mEq/L, which is near to borderline range (40–60 mEq/l) (Table 1). The mean serum FSH level was significantly elevated from normal range (1.5–12.4 mIU/ml) in patients with oligospermia (16.53 ± 9.68 mIU/ml) and non-obstructive azoospermia (26.90 ± 17.57 mIU/ml), on the other hand serum LH level was augmented only in non-obstructive azoospermic patients (10.87 ± 7.79 mIU/ml) (Table 1). Among the 60 patients of obstructive azoospermia, 12 had bilateral obstruction in seminal vesicle, 4 had nonpalpable epididymis and only one had a single kidney. However, the specific association between CFTR gene mutations and different forms of urogenital abnormalities such as the absence of kidney, epididymis and seminal vesicle could not be established.

H. Sharma et al. / Gene 548 (2014) 43–47

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Table 1 Clinical observation in Indian infertile male patients. Clinical observations

Non-CBAVD obstructive azoospermia (n = 60)

Oligospermia (n = 64)

Non-obstructive azoospermia (n = 86)

Mean age(Years) Mean sweat chloride (mEq/l)

29.27 ± 5.4 39.81 ± 8.03

33.07 ± 5.5⁎ Not done

29.45 ± 5.0 Not done

1.26 ± 0.53⁎ Alkaline 0

2.75 ± 0.82 Alkaline b10/ml

2.60 ± 0.89 Alkaline 0

Normal spermatogenesis

Abnormal spermatogenesis

Abnormal spermatogenesis/Sertoli cell only syndrome

14.65 ± 3.16

14.01 ± 5.64

15.56 ± 8.61

5.6 ± 3.44

16.35 ± 9.68⁎

26.90 ± 17.57⁎

5.07 ± 2.90

5.58 ± 2.65

10.87 ± 7.79

Semen analysis Volume (ml) pH Sperm concentration (million/ml) (normal value ≥ 20 million/ml) FNAC

Hormone assay Testosterone (nmol/l) (9.9–27.8 nmol/l)a FSH (mIU/ml) (1.5–12.4 mIU/ml)a LH (mIU/ml) (1.7–8.6 mIU/ml)a a Reference range in men. ⁎ P b 0.05 is considered as significant.

3.2. Mutations identified in Indian infertile males with non-CBAVD obstructive azoospermia Mutation F508del was found in heterozygous form in 7 patients with an allelic frequency of 5.8% in 60 patients with non-CBAVD obstructive azoospermia (Table 2). An intronic variant IVS(8)-5T mild CFTR mutation was found with an allelic frequency of 15.83%. Notably, this mild mutation was observed in homozygous form in 4 patients, however, in 1 patient IVS(8)-5T was found in the compound heterozygous form with F508del. Statistical analysis revealed that the heterozygous frequency of F508del mutation and IVS (8)-5T allele in patients with non-CBAVD obstructive azoospermia is significantly higher in comparison to those in healthy fertile male population (P b 0.001) (Table 3). The patients identified as having either single mutation or no mutations were further subjected to SSCP analysis. SSCP analysis and subsequent DNA sequencing revealed two novel mutations viz., R1358I and K1351R (Table 2). Notably K1351R mutation was found in association with IVS8-5T allele in one patient.

than control group viz., 3.5% (P b 0.001) (Tables 2 and 3). None of the other mutations except IVS8-5T identified in infertile male patients were present in any of the fertile males enrolled in the healthy control group.

3.4. Pathological predictions of novel substitution mutations The PolyPhen-2 output prediction score (http://genetics.bwh. harvard.edu/pph2/) for R1358I was more than 0.05 (threshold for pathological mutation) and therefore categorized as deleterious mutation which may probably affect CFTR structure and functions (Table 4). Another algorithm mmb program (http://mmb.pcb.ub.edu/) also validates predictions done by PolyPhen-2 and revealed R1358I as pathological mutation. The output prediction score with iStable program (http://predictor.nchu.edu.tw/iStable/indexSeq.php) for R1358I was also more than 0.5 (threshold for stability), thus predicting that substitution of arginine to isoleucine at 1358 position may affect the structural stability of protein (Table 4).

3.3. Mutations identified in Indian infertile males with spermatogenic failure (oligospermia/non-obstructive azoospermia)

4. Discussion

The mutation analysis of the entire coding region of CFTR gene from 150 infertile Indian male patients with oligospermia or non-obstructive azoospermia due to defective spermatogenesis revealed the presence of F508del mutation in exon 11 and IVS(8)-5T allele in intron 8. Mutation F508 del was detected in 11 out of 150 (7.3%) patients with an allelic frequency of 3.6%. Seven patients had IVS(8)-5T mutation in homozygous state while it was identified in heterozygous form in 13 patients. The allelic frequency of IVS8-5T mutation was 9%, which is significantly higher

It is a well established fact that the infertility resulting from CBAVD is associated with a high frequency of CFTR mutations therefore screening of CFTR mutations is advised for all patients with CBAVD. Till date we lack any evidence for CFTR gene mutation in infertile Indian males without CBAVD. It is noteworthy, that this is the first study from Indian population where we have demonstrated any association between CFTR mutations and infertility due to spermatogenic failure and obstructive azoospermia without CBAVD.

Table 2 CFTR mutation identified in the Indian infertile male patients. Mutations

Consequences

Exon/intron

No. of alleles

Non-CBAVD obstructive azoospermia (n = 60) T5 Reduction of oligo T tract to 5T at 1342-6 F508del Deletion of 3 bp (CTTor TTT) between 1652 and 1655 a R1358I G to A at 4073 a K1351R A to G at 4052

Aberrant splicing Deletion of phenylalanine at 508 Arginine to isoleucine at 1358 Lysine to arginine at 1351

Intron 8 Exon 11 Exon 25 Exon 25

19 7 1 1

Oligospermia/non-obstructive azoospermia (n = 150) T5 Reduction of oligo T tract to 5T at 1342-6 F508del Deletion of 3 bp (CTTor TTT) between 1652 and 1655

Aberrant splicing Deletion of phenylalanine at 508

Intron 8 Exon 10

27 11

a

Nucleotide change

Indicates novel substitution mutations.

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Table 3 Frequency of CFTR mutations identified. S. no.

1 2 3 4 5

Mutation

F508del IVS(8)-5T homozygous IVS(8)-5T heterozygous R1358I K1351R

Non-CBAVD obstructive azoospermia (OA) (n = 60)

7 (11.6%) 4 (6.6%) 11 (18.3%) 1 (1.6%) 1 (1.6%)

Spermatogenic failure (SF) (n = 150)

11 (7.3%) 7 (4.6%) 13 (8.6%) 0 0

Healthy control (ctrl) (n = 100)

0 0 7 (7%) 0 0

Chi square-test P value⁎ OA vs ctrl

SF vs ctrl

OA vs SF

0.0008 0.01 0.00 NS NS

0.008 0.04 NS – –

NS NS 0.0001 NS NS

⁎ P b 0.05 is considered as significant.

In the present study, we have observed heterozygous CFTR mutations (coding regions) in 19 (9.04%) out of the 210 infertile male patients with defective spermatogenesis and obstructive azoospermia without CBAVD. This observed frequency is very similar to that observed in non-CBAVD infertile German male population (9.5–5.7%) (Schulz et al., 2006; Van der ven et al., 1996) but lower than that found in an Italian population (14.3%) (Tamburino et al., 2008). India is the second most populated country in the world and as estimated 15–20% of the married couples suffer from infertility. The most common cause is spermatogenic failure or non-CBAVD obstructive azoospermia, a small fraction of these infertile couples opt for assisted reproduction technology (ART) to have their biological child (Suganti et al., 2014). Therefore, it becomes critically important to analyze the risk factor of harboring CFTR mutations in the offsprings of these infertile couples born through ART and to suggest a panel of mutation for genetic screening in such pathologic condition. In this study, F508del is identified as the most common CFTR mutation in patients with oligospermia or non-obstructive azoospermia (3.6% allelic frequency) as well as with obstructive azoospermia without CBAVD (5.8% allelic frequency). Moreover, IVS(8)-5T allele which is considered as a mild form of CFTR mutation was also identified at significantly higher frequency in infertile patients as compared to those in the healthy control. Intriguingly, extensive screening of 27 exons of the CFTR gene in the Indian infertile males leads to the identification of 2 novel substitutions which are reported only in Indian population. Both of these novel substitutions were identified in exon 25 comprising NBD2 domain of the CFTR protein. Altogether exon 11 comprising of the NBD1 region of CFTR and exon 25 were identified as hot spot regions for screening of CFTR mutation in Indian infertile males. Since the frequency of novel substitution identified in Indian infertile males is very less, therefore, we can only include two most common mutations viz., F508del and IVS(8)-5T in screening panel. In the absence of any of these common mutations, complete screening for exon 25 and exon 11 or if possible all 27 exons of the CFTR gene is suggested for infertile males without CBAVD opting for ART. According to few investigations, F508 del is identified as the most common CFTR mutation in the Indian CF population (19–44%) (Kabra

Table 4 Pathological and stability prediction of novel substitution mutations identified in Indian non-CBAVD obstructive azoospermia patients. Pathological/structural prediction

Polyphen score Pathological prediction iStable score Stability prediction

Novel mutants identified R1358I

K1351R

1.00 Probable damaging 0.716 Decrease

0.03 Benign 0.551 Unaffected

Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/) was used to generate the output score of novel sequence variants (threshold for pathological mutation was 0.05). Results from Polyphen-2 were validated by molecular modeling and bioinformatics (mmb) (http://mmb2.pcb.ub.es/pmut/temp/pmut1380564661173/) program. iStable software (http://predictor.nchu.edu.tw/iStable/indexPDB.php) was used to predict stability of mutated protein (threshold for stability was 0.5).

et al., 1996, 2003; Sharma et al., 2009a,b) and the reported carrier frequency of F508 del mutation in healthy individuals is 0.42–1.5% (Kapoor et al., 2006; Muthuswamy et al., 2014). Although, in our study we did not find any carrier of F508 del mutation among 100 healthy fertile control subjects screened, but recently Muthuswamy et al. (2014) have reported that the carrier frequency of F508 del mutation in the north Indian population is 3/200 (1.5%). Even if we consider this carrier frequency as the reference, we can infer that in the Indian infertile males with non-CBAVD obstructive azoospermia or spermatogenic failure the frequency of F508 del mutation is significantly higher to that of the normal healthy population (statistically checked P b 0.05). Although, it is a preliminary study and further study of large patient cohorts is required to establish a spectrum of mutations in such infertile male patients, but nevertheless, this study clinically signifies the importance of screening of the CFTR mutations in the Indian infertile males without CBAVD. Elevated level of sweat chloride (N60 mEq/l) which is considered as a hallmark symptom of classical CF was not observed in our patients with obstructive azoospermia. Most of the mutations identified in infertile males are in heterozygous condition and it is believed that heterozygous mutations of CFTR do not produce the CF phenotype. Some clinical studies have reported that the frequency of mutations or IVS(8)-5T variant in one allele is higher in non-CBAVD azoospermia patients compared with those in fertile males or the general population, suggesting that heterozygous mutations in CFTR without affecting its normal chloride conducting ability may increase the risk of male infertility associated with defective spermatogenesis by some undefined mechanism (Mocanu et al., 2010; Safinejad et al., 2011; Stuppia et al., 2005; Van der ven et al., 1996). Although, the reports available on frequency and association of CFTR mutation with male infertility are contradictory for decades, the accumulating evidences from animal and cellular models are indicative of the involvement of CFTR in a number of reproductive events including spermatogenesis and non-CBAVD azoospermia (Chen et al., 2012). Recently Xu et al. (2011) have shown the evidence for CFTR dependent regulation of CREB in human Sertoli cells and suggest that the impairment in this regulation may result in defective spermatogenesis as seen in non-obstructive azoospermia. This finding suggests the possible mechanism behind the role of CFTR in spermatogenesis. The present study also verifies the observed link between increased frequency of CFTR gene mutations in patients with non-obstructive azoospermia and oligospermia. In a large proportion of non-CBAVD infertile males, CFTR mutations were not identified which could be due to the presence of either the defects in introns or the promoter region of CFTR. The probable role of other environmental and genetic factors contributing to male infertility with spermatogenic failure and obstructive azoospermia without CBAVD may also need to be investigated. Taken together, we conclude that the frequency of CFTR mutations in the Indian cohort with infertility due to defective spermatogenesis or non-CBAVD obstructive azoospermia is significantly higher than that in the normal population, indicating the possibility that mutated CFTR in heterozygous state may also cause male infertility other than CBAVD. Therefore, particularly in context with the Indian population,

H. Sharma et al. / Gene 548 (2014) 43–47

we suggest that genetic counseling and screening of CFTR mutations may be recommended in Indian infertile males opting for ART. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgment The authors acknowledge the Indian Council of Medical Research, New Delhi, India for funding this research project (grant sanction no. 54/10/2008-BMS). We are also thankful to the infertile subjects and controls who participated in the study. References Boockfor, F.R., Morris, R.A., DeSimone, D.C., Hunt, D.M., Walsh, K.B., 1998. Sertoli cell expression of the cystic fibrosis transmembrane conductance regulator. Am. J. Physiol. 274, C922–C930. Castellani, C., Picci, L., Tamanini, A., Girardi, P., Rizzotti, P., Assael, B.M., 2009. Association between carrier screening and incidence of cystic fibrosis. J. Am. Med. Assoc. 302, 2573–2579. Chen, H., Ruan, Y.C., Xu, W.M., Chen, J., Chan, H.C., 2012. Regulation of male fertility by CFTR and implications in male infertility. Hum. Reprod. Update 6, 703–713. Chillon, M., Casals, T., Mercier, B., Brassas, L., Lissens, W., Silber, S., Romey, M.C., RuizRomero, J., Verlingue, C., Clustres, M., 1995. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N. Engl. J. Med. 332, 1475–1480. Cohn, J.A., Friedman, K.J., Noone, P.G., Knowles, M.R., Silverman, L.M., Jowell, P.S., 1998. Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis. N. Engl. J. Med. 339, 653–658. Cuppens, H., Cassiman, J.J., 2004. CFTR mutations and polymorphisms in male infertility. Int. J. Androl. 27, 251–256. Daly, A.K., Steen, V.M., Fairbrother, K.S., Idle, J.R., 1996. CYP2D6 multiallelism. Methods Enzymol. 272, 199–210. Dohle, G.R., Halley, D.J., Van Hemel, J.O., van den Ouwel, A.M., Pieters, M.H., Weber, R.F., Govaerts, L.C., 2002. Genetic risk factors in infertile men with severe oligozoospermia and azoospermia. Hum. Reprod. 17, 13–16. Ferrie, R.M., Schwarz, M.J., Robertson, N.H., Vaudin, S., Super, M., Malone, G., Little, S., 1992. Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene. Am. J. Hum. Genet. 51, 251–262. Gibson, L.E., Cooke, R.E., 1959. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 23, 545–549. Girodon, E., Cazeneuve, C., Lebargy, F., Chinet, T., Costes, B., Ghanem, N., Martin, J., Lemay, S., Scheid, P., Housset, B., Bignon, J., Goossens, M., 1997. CFTR gene mutations in adults with disseminated bronchiectasis. Eur. J. Hum. Genet. 5, 149–155. Gong, X.D., Li, J.C., Cheung, K.H., Leung, G.P., Chew, S.B., Wong, P.Y., 2001. Expression of the cystic fibrosis transmembrane conductance regulator in rat spermatids: implication for the site of action of antispermatogenic agents. Mol. Hum. Reprod. 7, 705–713. Hihnala, S., Kujala, M., Toppari, J., Kere, J., Holmberg, C., Höglund, P., 2006. Expression of SLC26A3, CFTR and NHE3 in the human male reproductive tract: role in male sub fertility caused by congenital chloride diarrhoea. Mol. Hum. Reprod. 12, 107–111. Jakubiczka, S., Bettecken, T., Stumm, M., Nickel, I., Müsebeck, J., Krebs, P., Fischer, C., Kleinstein, J., Wieacker, P., 1999. Frequency of CFTR gene mutations in males participating in an ICSI programme. Hum. Reprod. 14, 1833–1834. Kabra, M., Ghosh, M., Kabra, S.K., Khanna, A., Verma, I.C., 1996. Delta F 508 molecular mutation in Indian children with cystic fibrosis. Indian J. Med. Res. 104, 355–358. Kabra, S.K., Kabra, M., Lodha, R., Shastri, S., Ghosh, M., Pandey, R.M., Kapil, A., Aggarwal, G., Kapoor, V., 2003. Clinical profile and frequency of delta f508 mutation in Indian children with cystic fibrosis. Indian Pediatr. 40, 612–619. Kapoor, V., Shastri, S.S., Kabra, M., Kabra, S.K., Ramachandran, V., Arora, S., Balakrishnan, P., Deorari, A.K., Paul, V.K., 2006. Carrier frequency of F508del mutation of cystic fibrosis in Indian population. J. Cyst. Fibros. 5, 43–46. Mak, V., Zielenski, J., Tsui, L.C., Durie, P., Zini, A., Martin, S., Longley, T.B., Jarvi, K.A., 2000. Cystic fibrosis gene mutations and infertile men with primary testicular failure. Hum. Reprod. 15, 436–439.

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