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Correlation Between CFTR Gene Mutations in Iranian Men With Congenital Absence of the Vas Deferens and Anatomical Genital Phenotype

MOHAMAD ALI SADIGHI GILANI

AHMAD VOSOUGH DIZAJ

From the ^* Department of Reproductive Genetics and the Department of Male Infertility, Reproductive Biomedicine Research Center of Royan Institute, Tehran, Iran.

Correspondence to: Ramin Radpour, Department of Reproductive Genetics, Reproductive Biomedicine Research Center of Royan Institute, PO Box 19395-4644, Tehran, Iran (e-mail: rradpour{at}royaninstitute.org).

Received for publication April 6, 2007; accepted for publication July 30, 2007.

	Abstract

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Congenital bilateral absence of the vas deferens (CBAVD) and congenital unilateral absence of the vas deferens (CUAVD) are 2 causes of male sterility; these phenotypes are found in 1%–2% of men investigated for infertility and approximately 10% of men with azoospermia. To study the correlation between genital phenotype and cystic fibrosis genotype in men lacking at least 1 vas deferens, we evaluated the role of different CFTR gene mutations in the morphologic genital phenotype of 119 infertile men with bilateral or unilateral absence of the vas deferens (112 CBAVD and 7 CUAVD patients). Renal, scrotal, and transrectal ultrasonography were systematically performed. CFTR mutations and (TG)m(T)n polymorphism were analyzed, and epididymal and seminal vesicular abnormalities and testicular volume were compared among men with 2, 1, or no CFTR gene mutation, with or without the 5T allele. Our results showed that patients with CBAVD and renal agenesis have the same reproductive tract abnormalities as those with CUAVD, and reproductive tract abnormalities were independent of the subtypes of CFTR genotype in patients with absence of the vas deferens and CFTR gene mutations. Seminal vesicles did not differ between patients with or without CFTR gene mutation, but epididymal abnormalities were more frequent in CBAVD men without the mutation. Low testicular volume was observed in CBAVD men without the CFTR and IVS8-5T mutations, so we can hypothesize that a testicular factor (genetic or environmental) rather than CFTR gene mutations plays a role in determining the phenotype. Further studies using common diagnostic criteria are required to confirm our observations.

     Key words: CBAVD, congenital bilateral absence of the vas deferens, IVS8-5T, male infertility

More than 1500 cystic fibrosis (CF)–causing CFTR mutations have been identified (Cystic Fibrosis Mutation Database, 2007), About 35% of Iranian males with CBAVD have a single detectable CFTR mutation or polymorphism, whereas about 40% have 2 mutations (common CFTR mutations or IVS8-5T polymorphism) (Radpour et al, 2007). About 88% of CBAVD patients in which a mutation is found on both CFTR genes carry 1 severe mutation on 1 CFTR gene and a mild mutation on the second CFTR gene, and about 12% carry mild mutations on both CFTR genes (Claustres et al, 2000). The most frequent CFTR mutation conferring a mild phenotype found in CBAVD patients is the 5T polymorphism (Chillon et al, 1995). 5T is one of the alleles found at the polymorphic Tn locus in intron 8 of the CFTR gene. The 5T polymorphism can be found in combination with either a TG11, TG12, or TG13 allele (11, 12, or 13 TG repeats, respectively).

Clinical symptoms of CBAVD are bilateral nonpalpable vas deferens, absence of the distal part of or the whole epididymis, and hypotrophy or absence of the seminal vesicles, leading to azoospermia with low semen plasma volume and acidic pH. Because the severity of CF is to some extent related to CFTR genotype, we tested this correlation between CBAVD and CUAVD with CFTR genotype. To study the correlation between genital phenotype and CF genotype in men lacking at least 1 vas deferens, we evaluated the role of different CFTR gene mutations in the morphologic genital phenotype of 119 infertile men with bilateral or unilateral absence of the vas deferens (112 CBAVD and 7 CUAVD patients). Also, epididymal and seminal vesicular abnormalities and testicular volume were compared among men with 2, 1, or no CFTR gene mutation, with or without the 5T allele.

	Materials and Methods

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Samples

Blood samples were collected from 119 unrelated Iranian males visiting the Reproductive Biomedicine Research Center of Royan Institute, Iran, with azoospermia and bilateral or unilateral absence of the vas deferens. All patients had a complete physical examination by the same physician, with particular attention to the testes, vasa deferentia, epididymides, and prostate gland (evaluated by digital rectal examination). The diagnosis of CBAVD or CUAVD patients was initially suggested by impalpable scrotal vas on physical examination and transabdominal/-rectal ultrasonography (Table 1), subsequently confirmed by cytobiochemical characteristics (Table 2)—azoospermia with low semen volume (<1.5 mL) and decrease of fructose (vesicular marker) and carnitine (epididymal marker) concentrations—followed by hormonal analysis (Table 3) according to World Health Organization criteria (1999). Testicular volume was evaluated by orchidometry. Also we studied 84 fertile males from the general population in Iran as control subjects.

CFTR Mutation Scanning

As previously reported, DNA samples were analyzed, and all 27 exons of CFTR were amplified by polymerase chain reaction (PCR) and studied by denaturing gradient–gel electrophoresis or by single-strand conformation analysis (Radpour et al, 2006a). Long-range PCR was performed to analyze the TGmTn site (Radpour et al, 2007). The results were confirmed by sequencing of PCR products. Sequencing results were compared with the wild-type CFTR gene sequence published in the Cystic Fibrosis Mutation Database (http://www.genet.sickkids.on.ca/cftr/).

Statistical Analysis

Six subgroups were analyzed to compare reproductive tract abnormalities: 2 CFTR gene mutations, 1 CFTR gene mutation and 5T allele, 1 CFTR gene mutation, 2 5T alleles, 5T alone, and no mutation. A Punnett square was constructed with the raw data and compared with the predicted frequencies of each reported class with 5T and other mutations. Differences between percentages were tested with the ² test. Means (±SD) were compared with the Student's t test (SPSS software version 14.0; SPSS Inc, Chicago, Ill). All P values were 2-sided; P < .05 was considered statistically significant.

	Results

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After clinical investigations, 119 infertile men lacked at least 1 vas deferens, as determined by scrotal palpation. Absence of the vas deferens was confirmed by absence of the distal portion of the vas deferens (ampulla) on transrectal ultrasonography. Of the 119 patients, 112 had isolated CBAVD, and none of them had unilateral renal agenesis. Seven patients had CUAVD, of whom 1 had unilateral renal agenesis. Scrotal ultrasonography could not find dilatation of the rete testis and intratesticular calcification in studied patients.

The analysis of the entire coding sequences of CFTR gene, allowed us to identify 19 different mutations in Iranian CBAVD patients (Radpour et al, 2007). These mutations have been described previously in Iranian patients with CBAVD (Radpour et al, 2006a,b). Of these, 5 cases were homozygous or compound heterozygous (+/+), 67 were positive for only 1 mutation (+/-), and 49 cases were negative for both mutations (-/-) (IVS8-5T was not involved). The result of our study reflects the high allelic heterogeneity of CFTR gene mutations, although 2 mutations, IVS8-5T and F508del, were found to be more common in Iranian CBAVD patients. Also a Punnett square was constructed with the raw data and compared with the predicted frequencies of each reported class with 5T and the other mutations. The frequencies of no mutation vs 5T, another mutation, or 5T with another mutation were predicted for CBAVD with a 62% mutation pickup rate, and 38% of cases were not detectable (Figure).

The 119 patients (112 CBAVD and 7 CUAVD) were classified into 6 subgroups according to genotype to compare morphologic genital findings (Tables 4 and 5). Subgroups did not differ in seminal vesicle status and symmetry of vesicular and epididymal abnormalities. Two patients with 2 CFTR gene mutations lacked at least 1 seminal vesicle, and 2 of them were dilated. Thirty-five patients had only 1 CFTR mutation and no 5T allele; 12 lacked at least 1 seminal vesicle. From 11 patients with 2 5T alleles, seminal vesicles were absent in 4 patients, 3 were hypotrophic, and 1 was dilated. Both seminal vesicles were absent in 3 patients with the 5T allele only. Of the 22 men in whom no mutation was detected, seminal vesicles were absent or hypotrophic in 16 cases (Table 4). Of the 7 men with unilateral absence of the vas deferens, 2 had the 5T allele and no other CFTR gene mutation (Table 5). Epididymal abnormalities in CBAVD patients were significantly more frequent in the group without mutation than in the group with CFTR gene mutation, including the 5T allele (82% compared with 45%, respectively; P = .01, ² test). Also, testicular volume in CBAVD patients with no CFTR mutations was significantly smaller than in men with CFTR or IVS8-5T mutations (Table 4) (15.3 ± 4.5 mL compared with 11.2 ± 3.2 mL, respectively; P < .001, Student's t test).

View larger version (14K): [in this window] [in a new window]   Figure. Punnett square for comparing the raw data to the predicted frequencies of each reported class with 5T and other mutations. A: All kind of CFTR mutations + IVS8-5T polymorphic allele joined together as mutated allele (M) and compared with normal alleles. B: CFTR mutations and IVS8-5T compared with normal alleles separately. Actual number of cases are given in square brackets and the expected number of patients among 112 in parentheses below the calculated frequency. *Statistically different between observed and predicted allele frequencies. M indicates CFTR mutated allele; 5T, IVS8-5T polymorphic allele; and -, normal allele (no mutation).

Taken together, seminal vesicle abnormalities did not differ between patients with or without CFTR gene mutations, but epididymal abnormalities were more frequent in CBAVD men without the mutation. Low testicular volume was observed in CBAVD men without the CFTR and IVS8-5T mutations.

	Discussion

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Men with CBAVD but without CFTR gene mutations have a high incidence of urinary tract malformations (Dork et al, 1997). The group with urinary tract anomalies represents a separate clinical entity not related to CF and with different embryological pathogenesis. Some forms of infertility found in otherwise healthy men have also been reported to be associated with CFTR mutations, especially obstructive azoospermic conditions such as CBAVD, CUAVD, epididymal obstruction, and bilateral ejaculatory duct obstruction with concomitant seminal vesicle anomalies (Welsh and Smith, 1995).

We found that patients with CBAVD and a CFTR mutation had the same seminal vesicle phenotype as patients without CFTR mutation. Also, the same result was obtained in CUAVD patients. Epididymal abnormalities were more frequent in men without mutation. These data are similar to previous findings in patients with CBAVD (Jarvi et al, 1998; Robert et al, 2002). Among men studied with scrotal ultrasonography, the frequency of intrascrotal abnormalities did not differ among subgroups of men with or without CFTR gene mutations. Daudin et al (2000) found no correlation between epididymal and seminal vesicle morphology and between CFTR gene mutations and the 5T allele in 32 patients. In contrast, our findings showed epididymal abnormalities were more frequent in CBAVD men without the mutation (P = .001). Because the number of CUAVD patients in our study was few, further studies are required for confirming genotype/phenotype analysis in Iranian CUAVD patients. Construction of Punnett squares of each reported class with 5T and other mutations showed that more significant differences exist between observed and predicted allele frequencies (Figure), and this finding supports the existence of another unlinked gene that is causing additional phenotypic abnormalities in addition to the existing abnormalities found within the other categories. In the majority of cases, CBAVD can be considered a genital form of CF, presenting without the other clinical features of CF. Generally, 20% have 2, 60% have 1, and 20% have no CFTR mutations. (Claustres, 2005). In our study, 19.64% of patients had no CFTR mutations (Table 4).

Analyses of the correlation between phenotype and genotype showed that the CFTR mutations could be grouped into 2 categories, mild or severe, with respect to pancreatic function (Kristidis et al, 1992). The severe mutations are associated with pancreatic insufficiency, whereas the mild mutations, leading to a higher residual CFTR activity, confer pancreatic sufficiency (Kristidis et al, 1992). A CF patient is likely to be pancreatic sufficient if he has 1 or 2 mild mutations. Several studies have shown that some genotypes are associated with a severe phenotype, whereas others have a milder clinical effect and evolution. In this study we could not find any reliable relation between CFTR gene mutations and CBAVD or CUAVD phenotype, and this is probably explained by defects in the genital ducts, from CFTR dysfunction, that occur after the splitting of the wolffian duct into its reproductive and ureteral parts at 7 weeks of gestation.

Given that CFTR mutations are detected in men with CBAVD and, with a lower frequency, in men with CUAVD, it appears that a normal amount of a functional CFTR protein is required to ensure proper development of the vas deferens. However, the lack of CFTR mutations detection in CBAVD with renal abnormalities also suggests that the mesonephric duct plays an important role in the development of the vas deferens. Therefore, it can hypothesize that the CFTR protein is required at some specific embryonic stage of the development of the vas deferens. The detection of CFTR mRNA in human fetuses suggests that CFTR acts as a chloride channel early in the development of the reproductive tissues (Tizzano et al, 1993). Also the epididymis and the vas deferens form a tortuous ductal system that is 7 m long but has a diameter less than 0.5 mm. The absence or dysfunction of CFTR would make these organs very vulnerable to luminal concentration defects, especially in the distal portion of the vas deferens, where CFTR expression level is low.

Testicular volume is small in about 5%–10% of patients with CBAVD (Jarvi et al, 1998; Daudin et al, 2000). Robert et al (2002) found a correlation between testicular volume and no CFTR mutation, for which testicular volume was smallest in these groups; this result was found in 47 patients. Our comprehensive study of 119 obstructive azoospermia confirmed the correlation between testicular volume and no CFTR or IVS8-5T mutations, for which testicular volume in CBAVD patients without detectable CFTR mutations were significantly low (P = .001). Therefore, we can hypothesize that a testicular factor (genetic or environmental) rather than CFTR gene mutations plays a role in determining the phenotype. Because of this contradiction, further studies and standardization of testicular volume are needed to confirm these observations.

Our results showed that patients with CBAVD and renal agenesis have the same reproductive tract abnormalities as those with CUAVD, and reproductive tract abnormalities were independent of the subtypes of CFTR genotype in patients with absence of the vas deferens and CFTR gene mutations. But only CBAVD men without CFTR gene mutations had more epididymal abnormalities. CBAVD men without CFTR gene mutations had smaller testicular volume. These different patterns could share some of the same pathogenic factors. Further studies with common diagnostic criteria are required to confirm the results.

	Acknowledgments

 
We are indebted to the CBAVD patients for their cooperation. The Online Mendelian Inheritance of Man database (http://www.ncbi.nlm.nih.gov/omim/) was used to obtain data for this article (CBAVD [MIM #277180], CFTR [MIM *602421], and CF [MIM #219700]).

	Footnotes

 
This research was supported by grants from the Reproductive Biomedicine Research Center of Royan Institute of Iran.

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