Mukoviszidose – eine pleiotrope Ionenkanalerkrankung mit wesentlicher Lungenbeteiligung

The monogenic disease cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is inherited in an autosomal recessive fashion. Successful diagnosis of the disease is achieved by patient history, clinical assessment, genetic analysis of the CFTR gene and by in vivo measurement and ex vivo characterization of the basic defect in patient’s samples. Frequently, differential diagnosis of CFTR-related disorders such as congenital bilateral absence of the vas deferens (CBAVD), pancreatitis and bronchiectasis needs to be performed. Molecular therapeutics has been developed for some CFTR mutations and these will facilitate direct clinical use of the information provided by CFTR mutation analysis.     

Analysis of the whole CFTR coding regions and splice junctions in azoospermic men with congenital bilateral aplasia of epididymis or vas deferens

Several recent studies have demonstrated the presence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in healthy males with infertility caused by congenital absence of the vas deferens (CBAVD), previously recognized as an idiopathic genetic condition distinct from CF. In order to document further the genetic commonality of these two disorders, we undertook a double screening of the entire coding and flanking sequences of the CFTR gene, by using single-strand conformational polymorphism analysis and denaturing gradient gel electrophoresis in 12 unrelated infertile men with abnormalities of the vas deferens and/or epididymis. This strategy allowed us to identify 11 DNA sequence alterations considered as CF-causing mutations and several variations. Despite this double analysis, only two patients out of eight with CBAVD could be demonstrated as compound heterozygotes for CF mutations.     

'CFTR-opathies': disease phenotypes associated with cystic fibrosis transmembrane regulator gene mutations

Cystic fibrosis is a genetic disease that is associated with abnormal sweat electrolytes, sino-pulmonary disease, exocrine pancreatic insufficiency, and male infertility. Insights into genotype/phenotype relations have recently been gained in this disorder. The strongest relationship exists between 'severe' mutations in the gene that encodes the cystic fibrosis transmembrane regulator (CFTR) and pancreatic insufficiency. The relationship between 'mild' mutations, associated with residual CFTR function, and expression of disease is less precise. Atypical 'mild' mutations in the CFTR gene have been linked to late-onset pulmonary disease, congenital bilateral absence of the vas deferens, and idiopathic pancreatitis. Less commonly, sinusitis, allergic bronchopulmonary aspergillosis, and possibly even asthma may also be associated with mutations in the CFTR gene, but those syndromes predominantly reflect non-CFTR gene modifiers and environmental influences.     

Cystic fibrosis as a cause of infertility

Cystic fibrosis (CF) is one of the autosomal recessive diseases, caused by mutations in a gene known as cystic fibrosis transmembrane regulator (CFTR). The majority of adult males with CF (99%) is characterized by congenital bilateral absence of vas deferens (CBAVD). CBAVD is encountered in 1-2% of infertile males without CF. Females with CF are found to be less fertile than normal healthy women. In females with CF, delayed puberty and amenorrhoea are common due to malnutrition. CFTR mutations are also associated with congenital absence of the uterus and vagina (CAUV). The National Institutes of Health recommend genetic counseling for any couple seeking assisted reproductive techniques with a CF male or obstructive azoospermia which is positive for a CF mutation.     

Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens

Congenital absence of the vas deferens (CAVD) is a frequent cause for obstructive azoospermia and accounts for 1%–2% of male infertility. A high incidence of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has recently been reported in males with CAVD. We have investigated a cohort of 106 German patients with congenital bilateral or unilateral absence of the vas deferens for mutations in the coding region, flanking intron regions and promotor sequences of the CFTR gene. Of the CAVD patients, 75% carried CFTR mutations or disease-associated CFTR variants, such as the “5T” allele, on both chromosomes. The distribution of mutation genotypes clearly differed from that observed in cystic fibrosis. None of the CAVD patients was homozygous for ΔF508 and none was compound heterozygous for ΔF508 and a nonsense or frameshift mutation. Instead, homozygosity was found for a few mild missense or splicing mutations, and the majority of CAVD mutations were missense substitutions. Twenty-one German CAVD patients were compound heterozygous for ΔF508 and R117H, which was the most frequent CAVD genotype in our study group. Haplotype analysis indicated a common origin for R117H in our population, whereas another frequent CAVD mutation, viz. the “5T allele” was a recurrent mutation on different intragenic haplotypes and multiple ethnic backgrounds. We identified a total of 46 different mutations and variants, of which 15 mutations have not previously been reported. Thirteen novel missense mutations and one unique amino-acid insertion may be confined to the CAVD phenotype. A few splice or missense variants, such as F508C or 1716 G→A, are proposed here as possible candidate CAVD mutations with an apparently reduced penetrance. Clinical examination of patients with CFTR mutations on both chromosomes revealed elevated sweat chloride concentrations and discrete symptoms of respiratory disease in a subset of patients. Thus, our collaborative study shows that CAVD without renal malformation is a primary genital form of cystic fibrosis in the vast majority of German patients and links the particular expression of clinical symptoms in CAVD with a distinct subset of CFTR mutation genotypes. Received: 15 April 1997 / Accepted: 29 April 1997     

Congenital absence of the vas deferens: a mild form of cystic fibrosis

Genetic diseases presenting with different phenotypes are generally classified as distinct disorders before their molecular defect is revealed, as exemplified by the recent advance in understanding of the molecular biology of cystic fibrosis and an obstructive form of infertility, known as congenital absence of the vas deferens. The majority of men with congenital absence of the vas deferens have a defect in both copies of the CFTR gene and therefore represent a distinct phenotypic form of cystic fibrosis. These developments help us to gain new insight into the genetic basis of phenotypic variability and the possible contributing mechanisms in cystic fibrosis. Some of the lessons learned from the relationship between cystic fibrosis and congenital absence of the vas deferens may be useful in the understanding of other genetic disorders.     

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

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.     

Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? Analyses of the CFTR gene in 67 patients.

Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of sterility in men. Although the genetic basis of this condition is still unclear, it has been shown recently that some of these patients carry mutations in their cystic fibrosis transmembrane conductance regulator (CFTR) genes. To extend this observation, we have analyzed the entire coding sequence of the CFTR gene in a cohort of 67 men with CBAVD, who are otherwise healthy. We have identified four novel missense mutations (A800G, G149R, R258G, and E193K). We have shown that 42% of subjects were carriers of one CFTR allele and that 24% are compound heterozygous for CFTR alleles. Thus, we have been unable to identify 76% of these patients as carrying two CFTR mutations. Furthermore, we have described the segregation of CFTR haplotypes in the family of one CBAVD male; in this family are two male siblings, with identical CFTR loci but displaying different phenotypes, one of them being fertile and the other sterile. The data presented in this family, indicating a discordance between the CBAVD phenotype and a marked carrier (delta F508) chromosome, support the involvement of another gene(s), in the etiology of CBAVD.     

Absence de corrélation génotype-phénotype dans les absences de canaux déférents

Absence of the vas deferens is a rare cause of male infertility, associated with mutations in the cystic fibrosis transmembrane regulator (CFTR) gene in about 80% of cases. Only limited published data are available concerning the correlation between genotype and reproductive tract abnormalities observed in this disease: presence or absence of seminal vesicles and parts of the epididymis, symmetrical or asymmetrical lesions, testicular volumes. We screened 47 patients for the 13 most common CFTR mutations on the cystic fibrosis gene and for the 5-thymidine variant of the polythymidine tract of intron 8. Renal, scrotal and transrectal ultrasonography was performed in each patient to explore the testes and reproductive tract. All patients presented absence of the ampullae of the vas deferens. Forty patients presented bilateral absence of the vas deferens and 7 presented unilateral absence of the vas deferens. At least one mutation of the cystic fibrosis gene was present in 64% of cases: 47% had the ΔF 508 mutation and 63% had the 5T allele. No mutation was detected in seventeen patients, including 3 patients with unilateral renal agenesis and 3 patients with unilateral absence of the vas deferens. No differences were observed for seminal vesicles and symmetry of vesicular and epididymal abnormalities between patients with or without CFTR gene mutations, but epididymal abnormalities were significantly more frequent in the group without mutation (p=0.01). Testicular volumes were significantly lower in the patients without mutation or with the 5T allele only, than in the patients with at least one CFTR gene mutation: 10.7±4.1 ml versus 15.1±4.5 ml, respectively (p<0.001). In conclusion, in cases of isolated absence of the vas deferens, there is no difference in sperm duct abnormalities between patients with or without CFTR gene mutation. These results suggest that other genetic or environmental determinants are required to explain a common pathogenesis for these malformations. The decreased testicular volume of patients without CFTR gene mutation or with the 5T allele only suggests the existence of an unidentified secretory or mixed factor involved in these forms of absence of the vas deferens.     

Extensive analysis of 40 infertile patients with congenital absence of the vas deferens: in 50% of cases only one CFTR allele could be detected

Mutations in the cystic fibrosis (CF) conductance transmembrane regulator (CFTR) gene have been detected in patients with CF and in males with infertility attributable to congenital bilateral absence of the vas deferens (CBAVD). Thirty individuals with CBAVD and 10 with congenital unilateral absence of the vas deferens (CUAVD) were analyzed by single-strand conformation analysis and denaturing gradient gel electrophoresis for mutations in most of the CFTR gene. All 40 individuals were pancreatic sufficient, but twenty patients had recurrent or sporadic respiratory infections, asthma/asthmatic bronchitis, and/or rhino-sinusitis. Agenesia or displasia of one or both seminal vesicles was detected in 30 men and other urogenital malformations were present in six subjects. Among the 40 samples, we identified 13 different CFTR mutations, two of which were previously unknown. One new mutation in exon 4 was the deletion of glutamic acid at codon 115 (E115). A second new mutation was found in exon 17b, viz., an AC substitution at position 3311, changing lysine to threonine at codon 1060 (K1060T). CFTR mutations were detected in 22 out of 30 (73.3%) CBAVD patients and in one out of 10 (10%) CUAVD individuals, showing a significantly lower incidence of CFTR mutations in CBAVD/CUAVD patients (P 0.0001), compared with that found in the CF patient population. Only three CBAVD patients were found with more than one CFTR mutation (F508/L206W, F508/R74W+D1270N, Rl 17H/712-1GT), highlighting L206W, R74W/ D1270N, and R117H as benign CF mutations. Sweat electrolyte values were increased in 76.6% of CBAVD patients, but three individuals without CFTR mutations had normal sweat electrolyte levels (10% of the total CBAVD patients), suggesting that factors other than CFTR mutations are involved in CBAVD. The failure to identify a second mutation in exons and their flanking regions of the CFTR gene suggests that these mutations could be located in introns or in the promoter region of CFTR. Such mutations could result in CFTR levels below the minimum 6%–10% necessary for normal protein function.     

Effects of cystic fibrosis and congenital bilateral absence of the vas deferens-associated mutations on cystic fibrosis transmembrane conductance regulator-mediated regulation of separate channels

The protein defective in cystic fibrosis (CF), the CF transmembrane-conductance regulator (CFTR), functions as an epithelial chloride channel and as a regulator of separate ion channels. Although the consequences that disease-causing mutations have on the chloride-channel function have been studied extensively, little is known about the effects that mutations have on the regulatory function. To address this issue, we transiently expressed CFTR-bearing mutations associated with CF or its milder phenotype, congenital bilateral absence of the vas deferens, and determined whether mutant CFTR could regulate outwardly rectifying chloride channels (ORCCs). CFTR bearing a CF-associated mutation in the first nucleotide-binding domain (NBD1), DeltaF508, functioned as a chloride channel but did not regulate ORCCs. However, CFTR bearing disease-associated mutations in other domains retained both functions, regardless of the associated phenotype. Thus, a relationship between loss of CFTR regulatory function and disease severity is evident for NBD1, a region of CFTR that appears important for regulation of separate channels.     

Heterogeneity in the severity of cystic fibrosis and the role of CFTR gene mutations

Cystic fibrosis is a common, fatal disorder caused by abnormalities in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a chloride channel that regulates secretion in many exocrine tissues. The presentation of cystic fibrosis is highly variable as measured by the age of onset of disease, the presence of pancreatic insufficiency, or the progression of lung disease. Over 400 mutations in the CFTR gene have been described in cystic fibrosis patients and considerable effort has focused on the correlation between specific mutations and genotypes and clinical characteristics. Individual tissues display variation in their sensitivity to CFTR mutations. The vas deferens is functionally disrupted in nearly all males, whereas mild and severe pancreatic involvement is determined by the patient's genotype. The severity of pulmonary disease is poorly correlated with genotype, suggesting that there are other important genetic and/or environmental factors that contribute to lung infections and the subsequent disruption of lung function.     

Cse1l is a negative regulator of CFTR-dependent fluid secretion

Transport of chloride through the cystic fibrosis transmembrane conductance regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates [1, 2]. Loss of CFTR function leads to cystic fibrosis [1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine, and vas deferens. Conversely, uncontrolled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera [5, 6] and other secretory diarrheas [7] as well as polycystic kidney disease [8-10]. Understanding how CFTR activity is regulated in?vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation, and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the uncontrolled activation of the CFTR channel. Analyses in zebrafish larvae and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much-needed model system to study CFTR regulation in?vivo.     

CFTR Haplotype Analysis Reveals Genetic Heterogeneity in the Etiology of Congenital Bilateral Aplasia of the Vas Deferens

Congenital bilateral aplasia of the vas deferens (CBAVD) was suggested to be a mild form of cystic fibrosis (CF). Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in males with CBAVD revealed that in some males CBAVD is caused by two defective CFTR alleles. The genetic basis of CBAVD in the other males and its association with CF remained unclear. We undertook this study to test the hypothesis of commonality of CBAVD and CF by haplotype analysis, in the CFTR locus, of males suffering from CBAVD and of their families. According to the hypothesis of commonality of CBAVD and CF, two brothers with CBAVD are expected to carry the same two CFTR alleles, while their fertile brothers are expected to carry at least one different allele. Eleven families were studied, of which two families, with unidentified CFTR mutations, did not support this hypothesis. In these families two brothers with CBAVD inherited different CFTR alleles. Their fertile brothers inherited the same CFTR alleles as their brothers with CBAVD. These results provide evidence for genetic heterogeneity in CBAVD. Though in some families CBAVD is associated with two CFTR mutations, we suggest that in others it is caused by other mechanisms, such as mutations at other loci or homozygosity or heterozygosity for partially penetrant CFTR mutations.     

Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign

An abbreviated tract of five thymidines (5T) in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is found in approximately 10% of individuals in the general population. When found in trans with a severe CFTR mutation, 5T can result in male infertility, nonclassic cystic fibrosis, or a normal phenotype. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, we determined TG repeat number in 98 patients with male infertility due to congenital absence of the vas deferens, 9 patients with nonclassic CF, and 27 unaffected individuals (fertile men). Each of the individuals in this study had a severe CFTR mutation on one CFTR gene and 5T on the other. Of the unaffected individuals, 78% (21 of 27) had 5T adjacent to 11 TG repeats, compared with 9% (10 of 107) of affected individuals. Conversely, 91% (97 of 107) of affected individuals had 12 or 13 TG repeats, versus only 22% (6 of 27) of unaffected individuals (P<.00001). Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats (odds ratio 34.0, 95% CI 11.1-103.7, P<.00001). Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles.     

Splicing factors induce cystic fibrosis transmembrane regulator exon 9 skipping through a nonevolutionary conserved intronic element

In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens, variations in the TG(m) and T(n) polymorphic repeats at the 3' end of intron 8 of the cystic fibrosis transmembrane regulator (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have previously shown a direct relationship between the TG(m)T(n) polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG(m) and T(n) polymorphic repeats. The comparative and functional analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG(m) and T(n) polymorphism at the 3' splice region, enhancer and silencer exonic elements, and the intronic splicing silencer in the proximal 5' intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG(m)T(n) locus in monosymptomatic forms of cystic fibrosis.     

Imagerie des glandes annexes de la voie séminale

Ultrasonography (US) is presently routine part of the investigation of infertile men with low volume ejaculate. It provides excellent depiction of the congenital or acquired obstructive lesions of the reproductive system, particulary in its distal part. For 10 years, we performed a US examination of the kidneys, the scrotum contents and transrectal US (TRUS) in every case a excretory cause of male hypofertility could be suspected. In selected patients, MR imaging (with endorectal coil) appears usefull. We describe the normal and abnormal anatomy of the epididymis, vas deferens, seminal vesicle (SV) and ejaculatory ducts in male infertility with US and MRI. Dilatation of the small ducts in the epididymis and sometimes in the testis at scrotal US is suggestive of a downstream obstacle. Sometimes these dilatation could appear as small hypoechoic areas in the epididymis. In contrast, post-inflammatory changes are hyperechoic but often associated with dilatations. TRUS permits to confirm the absence of the vas deferens in congenital bilateral absence of vas deferens (CBAVD): it shows the absence of the ampullae and severe abnormalities of the SV. In case of unilateral absence of the vas deferens, the association with a homolateral kidney agenesis does not lead to the screening for mutations in the cystic fibrosis gene, but suggests a wolffian duct abnormality. MRI (with endorectal coil) is indicated when TRUS is unconclusive. Scrotal US permits to guide semen aspiration and TRUS is indicated to eliminate a distal obstruction when a surgical anastomosis is planed. TRUS (as well as scrotal US) can suggest an obstacle when dilatation of one or several segments of the seminal tract is observed. Sometimes the cause is obvious with imaging: CBAVD, prostatic cyst, inflammatory and post-infectious changes, lithiasis etc. However, it remains many cases where US is only one component of the therapeutic decision, besides clinical examination, sperm count, FSH level and biochemical sperm markers.     

High‐resolution imaging of the actin cytoskeleton and epithelial sodium channel,CFTR, and aquaporin‐9 localization in the vas deferens

Vas deferens is a conduit for sperm and fluid from the epididymis to the urethra. The duct is surrounded by a thick smooth muscle layer. To map the actin cytoskeleton of the duct and its epithelium, we reacted sections of the proximal and distal regions with fluorescent phalloidin. Confocal microscopic imaging showed that the cylinder‐shaped epithelium of the proximal region has a thick apical border of actin filaments that form microvilli. The epithelium of the distal region is covered with tall stereocilia (13–18 µm) that extend from the apical border into the lumen. In both regions, the lateral and basal cell borders showed a thin lining of actin cytoskeleton. The vas deferens epithelium contains various channels to regulate the fluid composition in the lumen. We mapped the localization of the epithelial sodium channel (ENaC), aquaporin‐9 (AQP9), and cystic fibrosis transmembrane conductance regulator (CFTR) in the rat and mouse vas deferens. ENaC and AQP9 immunofluorescence were localized on the luminal surface and stereocilia and also in the basal and smooth muscle layers. CFTR immunofluorescence appeared only on the luminal surface and in smooth muscle layers. The localization of all three channels on the apical surface of the columnar epithelial cells provides clear evidence that these channels are involved concurrently in the regulation of fluid and electrolyte balance in the lumen of the vas deferens. ENaC allows the flow of Na⁺ ions from the lumen into the cytoplasm, and the osmotic gradient generated provides the driving force for the passive flow of water through AQP channels.     

Cystic fibrosis in Uruguay

We conducted clinical and genetic analyses of 52 cystic fibrosis (CF) patients in Uruguay, which is about half of the known affected individuals in the country. A relatively high proportion had a mild presentation, characterized by pancreatic sufficiency (28%), a strong pulmonary component (97%), and borderline sweat electrolyte measurements (25%). Mutational analysis of CF chromosomes demonstrated a relatively low incidence of the DeltaF508 allele (40%) and a large number of other cystic fibrosis conductance regulator mutations, with an overall detection rate of about 71%. Fifteen different mutations were detected in our patients: DeltaF508, G542X, R1162X, G85E, N1303K, R334W, R75Q, R74W, D1270N, W1282X, DeltaI507, 2789+5G-->A, R1066C, -816C/T, R553X, as well as RNA splicing variant IVS8-5T. This group of Uruguayan CF patients has some characteristics in common with other populations of similar origin (Hispanics), as well as some unique characteristics.