1 Department of Andrology, 2 Paediatrics, 3 Pulmonary Diseases, 4 Clinical Genetics and 5 Urology, University Hospital Dijkzigt and Erasmus University, Rotterdam, The Netherlands
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Abstract |
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Key words: CFTR gene analysis/clinical symptoms/congenital bilateral absence of the vas deferens/cystic fibrosis/intestinal current measurement
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Introduction |
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The aetiology of CBAVD is unknown. Most male cystic fibrosis (CF) patients have CBAVD, and it was suggested that CBAVD represents an incomplete form of CF (Holsclaw et al., 1971). Since the identification of the cystic fibrosis transmembrane regulator (CFTR) gene (Riordan et al., 1989
), mutations have been found in 62% of cases of CBAVD (Patricio et al., 1993
; Mercier et al., 1995
; De Braekeleer and Ferec, 1996
).
CBAVD patients have been reported to carry two, one or no CFTR gene mutations, one of them being F508, the most frequent CF mutation. Recently, the R117H mutation, a rare mutation in CF patients, was found to occur frequently in CBAVD patients (Gervais et al., 1993
). Furthermore, the 5T variant of the polypyrimidine stretch in intron 8, which is thought to influence splicing, was shown to occur more frequently in CBAVD as compared to controls (Chu et al., 1993
; Chillon et al., 1995
). In cases where CBAVD is associated with urogenital malformation, CFTR gene mutations appear to be absent, suggesting a different aetiology (Dumur et al., 1996
).
The aim of this study was to investigate whether patients with CBAVD have other CF related non-genital manifestations, and if so, how to improve genetic counselling in case of demand for MESA/ICSI treatment.
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Materials and methods |
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A medical history was obtained focused on symptoms common in CF, such as rhino-sinusitis, nasal polyps, obstructive lung disease, recurrent pulmonary infections, gastro-intestinal malabsorption, fat intolerance, oily stools, cholelithiasis, liver dysfunction and intestinal obstruction. Family history was documented for CF and other genetic abnormalities.
Physical examination
This included urogenital and pulmonary investigation and measurement of weight, height and nutritional state. Pulmonary function tests included forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1). A chest X-ray was performed. The Shwachman score was determined for all patients (Shwachman, 1990). Sonography of the kidneys and transrectal ultrasound was performed to detect urogenital malformations.
Laboratory testing
Laboratory testing included bacterial cultures in sputum, measurements of gonadotrophins [luteinizing hormone (LH) and follicle stimulating hormone (FSH)], liver function, serum glucose level and faecal chymotrypsin. Bilateral sweat tests were performed and, in the absence of normal values for adults, judged as abnormal if chloride >50 mmol/l.
Electrophysiological study
Interstitial current measurement on rectal tissue were performed (Veeze et al., 1994). Rectal suction biopsies were mounted in an Ussing chamber with an exposed area of 1.13 mm2. Sodium channels were blocked by adding amiloride (104 mol/l). Endogenous prostaglandin synthesis that is possibly linked to cAMP-mediated chloride secretion was inhibited by adding indomethacin (105 mol/l). Carbachol (104 mol/l) was added for cholinergic activation of chloride secretion.
In healthy controls, a carbachol-provoked change in interstitial current measurement values was found (control, Figure 1). The inward current in controls reflects transcellular chloride transport (serosa to mucosa) through the NaKCl co-transporter in the basolateral membrane and the CFTRchloride channel in the apical membrane. In the majority of CF patients, a carbachol-induced outward current response (type I negative, Figure 1
) occurs. This reversed response probably results from apical potassium secretion that is unmasked in the case of absent or largely reduced chloride secretion.
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Interstitial current measurements are especially indicated in individuals with borderline or high normal sweat test results and an inconclusive CFTR mutation analysis, who cannot otherwise be distinguished from CF-carriers.
DNA analysis
DNA was isolated from peripheral leukocytes. CFTR mutation analysis was performed for 10 mutations: we analysed for the mutations R117H, A455E, F508, 17171G
A, G542X, R553X, R1162X, S1251N, W1282X, and N1303K. The length of the T-stretch in intron 8 was determined (Kiesewetter et al., 1993
). Only the allele specific oligonucleotide for the identification of the 9 T-stretch was changed into: 5'-TGTGTG TTT TTT TTT AAC AG-3', using a hybridization temperature of 37°C for all allele specific oligonucleotides.
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Results |
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Interstitial current measurement showed either a typical CF response (Figure 1, type I) (n = 4), a low residual chloride secretion (Figure 1
, type II) (n = 1) or a high residual secretion (Figure 1
, type III) (n = 6). The test was found inconclusive in one case and normal (Figure 1
, control) in 10 patients.
CFTR gene analysis showed one or two mutations in 14/21 cases. In eight patients two different mutations (compound heterozygosity) were found; in six patients only one mutation could be identified. In seven cases, no common CFTR gene mutation could be detected: four out of seven of these were non-Caucasians. A 5T allele in one copy of the CFTR gene was found in four cases, three times in combination with a mutation in the other allele.
The F508 mutation was found in eight patients, R117H in six, A445E in three and 17171G
A and R553X both in one. Three partners were found to have a single CFTR gene mutation (R117H, R117H,
F508).
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Discussion |
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In CF, conductive chloride transport is defective in epithelial tissues, resulting in viscous secretions associated with pulmonary infections, malabsorption and intestinal obstruction. The severity of the disease varies widely: homozygosity for the F508 mutation was found to be associated with pancreatic insufficiency, early manifestations, poor lung function and high mortality (Kerem et al., 1990
). Other mutations, like R117H, are associated with a milder form of CF where conductive chloride transport is defective, but not absent (Gervais et al., 1993
).
The CFTR gene mutations occur frequently in CBAVD (Patricio et al., 1993; Oates and Amos, 1994
; de Braekeleer et al., 1996), but the molecular basis of CBAVD is not completely understood. Mutations with a low frequency in classic CF, such as R117H, were found to occur regularly in CBAVD (Gervais et al., 1993
). Homozygosity for
F508 or compound heterozygosity for two severe mutations were not found in cases of CBAVD. It has been suggested that CBAVD patients are compound heterozygotes for a severe mutation on one allele in combination with a mild CFTR gene mutation on the other allele. In the majority of cases, however, only one CFTR gene mutation could be detected in CBAVD. Recently alterations in the non-coding regions of the gene, such as the polypyrimidine stretch in intron 8, in combination with a mutation in the other allele, were found to cause abnormal levels of CFTR protein (Chu et al., 1993
). Impaired CFTR protein function may cause defective, but not absent chloride excretion, resulting in absence of the vas deferens, but not in pulmonary or pancreatic insufficiency (Anguiano et al., 1992
). The epididymis may be more susceptible to defective chloride transport, resulting in an early regression of the mesonephric duct. In contrast, only 6% of CFTR protein function is necessary for normal pancreatic function (Tizzano et al., 1994
). Also, the wide variability of symptoms related to various combinations of CFTR mutations suggests a possible role for unlinked genetic factors in the expression of these mutations.
In this study, 21 patients with CBAVD were investigated for non-genital manifestations of CF: in six patients mild CF symptoms were present. Slightly abnormal liver and pancreatic function were detected in seven, sweat tests showed high levels of chloride in four patients. Electrophysiology of rectal suction biopsies, not previously performed in CBAVD, showed defective chloride excretion in 11 patients. Three of these patients showed very low sweat test results, indicating different tissue expression of impaired CFTR function.
CBAVD appears to be a heterogeneous clinical and genetic condition: two CFTR gene mutations were detected in eight patients, five of them showing CF characteristics on interstitial current measurement. In these men the CBAVD might represent a mild form of CF. Of the patients carrying a single CFTR mutation, four also showed defective chloride excretion on interstitial current measurement, suggesting mutations going undetected with the current screening technology. So far, no convincing evidence has been brought forward that the presence of a single CFTR mutation (i.e. simple heterozygosity) has any phenotypic consequences (Meschede et al., 1997). Therefore, in the case of CBAVD and defective chloride excretion further analysis of the CFTR gene is required to detect rare variant mutations.
In most cases of CBAVD, residual or normal chloride excretion was found in combination with either an abnormal sweat test or CFTR gene mutations. Only in five cases of CBAVD no abnormalities could be found, four of these men being non-Caucasians. These results suggest that there is a wide spectrum of phenotypic expression of cystic fibrosis, with pancreatic and pulmonary insufficiency at one end and CBAVD at the other.
Since the introduction of microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection (Silber et al., 1994) infertility due to CBAVD has been treated successfully, resulting in ongoing pregnancy. Biological parenthood is now a realistic option for males with CBAVD, producing ongoing pregnancies in 2030% of cases (Dohle et al., 1998
).
For couples with CBAVD-related infertility CFTR mutation analysis and genetic counselling of the patient and his partner is essential before MESA/ICSI procedures are performed (Pauer et al., 1997). Although the a priori carrier risk for a CFTR gene mutations is only 34%, three partners of the male CBAVD group had a single CFTR gene mutation. In these cases the risk of offspring with a (mild or severe) form of CF could be 50%.
As there is no straightforward relationship between the genotype and the phenotype for most CFTR gene mutations, genetic counselling in these situations is complex, as no precise predictions on rare compound phenotypes of CF are possible. Considering all the medical and psychological burdens of MESA and ICSI procedures, followed by chorionic biopsy for early prenatal diagnosis of CF, reproduction becomes complicated for these couples. Pre-implantation screening of embryos would be an alternative technique for prenatal diagnosis, but does not solve all ethical problems. In case of a CFTR mutation in the partner and no detectable mutation in the CBAVD male, a positive interstitial current measurement test in the patient will indicate rare variant alleles of the CF gene. However, if no CFTR gene mutations are found in the female partner, the risk of offspring with CF is at the most 1:400. Prenatal or preimplantation screening for CF is not possible in these cases.
In conclusion, CBAVD appears to be a heterogeneous condition with respect to CF symptoms, tissue expression of defective chloride excretion and CFTR gene mutation analysis. Only in a small subset of men with CBAVD could no abnormalities be detected.
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Notes |
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References |
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Submitted on June 29, 1998; accepted on November 4, 1998.