Chimeric beta-defensin analogs, including the novel 3NI analog, display salt-resistant antimicrobial activity and lack toxicity in human epithelial cell lines.

Human beta-defensins (hBDs) are crucial peptides for the innate immune response and are thus prime candidates as therapeutic agents directed against infective diseases. Based on the properties of wild-type hBD1 and hBD3 and of previously synthesized analogs (1C, 3I, and 3N), we have designed a new analog, 3NI, and investigated its potential as an antimicrobial drug. Specifically, we evaluated the antimicrobial activities of 3NI versus those of hBD1, hBD3, 1C, 3I, and 3N. Our results show that 3NI exerted greater antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis than did hBD1 and hBD3, even with elevated salt concentrations. Moreover, its antiviral activity against herpes simplex virus 1 was greater than that of hBD1 and similar to that of hBD3. Subsequently, we investigated the cytotoxic effects of all peptides in three human epithelial carcinoma cell lines: A549 from lung, CaCo-2 from colon, and Capan-1 from pancreas. None of the analogs significantly reduced cell viability versus wild-type hBD1 and hBD3. They did not induce genotoxicity or cause an increase in the number of apoptotic cells. Using confocal microscopy, we also investigated the localization of the peptides during their incubation with epithelial cells and found that they were distributed on the cell surface, from which they were internalized. Finally, we show that hBD1 and hBD3 are characterized by high resistance to serum degradation. In conclusion, the new analog 3NI seems to be a promising anti-infective agent, particularly given its high salt resistance--a feature that is relevant in diseases such as cystic fibrosis.

Genes that determine immunology and inflammation modify the basic defect of impaired ion conductance in cystic fibrosis epithelia.

BACKGROUND: The cystic fibrosis (CF) basic defect, caused by dysfunction of the apical chloride channel CFTR in the gastrointestinal and respiratory tract epithelia, has not been employed so far to support the role of CF modifier genes. METHODS: Patients were selected from 101 families with a total of 171 F508del-CFTR homozygous CF patients to identify CF modifying genes. A candidate gene based association study of 52 genes on 16 different chromosomes with a total of 182 genetic markers was performed. Differences in haplotype and/or diplotype distribution between case and reference CF subpopulations were analysed. RESULTS: Variants at immunologically relevant genes were associated with the manifestation of the CF basic defect (0.01

Benchmarks for cystic fibrosis carrier screening: a European consensus document.

This paper presents an overview of the conclusions from an international conference convened to address current issues related to the provision of Cystic Fibrosis carrier screening within Europe. Consensus was not aimed at stating whether such a programme should be implemented. Instead the focus was to provide a framework for countries and agencies who are considering or planning its establishment. The general principles and target population of Cystic Fibrosis carrier screening, advantages and disadvantages, health economics, monitoring and future evaluative and research directions were covered. A range of screening strategies have been assessed and compared: pre-conceptional and prenatal screening; individual and couple screening; sequential and simultaneous sampling or testing. Furthermore, technical issues were examined with respect to the choice of the panel of mutations, its detection rate, sensitivity, management of intermediate 'at-risk' couples, screening approach to different populations and ethnic minorities, and assurance of laboratory quality control. The consensus statement also aims to establish the benchmarks for communicating with health care providers, the general public and potential and actual participants before and after the genetic test.

Genomic copy number determines functional expression of {beta}-defensin 2 in airway epithelial cells and associates with chronic obstructive pulmonary disease.

RATIONALE: Copy number variations of the cluster of beta-defensin genes have been associated with psoriasis and inflammatory bowel disease. Controversy still exists on whether the beta-defensins genes determine susceptibility for chronic obstructive pulmonary disease (COPD). OBJECTIVES: We investigated whether genomic copy number variations of the beta-defensin gene cluster have a functional role in airway epithelial cells and associate with the presence of COPD. METHODS: Baseline and inflammatory induced transcript expression of DEFB4 was studied in nasal epithelial cell cultures and its effect on Pseudomonas aeruginosa inhibition was assessed. Subsequently, relevant functional cut-offs for copy numbers were used to explore associations with COPD in two independent case-control studies. MEASUREMENTS AND MAIN RESULTS: Copy number variation in the beta-defensin encoding genes correlated with baseline mRNA DEFB4 expression levels (R(2) = 0.96; P = 0.02), with a plateau effect from five copies or more. Only when higher copy numbers of beta-defensin genes were present, transcription was significantly up-regulated by tumor necrosis factor-alpha (P < 0.0001), which resulted in better antimicrobial activity in vitro. When comparing healthy smokers with COPD patients, a copy number greater than or equal to 5 was associated with increased risk for COPD with an adjusted odds ratio of 1.8 (confidence interval, 1.1-2.8; P = 0.02), which was confirmed by a second independent case-control study. CONCLUSIONS: Genomic copy number variation of beta-defensin encoding genes has a functional role in airway epithelial cells, which may contribute to the pathogenesis of COPD.

Mutations in the amiloride-sensitive epithelial sodium channel in patients with cystic fibrosis-like disease.

We investigated whether mutations in the genes that code for the different subunits of the amiloride-sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)-like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three-fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.-55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R-SCNN1A polymorphism was even found to result in a four-fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R-SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0-10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases.

Immunohistochemical evidence for Zic1 coexpression with beta-catenin in the myofibroblast of Dupuytren disease.

The active cellular component in Dupuytren disease (DD) is the alpha-smooth muscle actin (alpha-SMA) containing myofibroblast. The underlying regulatory processes in activation of myofibroblasts resemble the pathophysiology of certain types of cancer. Accumulation of beta-catenin has been shown in many fibroproliferative processes, including DD and, recent findings attributed a possible role to the Zic1 transcription factor. To assess Zic1 expression in DD and investigate its relation with the accumulation of beta-catenin, neighbouring tissue samples in 20 patients with DD were stained immunohistochemically with monoclonal antibodies for beta-catenin, alpha-SMA, and Zic1. Histological appearance was staged according to Luck. Cell-rich areas with accumulation of beta-catenin in myofibroblasts that stained for alpha-SMA and showed apparent Zic1 coexpression were obvious. This coexpression seemed independent of proliferative or involutional histological staging. We found only Zic1 expression in residual stages. A different pattern of expression of protein in the residual stage may support earlier suggestions of a cellular heterogeneity with the existence of different cell (sub-)populations in nodules and cords. On the other hand coexpression of Zic1 and beta-catenin may indicate a relation between Zic1 and the Wnt-pathway. Further studies are needed to elucidate cellular origin, potential heterogeneity and activity of the myofibroblasts in DD, and to define the exact role of Zic1 in fibroproliferative processes, wound healing, and cancer. The fibroblast in DD is an interesting model for future experiments.

Genetic analysis of Rwandan patients with cystic fibrosis-like symptoms: identification of novel cystic fibrosis transmembrane conductance regulator and epithelial sodium channel gene variants.

BACKGROUND: The defect in chloride and sodium transport in cystic fibrosis (CF) patients is a consequence of CF transmembrane conductance regulator (CFTR) loss of function and an abnormal interaction between CFTR and the epithelial sodium channel (ENaC). A few patients were described with CF-like symptoms, a single CFTR mutation, and an ENaC mutation. METHODS: To study African patients with CF-like symptoms and to relate the disease to gene mutations of both CFTR and ENaC genes, we collected clinical data and DNA samples from 60 African patients with a CF phenotype. The CFTR gene was first analyzed in all patients by denaturing high-performance liquid chromatography followed by direct sequencing; whereas, the sodium channel non-voltage-gated 1 alpha (SCNN1A), sodium channel non-voltage-gated 1 beta (SCNN1B), and sodium channel non-voltage-gated 1 gamma (SCNN1G) subunits of the ENaC gene were analyzed by sequencing in the five patients who carried only one CF mutation. The frequency of all identified ENaC variants was established in a control group of 200 healthy individuals and in the 55 CF-like patients without any CFTR mutation. RESULTS: Three CFTR mutants, including one previously undescribed missense mutation (p.A204T), and a 5T/7T variant were identified in five patients. ENaC gene sequencing in these five patients detected the following eight ENaC variants: c.72T>C and p.V573I in SCNN1A; p.V348M, p.G442V, c.1473 + 28C>T, and p.T577T in SCNN1B; and p.S212S and c.1176 + 30G>C in SCNN1G. In the 55 CF-like patients without any CFTR mutation, we identified five of these eight ENaC variants, including the frequent p.G442V polymorphism, but we did not detect the presence of the p.V348M, p.T577T, and c.1176 + 30G>C ENaC variants. Moreover, these last three ENaC variants, p.V348M, p.T577T, and c.1176 + 30G>C, were not found in the control group. CONCLUSION: Our data suggest that CF-like syndrome in Africa could be associated with CFTR and ENaC mutations.

Beta-catenin overexpression in Dupuytren's disease is unrelated to disease recurrence.

Recurrence of Dupuytren's contracture is common yet unpredictable, compromising surgical outcome. The alpha-smooth muscle actin-containing myofibroblast is the active contractile cellular component. Based on recent reports on beta-catenin accumulation in Dupuytren's disease, we investigated a possible relation with disease recurrence. We divided a collection of 143 nodules into those from patients with recurrent or nonrecurrent nodules and with a minimal 3-year followup. We randomly selected 12 and 11 samples of each group, respectively. We looked at Dupuytren's diathesis, immunohistologic staining for beta-catenin and alpha-smooth muscle actin, and Luck's histologic stages (zones). The expression of selected Wnt genes was examined with TaqMan PCR in separate histologic zones. All samples showed cytoplasmic and nuclear beta-catenin accumulation in myofibroblasts in involutional zones. The risk score of Abe et al. and Dupuytren's diathesis were greater in the recurrent group. Greater Wnt5a expression in the beta-catenin-accumulating involutional zone was seen. We conclude intracellular beta-catenin accumulation, possibly regulated by upstream Wnt signaling pathway activation and confined in myofibroblasts in the involutional zone of Dupuytren's diathesis, is unrelated to disease recurrence. Clinical parameters for Dupuytren's diathesis remain the best way to predict recurrence risk.

Cystic fibrosis transmembrane conductance regulator gene polymorphisms in patients with primary sclerosing cholangitis.

BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) is a progressive cholestatic disease commonly associated with inflammatory bowel disease (IBD) and characterized by fibrosing inflammatory destruction of bile ducts. The histological features in the liver of PSC patients are similar to those observed in cystic fibrosis (CF). Our aim was to study whether variants in the CFTR gene are associated with the occurrence and/or evolution of PSC. METHODS: PSC patients (n=140) were genotyped for F508del, the TGmTn variants, and four additional polymorphic loci (1001+11 C>T, M470V, T854T and Q1463Q), and compared to 136 matched healthy controls. RESULTS: The 1540G-allele, encoding V470, was less frequent in PSC (52%) than in controls (64%, p=0.003), and was associated with protection against PSC in individuals without IBD (OR 0.25, 95% CI 0.12-0.52, p=0.0002). Also TG11-T7 was less frequent in PSC (53%) than in controls (61%, p=0.04), this haplotype was associated with reduced risk for PSC (OR 0.34, 95% CI 0.17-0.70, p=0.003) in individuals without IBD. CONCLUSIONS: In this cohort of PSC patients, several CFTR-variants affecting the functional properties of the CFTR protein seem to offer protection against the development of PSC, confirming our hypothesis that CFTR might be implicated in the pathogenesis of PSC.

Analysis of the CFTR gene in Iranian cystic fibrosis patients: identification of eight novel mutations.

BACKGROUND: Cystic fibrosis (CF) is the most common inherited disorder in Caucasian populations, with over 1400 mutations identified in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Mutations in the CFTR gene may be also causative for CBAVD (Congenital Bilateral Absence of the Vas Deferens). The type and distribution of mutations varies widely between different countries and/or ethnic groups, and is relatively unknown in Iran. We therefore performed a comprehensive analysis of the CFTR gene in Iranian CF patients. METHODS: 69 Iranian CF patients, and 1 CBAVD patient, were analysed for mutations in the complete coding region, and its exon/intron junctions, of their CFTR genes, using different methods, such as ARMS (amplification refractory mutation system)-PCR, SSCP (single stranded conformation polymorphism) analysis, restriction enzyme digestion analysis, direct sequencing, and MLPA (Multiplex Ligation-mediated Probe Amplification). RESULTS: CFTR mutation analysis revealed the identification of 37 mutations in 69 Iranian CF patients. Overall, 81.9% (113/138) CFTR genes derived from Iranian CF patients could be characterized for a disease-causing mutation. The CBAVD patient was found to be homozygous for the p.W1145R mutation. The most common mutations were p.F508del (DeltaF508) (18.1%), c.2183_2184delAAinsG (2183AA>G) (6.5%), p.S466X (5.8%), p.N1303K (4.3%), c.2789+5G>A (4.3%), p.G542X (3.6%), c.3120+1G>A (3.6%), p.R334W (2.9%) and c.3130delA (2.9%). These 9 types of mutant CFTR genes totaled for 52% of all CFTR genes derived from the 69 Iranian CF patients. Eight mutations, c.406-8T>C, p.A566D, c.2576delA, c.2752-1_2756delGGTGGCinsTTG, p.T1036I, p.W1145R, c.3850-24G>A, c.1342-?_1524+?del, were found for the first time in this study. CONCLUSIONS: We identified 37 CFTR mutations in 69 well characterized Iranian CF patients, obtaining a CFTR mutation detection rate of 81.9%, the highest detection rate obtained in the Iranian population so far. These findings will assist in genetic counseling, prenatal diagnosis and future screening of CF in Iran.

ZIC1 gene expression is controlled by DNA and histone methylation in mesenchymal proliferations.

RNA and protein analysis revealed the consistent upregulation of the neural transcription factors ZIC1 and ZIC4 in desmoid tumors and other fibroproliferative disorders. The 5' flanking region of the ZIC1 promoter was unmethylated in desmoid tumor fibroblasts, while a hypermethylated ZIC1 promoter was found in human and mouse cell lines not expressing the gene. In addition, expressing cells showed a H3K4me2 at the ZIC1 promoter, whereas non-expressing cells showed higher levels of H3K9me2 in the same region. To our knowledge, this is the first report describing ZIC1 expression in mesenchymal proliferations and a role for DNA methylation in the control of ZIC1 expression.

TGF-beta modulates beta-Catenin stability and signaling in mesenchymal proliferations.

Here for the first time we showed, despite the oncogenic mutations in beta-Catenin, that TGF-beta is a modulator of beta-Catenin levels in tumoral fibroblasts as well as non-tumoral fibroblasts. The results show that the TGF-beta pathway is active in desmoids cells and in in situ tumors. A dose dependent increase in beta-Catenin protein levels was observed after TGF-beta treatment in combination with an increased repression of GSK-3beta both in normal and tumoral fibroblasts. TGF-beta stimulation also led to an altered -- up to 5 fold -- transcriptional activity of beta-Catenin responsive promoters, such as IGFBP6 as well as increase of TOPflash activity. TGF-beta stimulation increased cell proliferation and BrdU incorporation 2.5 times. Taken together, we propose that TGF-beta is a modulator of beta-Catenin levels in tumoral fibroblasts and non-tumoral fibroblasts, despite the oncogenic mutations already present in this gene in tumoral fibroblasts of desmoid tumors. This modulation of beta-Catenin levels by TGF-beta may be involved in determining the tumoral phenotype of the cells.

Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney.

Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype has been documented in patients with CF. This study investigated the expression, subcellular distribution, and processing of CFTR in the kidney; used various mouse models to assess the role of CFTR in proximal tubule (PT) endocytosis; and tested the relevance of these findings in patients with CF. The level of CFTR mRNA in mouse kidney approached that found in lung. CFTR was located in the apical area of PT cells, with a maximal intensity in the straight part (S3) of the PT. Fractionation showed that CFTR co-distributed with the chloride/proton exchanger ClC-5 in PT endosomes. Cftr(-/-) mice showed impaired (125)I-beta(2)-microglobulin uptake, together with a decreased amount of the multiligand receptor cubilin in the S3 segment and a significant loss of cubilin and its low molecular weight (LMW) ligands into the urine. Defective receptor-mediated endocytosis was found less consistently in Cftr(DeltaF/DeltaF) mice, characterized by a large phenotypic heterogeneity and moderate versus mice that lacked ClC-5. A significant LMW proteinuria (and particularly transferrinuria) also was documented in a cohort of patients with CF but not in patients with asthma and chronic lung inflammation. In conclusion, CFTR inactivation leads to a moderate defect in receptor-mediated PT endocytosis, associated with a cubilin defect and a significant LMW proteinuria in mouse and human. The magnitude of the endocytosis defect that is caused by CFTR versus ClC-5 loss likely reflects functional heterogeneity along the PT.

The TNFalpha receptor TNFRSF1A and genes encoding the amiloride-sensitive sodium channel ENaC as modulators in cystic fibrosis.

The CFTR mutations in cystic fibrosis (CF) lead to ion transport anomalities which predispose to chronic infection and inflammation of CF airways as the major determinants for morbidity and mortality in CF. Discordant clinical phenotypes of siblings with identical CFTR mutations and the large variability of clinical manifestations of patients who are homozygous for the most common mutation F508del suggest that both environment and genes other than CFTR contribute substantially to CF disease. The prime candidates for genetic modifiers in CF are elements of host defence such as the TNFalpha receptor and of ion transport such as the amiloride-sensitive epithelial sodium channel ENaC, both of which are encoded side by side on 12p13 (TNFRSF1A, SCNN1A) and 16p12 (SCNN1B, SCNN1G). Thirty-seven families with F508del-CFTR homozygous siblings exhibiting extreme clinical phenotypes that had been selected from the 467 pairs of the European CF Twin and Sibling Study were genotyped at 12p13 and 16p12 markers. The ENaC was identified as a modulator of CF by transmission disequilibrium at SCNN1G and association with CF phenotype intrapair discordance at SCNN1B. Family-based and case-control analyses and sequencing of SCNN1A and TNFRSF1A uncovered an association of the TNFRSF1A intron 1 haplotype with disease severity. Carriers of risk haplotypes were underrepresented suggesting a strong impact of both loci on survival. The finding that TNFRSF1A, SCNN1B and SCNN1G are clinically relevant modulators of CF disease supports current concepts that the depletion of airway surface liquid and inadequate host inflammatory responses trigger pulmonary disease in CF.

Differential induction of human beta-defensin expression by periodontal commensals and pathogens in periodontal pocket epithelial cells.

BACKGROUND: To investigate the possible role of beta-defensins in gingival health and periodontal disease, we examined the effect of several stimuli on the expression of interleukin-8 (IL-8), human beta-defensin-1, -2, -3, and -4 (hBD) in primary human diseased gingival epithelial (HGE) cell cultures from periodontitis patients by quantitative TaqMan reverse transcription polymerase chain reaction (RT-PCR). METHODS: Several strains of the periodontopathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were added to the cells, as well as the oral commensal bacteria Fusobacterium nucleatum and Escherichia coli. The induction by the proinflammatory stimuli phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor-alpha (TNF-alpha) was also tested. RESULTS: In addition to the published observations (PMA induces hBD-2 and -4; TNF-alpha induces hBD-2 and -3), it was found that PMA can upregulate hBD-1 and hBD-3, whereas TNF-alpha can induce hBD-4. The commensal bacteria were significant inducers of hBD-2, hBD-3, and IL-8. The pathogen P. gingivalis induced hBD-1 and hBD-3 at different time points than the commensals, but no induction of IL-8 and hBD-2 could be observed. These data fit with the chemokine paralysis theory. A correlation was found between the pathogenicity of different serotypes of A. actinomycetemcomitans and the induction profiles of defensins and IL-8. CONCLUSION: The results suggest that a correlation can be found in diseased oral epithelium between the defensin profiles that are induced and the pathogenicity of the oral bacterial strains.

Interaction of the protein phosphatase 2A with the regulatory domain of the cystic fibrosis transmembrane conductance regulator channel.

A direct interaction of the regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator protein (CFTR) with PR65, a regulatory subunit of the protein phosphatase 2A (PP2A), was shown in yeast two hybrid, pull-down and co-immunoprecipitation experiments. The R domain could be dephosphorylated by PP2A in vitro. Overexpression of the interacting domain of PR65 in Caco-2 cells, as well as treatment with okadaic acid, showed a prolonged deactivation of the chloride channel. Taken together our results show a direct and functional interaction between CFTR and PP2A.

Distribution of human beta-defensin polymorphisms in various control and cystic fibrosis populations.

Human beta defensins contribute to the first line of defense against infection of the lung. Polymorphisms in these genes are therefore potential modifiers of the severity of lung disease in cystic fibrosis. Polymorphisms were sought in the human beta-defensin genes DEFB1, DEFB4, DEFB103A, and DEFB104 in healthy individuals and cystic fibrosis (CF) patients living in various European countries. DEFB1, DEFB4, and DEFB104 were very polymorphic, but DEFB103A was not. Within Europe, differences between control populations were found for some of the frequent polymorphisms in DEFB1, with significant differences between South-Italian and Czech populations. Moreover, frequent polymorphisms located in DEFB4 and DEFB104 were not in Hardy Weinberg equilibrium in all populations studied, while those in DEFB1 were in Hardy Weinberg equilibrium. Sequencing of a monochromosomal chromosome 8 mouse-human hybrid cell line revealed signals for multiple alleles for some loci in DEFB4 and DEFB104, but not for DEFB1. This indicated that more than one DEFB4 and DEFB104 gene was present on this chromosome 8, in agreement with recent findings that DEFB4 and DEFB104 are part of a repeat region. Individual DEFB4 and DEFB104 PCR amplification products of various samples were cloned and sequenced. The results showed that one DNA sample could contain more than two haplotypes, indicating that the various repeats on one chromosome were not identical. Given the higher complexity found in the genomic organization of the DEFB4 and DEFB104 genes, association studies with CF lung disease severity were performed only for frequent polymorphisms located in DEFB1. No association with the age of first infection by Pseudomonas aeruginosa or with the FEV1 percentage at the age of 11-13 years could be found.

Upregulation of Wilms' tumor gene 1 (WT1) in desmoid tumors.

Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors in which beta-catenin/TCF3 mediated Wnt signaling is activated. More than 80% of desmoid tumors contain activating mutations in beta-catenin. It has been shown that the Wnt signaling pathway interacts with Wilms' tumor gene 1 (WT1) in normal kidney development and plays a role in the genesis of some Wilms' tumors. About 15% of Wilms' tumors contain WT1 mutations and of these, about 50% contain beta-catenin mutations. This overlap in mutation pattern of WT1 and beta-catenin in Wilms' tumor suggests that these 2 genes may collaborate in the genesis of a subset of Wilms' tumors. To investigate whether this hypothesis could be extended to other Wnt-dependent tumor types, we searched for WT1 mutations and studied WT1 expression in beta-catenin mutant desmoid tumors. We investigated the expression of WT1 mRNA and protein in desmoid tumors. Medium to high abundant levels of WT1 mRNA were detected by TaqMan quantitative PCR in all tested desmoid cells, whereas adjacent normal fibroblasts showed less expression of WT1. Western blot analysis and immunohistochemistry confirmed this overexpression at the protein level. A mutational screen of the WT1 zinc-finger region by sequence analysis did not identify any mutations. Finally, we investigated a possible role of beta-catenin on WT1 regulation and vice versa. Overexpression of different beta-catenin mutants in the HEK293T cell line did not modulate WT1 promoter activity and WT1 did not affect beta-catenin /TCF transcriptional activity in this cell line. These results show that the wild-type WT1 gene is strongly overexpressed in beta-catenin mutant desmoid tumors and may play a role in tumorigenesis of desmoid tumors, similar to what has been suggested in some epithelial malignancies.

CFTR mutations and polymorphisms in male infertility.

Apart from cystic fibrosis, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are also involved in congenital bilateral absence of the vas deferens (CBAVD). A mutation is identified in about 80% of the CFTR genes derived from CBAVD patients; the genetic defect in the remainder is yet unknown. In contrast to CF patients, when CFTR is involved, at least one of the mutant CFTR genes of CBAVD patients harbors a mild mutation. A polyvariant mutant CFTR gene is the most frequent CBAVD causing mutant CFTR gene. Here, combinations of particular alleles at several polymorphic loci yield insufficient functional CFTR. The fact that most CBAVD patients, that carry mutations on both CFTR genes, have no lung disease is most probably explained by tissue specific alternative splicing, which is increased in vas deferens compared to bronchial tissue. It has also been reported that CBAVD may be involved in other forms of infertility than CBAVD, however this has not always been confirmed in other studies. Because of techniques such as intracytoplasmic sperm injection, CBAVD patients are now able to father children, however such couples have an increased risk of having a child with cystic fibrosis, and therefore genetic testing and counselling should be provided.