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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.

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.

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.

Using the patch-clamp method, we investigated a relationship between MDR1 expression and its effects on the CFTR channel function. Incubation of CaCo-2 cells with increasing concentrations of doxorubicin resulted in a reduction of CFTR chloride channel activity in a dose-dependent manner. This reduction was associated with a decrease of CFTR mRNA and simultaneous up-regulation of MDR1 mRNA in the presence of doxorubicin. Similar alteration of the CFTR function was observed in CaCo-2 cells transiently overexpressing MDR1. No alterations of the cAMP-dependent chloride currents were observed in COS-1 cells transiently co-expressing CFTR and MDR1 from strong CMV promoters. This indicated that repression of CFTR by MDR1 induction requires the presence of the native CFTR promoter.

The incidence of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in children with intermediate sweat chloride levels is unknown. The results of 2,349 sweat tests performed at two Belgian university hospitals were reviewed. Intermediate chloride concentrations were observed in 98 subjects (4.2%), 68 being younger than 18 years of age. Forty-three children could be traced and their parents agreed to take part in the study. Exhaustive analysis of the CFTR gene disclosed a total of 24 putative mutations (27.9%). Three subjects were found to carry only one CFTR mutation, whereas 10 harbored one mutation on both CFTR genes. These 10 children were investigated in detail. At the time of writing, the mean age (+/-SD) of this group is 8.9 years (+/-4.2 years). Nine children are pancreatic sufficient. Three have been asymptomatic for more than two years, whereas the others display, to different degrees, clinical features suggestive of CF. The sweat chloride concentration is slightly higher in this group (39.4 +/- 5.4 mM) than in subjects without CFTR mutation (35.2 +/- 4.4 mM, p < 0.05). The nasal potential difference was abnormal in five of the nine subjects tested. In this study, 23% of children displaying intermediate sweat chloride levels were found to carry a putative mutation on both CFTR genes.

Cystic fibrosis is a frequent autosomal recessive disorder that is caused by the malfunctioning of a small chloride channel, the cystic fibrosis transmembrane conductance regulator. The protein is found in the apical membrane of epithelial cells lining exocrine glands. Absence of this channel results in imbalance of ion concentrations across the cell membrane. As a result, fluids secreted through these glands become more viscous and, in the end, ducts become plugged and atrophic. Little is known about the pathways that link the malfunctioning of the CFTR protein with the observed clinical phenotype. Moreover, there is no strict correlation between specific CFTR mutations and the CF phenotype. This might be explained by the fact that environmental and additional genetic factors may influence the phenotype. The CFTR protein itself is regulated at the maturational level by chaperones and SNARE proteins and at the functional level by several protein kinases. Moreover, CFTR functions also as a regulator of other ion channels and of intracellular membrane transport processes. In order to be able to function as a protein with pleiotropic actions, CFTR seems to be linked with other proteins and with the cytoskeleton through interaction with PDZ-domain-containing proteins at the apical pole of the cell. Progress in cystic fibrosis research is substantial, but still leaves many questions unanswered.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits Ca(2+)-activated Cl- channels (CaCC) by an unknown mechanism. This inhibition does not require CFTR activation (activity-independent inhibition), but is potentiated when CFTR is activated (activity-dependent inhibition). In this study, we evaluated, in endothelial cells, possible structural determinants for this interaction. Bovine pulmonary artery endothelium (CPAE) cells, which do not express CFTR, were transfected transiently with three hybrid CFTR constructs. The functional interaction between CaCC and CFTR was assessed using the patch-clamp technique in the whole-cell configuration. CaCC was stimulated by application of adenosine 5'-triphosphate (ATP) to the bath solution. CFTR currents were evoked by application of a forskolin/3-isobutyl-l-methylxanthine (IBMX) cocktail. The inhibitory effect of CFTR was conserved when the PDZ (PSD-95/Discs large/ZO-1) binding motif was deleted (CFTR-delta PDZ). In contrast, both the CFTR activity-independent and -dependent inhibition of CaCC were abolished when the C-terminal part of the regulatory (R)-domain of CFTR was deleted (CFTR-delta R780-830). The activity-dependent inhibition of CaCC, but not the activity-independent inhibition, could be rescued by introducing the multiple drug resistance (MDR)-1 mini-linker in place of the deletion (CFTR-delta R-linker). It is concluded that the C-terminal part of the R-domain is an important determinant for CFTR-CaCC interaction.

Background: This study describes the functional interaction between the putative Ca2+ channel TRP4 and the cystic fibrosis transmembrane conductance regulator, CFTR, in mouse aorta endothelium (MAEC)., Results: MAEC cells express CFTR transcripts as shown by RT-PCR analysis. Application of a phosphorylating cocktail activated a Cl- current with characteristics similar to those of CFTR mediated currents in other cells types (slow activation by cAMP, absence of rectification, block by glibenclamide). The current is present in trp4 +/+ MAEC, but not in trp4 -/- cells, although the expression of CFTR seems unchanged in the trp4 deficient cells as judged from RT-PCR analysis., Conclusions: It is concluded that TRP4 is necessary for CFTR activation in endothelium, possibly by providing a scaffold for the formation of functional CFTR channels.

The morphology of the mouse vas deferens still undergoes major changes from birth to 40 days of age, such as differentiation of the mesenchymal cells into fibroblasts and muscle cells, differentiation of the epithelium into basal and columnar epithelial cells, development of stereocilia, and the appearance of smooth endoplasmic reticulum organised in fingerprint-like structures or parallel, flattened saccules. In mutant homozygous DeltaF508 (DeltaF/DeltaF) and knock-out (cf/cf) CFTR mice, strain 129/FvB and 129/C57BL-6, respectively, a similar development occurred until the age of 20 days. At 40 days, however, the lumen was filled with eosinophilic secretions, and sperm cells were absent in the majority of the animals examined, although sperm production in testis and epididymis appeared to be normal. CFTR was localised in the apical membrane and cytoplasm of the vas deferens epithelium from 40 days on but could not be detected in the vas deferens before 20 days or in mutant adult CFTR mice as expected. Western blots of membrane preparations showed that the mature form of CFTR was present in vas deferens and testis but absent in seminal vesicles. Our results suggest that the function of CFTR is probably essential after 20 days in the vas deferens and that its absence or dysfunction may result in a vas deferens with a differentiated epithelium but a collapsed lumen, which could at least temporarily delay the transport of spermatozoa. These observations contrast with those made in the overall majority of CF patients. Mol. Reprod. Dev. 55:125-135, 2000., (Copyright 2000 Wiley-Liss, Inc.)

To improve our insight into the structure and function of the CFTR R domain, deletion and hybrid constructs in which different parts of the R domain were deleted or replaced by the MDR1 linker domain, and vice versa, were made. Replacement of the linker domain by the R domain did not result in a decrease and replacement of the CFTR R domain by the linker domain did not result in an increase of maturation efficiency, when compared to the respective wild-type proteins. This indicates that the R domain is not responsible for the high degree of degradation observed for CFTR translation products in the ER, but rather the overall structure or sequences located outside the R domain. Replacing the C-terminal part of the R domain (amino acids 780-830) by the MDR1 linker domain resulted in the appearance of PKA-dependent whole cell chloride currents which were not significantly different from wild-type CFTR currents. This might indicate that the PKA sites present in the linker domain are functional and that not the exact sequence of the C-terminal part of the R domain is important, but rather the presence of PKA sites and the length. Moreover, when this hybrid construct was PKC-stimulated, chloride currents were activated. Although these PKC-induced currents were lower than the PKA-induced ones, this again indicates that the linker domain is functional in this hybrid construct. Taken together, these results suggest that the MDR1 linker domain can substitute for part of the regulatory domain of the CFTR protein.

Genotype-phenotype correlations in cystic fibrosis (CF) have been found for lung and pancreatic function, but not for paranasal sinus disease. Because such correlations may have pathophysiological and clinical implications, the correlation of mutations, in particular DeltaF508, with paranasal sinus disease was investigated in 113 CF patients with known genotype. The clinical importance of paranasal sinus disease was evaluated using three parameters: polyps, overall clinical severity of upper airway problems, and surgery. Polyps were evaluated by nasal endoscopy and graded on a five-point scale. Four severity groups were distinguished based on history, clinical records, and examination: no upper airway problems; more problems than in control subjects; severe, recurrent or chronic problems; and paranasal sinus surgery cases. DeltaF508 homozygosity correlated with clinical severity (p < 0.02) and with the presence of polyps on endoscopy (p < 0.05). The relative risk for paranasal sinus surgery in DeltaF508 homozygous CF patients was 2.33. In conclusion, there are genotype-phenotype correlations for paranasal sinus disease in CF. DeltaF508 homozygosity is a risk factor for paranasal sinus disease in CF.

1. To investigate whether the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with volume regulated anion channels (VRACs), we measured the volume-activated chloride current (ICl,swell) using the whole-cell patch-clamp technique in calf pulmonary artery endothelial (CPAE) cells and in COS cells transiently transfected with wild-type (WT) CFTR and the deletion mutant DeltaF508 CFTR. 2. ICl,swell was significantly reduced in CPAE cells expressing WT CFTR to 66.5 +/- 8.8 % (n = 13; mean +/- s. e.m.) of the control value (n = 11). This reduction was independent of activation of the CFTR channel. 3. Expression of DeltaF508 CFTR resulted in two groups of CPAE cells. In the first group IBMX and forskolin could activate a Cl- current. In these cells ICl,swell was reduced to 52.7 +/- 18.8 % (n = 5) of the control value (n = 21). In the second group IBMX and forskolin could not activate a current. The amplitude of ICl,swell in these cells was not significantly different from the control value (112.4 +/- 13.7 %, n = 11; 21 control cells). 4. Using the same method we showed that expression of WT CFTR in COS cells reduced ICl,swell to 62.1 +/- 11.9 % (n = 14) of the control value (n = 12) without any changes in the kinetics of the current. Non-stationary noise analysis suggested that there is no significant difference in the single channel conductance of VRAC between CFTR expressing and non-expressing COS cells. 5. We conclude that expression of WT CFTR down-regulates ICl, swell in CPAE and COS cells, suggesting an interaction between CFTR and VRAC independent of activation of CFTR.

The deficiency of adenylosuccinate lyase (ADSL, also termed adenylosuccinase) is an autosomal recessive disorder characterized by the accumulation in body fluids of succinylaminoimidazole-carboxamide riboside (SAICA-riboside) and succinyladenosine (S-Ado). Most ADSL-deficient children display marked psychomotor delay, often accompanied by epilepsy or autistic features, or both, although some patients may be less profoundly retarded. Occasionally, growth retardation and muscular wasting are also present. Up to now, nine missense mutations of the ADSL gene had been reported in six apparently unrelated sibships. In the present study of 10 additional patients with ADSL deficiency, nine point mutations, among which seven unreported missense mutations, and the first splicing error reported in this disorder, have been identified. These mutations have been characterized, taking into account the finding that the cDNA of human ADSL is 75 nucleotides longer at its 5'-end, and encodes a protein of 484 rather than 459 amino acids as previously reported. Five apparently unrelated patients were found to carry a R426H mutation. With the exceptions of the latter mutation, of a R190Q mutation that had been reported previously, and of a K246E mutation that was found in two unrelated patients, all other mutations were found only in a single family.

We analysed DNA samples from 26 Pakistani patients with cystic fibrosis (CF) living in the United Kingdom (14 from patients residing in the north west of England, who were referred directly to the North West Regional Molecular Genetics Laboratory, and 12 from other regional molecular genetics laboratories). Of 56 mutations seen in native U.K. CF patients, only DeltaF508, R709X, and 2184insA were detected in the Pakistani patients. Combined SSCP/Heteroduplex analysis, DGGE, and direct DNA cycle sequencing revealed five novel mutations: Y569D, Q98X, 296+12(T>C), 1161delC, and 621+2(T>C), which appear to be specific to Pakistani CF families. In addition, a novel polymorphism, 297-67(A/C), and three previously described rare mutations, 1525-1(G>A), R560S, and 1898+1(G>T), were detected. In the 14 Pakistani CF patients from the north west of England, DeltaF508 accounted for approximately 32% (9/28 chromosomes) and the overall detection rate of CF mutations in this group was approximately 86% (24/28 chromosomes).

CFTR transcripts have been qualitatively and quantitatively analysed in nasal epithelial and vas deferens cells by means of reverse transcription PCR. Alternative splicing of exon 9, which is known to occur in nasal epithelial cells, also occurred in vas deferens cells. The extent of this alternative splicing was determined by the allele present at the Tn locus at the end of intron 8 of the CFTR gene. However, the proportion of transcripts lacking exon 9 sequences was increased in vas deferens cells compared with nasal epithelial cells, independent of the Tn genotype. We postulate that this tissue specific difference in the proportion of CFTR transcripts lacking exon 9 sequences could contribute to the tissue specific disease phenotype observed in individuals with congenital bilateral absence of the vas deferens.

A quality control study was performed to determine the accuracy of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) mutation screening in 40 different genetic screening laboratories throughout Europe. A total of 9 different samples were investigated blindly by the participating laboratories. Only 25/40 laboratories, i.e. 62.5%, were able to type all samples correctly for the mutations for which they routinely screened. Only 2 of the 9 samples were correctly typed in all 40 laboratories. The lowest accuracy rate was 80% for 1 sample. 12.5% of the participating laboratories interpreted the F508C polymorphism as a true CF disease mutation and 23.5% interpreted the delta I507 mutation as a delta F508 mutation. For the delta F508 mutation, a false-negative result of 3.75% was obtained. It is clear that the accuracy of CFTR typing should be improved.

Holt-Oram syndrome is an autosomal dominant disorder with congenital heart defects and skeletal malformations of the upper extremities. A patient with a deletion of 14q23-24 and Holt-Oram syndrome has been described. In this report, however, genetic linkage to the 14q23-24 region is excluded in a multigeneration family with five available individuals affected with Holt-Oram syndrome. Familial Holt-Oram syndrome might be different from the syndrome with the 14q23-24 deletion.

Ten polymorphic loci, located in a 1 Mb interval across the cystic fibrosis locus, were analyzed on normal and mutant CFTR genes. A different distribution of haplotype backgrounds among normal and mutant CFTR genes was observed. With exception of the D7S8 locus, the three most common mutations, delta F508, G542X and N1303K, were found on an identical haplotype background. In agreement with the observed linkage equilibrium between the Q1463Q and D7S8 loci, both alleles at the D7S8 locus were found on delta F508 CFTR genes. However, the G542X and N1303K mutations, which have been estimated to be at least 35000 years old, were found to be associated with a single allele at the D7S8 locus. Absence of recombination between the D7S8 and Q1463Q loci was also observed on normal CFTR genes with this haplotype background. At the Tn locus in intron 8, allele 9 known to result in very efficient splicing was associated with the most frequent mutations. At the M470V locus, located in a conserved region of the first nucleotide binding fold, the amino acid methionine was found to be associated with the frequent mutations, in particular with mutations located in one of the two nucleotide binding folds which are generally known as severe mutations with regard to exocrine pancreatic function. On mutant CFTR gene, this locus was in complete association with the centromeric D9 locus, in the absence of a complete association with the intervening loci.(ABSTRACT TRUNCATED AT 250 WORDS)

Huntington's disease (HD) is an autosomal dominant disorder with choreic movements, psychiatric manifestations and cognitive dysfunction. Recently the IT15 gene on chromosome 4p has been identified containing an unstable and expanded trinucleotide repeat in patients with HD. We report on the characteristics of this repeat in 248 individuals from 41 Belgian HD families. The length of the expanded repeat was defined precisely and reproducibly on an ALF sequencer and correlated well with the age of onset (r = -0.72). Paternal transmission of the expanded repeat resulted on average in a significantly longer repeat length (+2.79 repeats) than maternal transmission (-0.29 repeats). (CAG)n repeat of a premutation (?) size was observed in this population with subsequent expansion in the disease range. Presymptomatic or prenatal testing using only linked markers may be problematic in these cases.

We have previously shown that about 85% of the mutations in 194 Belgian cystic fibrosis alleles could be detected by a reverse dot-blot assay. In the present study, 50 Belgian chromosomes were analyzed for mutations in the cystic fibrosis transmembrane conductance regulator gene by means of direct solid phase automatic sequencing of PCR products of individual exons. Twenty-six disease mutations and 14 polymorphisms were found. Twelve of these mutations and 3 polymorphisms were not described before. With the exception of one mutant allele carrying two mutations, these mutations were the only mutations found in the complete coding region and their exon/intron boundaries. The total sensitivity of mutant CF alleles that could be identified was 98.5%. Given the heterogeneity of these mutations, most of them very rare, CFTR mutation screening still remains rather complex in our population, and population screening, whether desirable or not, does not appear to be technically feasible with the methods currently available.

We have developed a heminested PCR (polymerase chain reaction) method, performed on single cells, for the analysis of the most common cystic fibrosis (CF) mutation (delta F508). As a quality control, the polymorphic exon 2 of the HLA DQA1 locus was co-amplified from the same cell. With a non-radioactive reverse dot-blot assay, the genotype of these two loci could be determined. Experiments on 98 single fibroblasts, heterozygous for the CFTR and the DQA1 locus, showed that amplification of either locus could be obtained in 97 per cent of the cases, but only 90 per cent showed heterozygosity for CF, 75 per cent showed heterozygosity for DQA1, and 74 per cent showed heterozygosity for both CF and DQA1. Contaminations detected only after DQA1 typing occurred in 3 per cent of our samples. Error rate calculations based on our experimental PCR data indicate that single blastomere diagnosis would lead to unacceptable errors, i.e., an affected fetus, in less than 1 per cent of the cases. The risk of undetected crossing-over or the dubious results that crossing-over could generate, would make isolated polar body diagnosis at the present time very difficult. The combined approach of PCR on polar bodies followed by confirmation of the diagnosis on blastomeres, however, should give a solid base for preimplantation diagnosis of monogenic disorders.

An assay is described in which 11 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be screened simultaneously. Six different exons of the CFTR gene are amplified in a single multiplex amplification. Biotinylated dUTP is incorporated into the different fragments during the amplification process. A sample of this mixture is then hybridized to 21 different poly-dT tailed oligonucleotide probes which are bound to a nylon membrane. In order to screen the different mutations in a single step hybridization, the length of the different oligonucleotides and the amount used in the assay were optimized. The detection is performed by binding avidin-alkaline phosphatase to the biotin, followed by a chemiluminescent reaction. By means of this fast and sensitive assay, about 85% of all the cystic fibrosis mutations in the Belgian population can be detected.

When screening for the presence of major cystic fibrosis mutations in Algerian cystic fibrosis families by heteroduplex formation, aberrant heteroduplexes were observed for exon 10 in one family. Here we describe the clinical and molecular findings in a severely affected child of this family, homozygous for the 1609delCA and for the M470V polymorphism.

The alpha 2 subunit of the VLA-2 receptor (CD49B) was mapped to human chromosome 5 by several independent approaches. First, the expression of the alpha 2 subunit at the protein level was investigated in a panel of human-mouse hybrid cell lines. Cell surface expression was detected by indirect immunofluorescence with monoclonal anti-alpha 2 antibody 12F1. Intracellular alpha 2 antigen was detected by immunostaining of whole cell extracts or of immunoprecipitated 12F1 antigen with the monoclonal antibodies 3H8 and 5C5. Second, the presence of human genomic alpha 2 sequences in the panel of human-mouse hybrids was detected by PCR, using primers derived from the published alpha 2 cDNA sequence. The specificity of the amplification product was shown by direct sequencing. The results of the PCR study were confirmed by amplifying a CD14 gene fragment, known to map to chromosome 5. Finally, in situ hybridization with a 3H-labeled 1040-bp cDNA probe, also obtained by PCR, confirmed and refined the localization of CD49B on chromosome 5 at q23-31.

Using Southern blotting and the polymerase chain reaction, the prevalence of the haplotypes for XV2c, CS7, KM19 and D9 on CF and on normal chromosomes could be determined in 35 Belgian families. A set of primers complementary to the DNA sequence of the CF gene around the delta F508 deletion was used to amplify this particular segment of the gene. In a total of 57 families, deletion screening showed that 69 out of 116 CF chromosomes (59.5%) carried the delta F508 deletion. Both the delta F508 deletion and another mutation(s) showed strong association with the haplotype 1-2-2-2.

The polymerase chain reaction (PCR) technique has provided a substantial improvement for the detection and analysis of known genetic polymorphisms. Here, we describe the application of this method for the detection of variable number of tandem repeat (VNTR) sequences. With the use of unique oligonucleotide primers, flanking the repeat sequence, and the thermostable Taq DNA polymerase, the hypervariable regions 3' of the Ha-ras gene, 3' of the apolipoprotein B gene, and 5' to the joining segments of the heavy-chain immunoglobulin gene could be amplified. Alleles up to 2,000 bp could be visualized directly on ethidium bromide-stained agarose gels. Larger alleles were seen only after traditional Southern blot analysis with an internal probe. The value of this new approach for the detection of VNTRs is illustrated in a case of paternity dispute.