Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been shown to cause cystic fibrosis (CF) and male infertility due to congenital bilateral absence of the vas deferens. We report the identification of a 6.8 kb deletion (del14a) and a nonsense mutation (S1455X) in the CFTR genes of a mother and her youngest daughter with isolated elevated sweat chloride concentrations. Detailed clinical evaluation of both individuals found no evidence of pulmonary or pancreatic disease characteristic of CF. A second child in this family with classic CF was homozygous for the del14a mutation, indicating that this mutation caused severe CFTR dysfunction. CFTR mRNA transcripts bearing the S1455X mutation were stable invivo, implying that this allele encoded a truncated version of CFTR missing the last 26 amino acids. Loss of this region did not affect processing of transiently expressed S1455X-CFTR compared with wild-type CFTR. When expressed in CF airway cells, this mutant generated cAMP-activated whole-cell chloride currents similar to wild-type CFTR. Preservation of chloride channel function of S1455X-CFTR was consistent with normal lung and pancreatic function in the mother and her daughter. These data indicate that mutations in CFTR can be associated with elevated sweat chloride concentrations in the absence of the CF phenotype, and suggest a previously unrecognized functional role in the sweat gland for the C-terminus of CFTR.
Cystic fibrosis (CF), a lethal autosomal recessive disorder common in the Caucasian population, is classically characterized by obstructive lung disease, pancreatic insufficiency and elevated sweat chloride values (1,2). These manifestations are believed to be the consequence of abnormal epithelial cell transport of electrolytes caused by dysfunction of the CF transmembrane conductance regulator (CFTR) (3-8). CFTR is a cAMP-activated chloride channel that is present in the apical membranes of secretory epithelia (9-11). In the sweat gland, CFTR is expressed in the apical membrane of secretory coil cells and apical and basolateral membranes of absorptive duct cells (12). CFTR is composed of 1480 amino acids organized into two membrane-associated domains, two regions that interact with ATP and a central regulatory domain that is phosphorylated by protein kinases (1,2). The functions of the N- and C-termini, if any, are unknown. More than 700 mutations have been identified in the CFTR gene (The CF Genetic Analysis Consortium, personal communication), some of which have been correlated with certain clinical manifestations, such as the severity of pancreatic disease, age of onset and level of sweat chloride abnormality (13-18). Mutations in CFTR have also been observed in males who do not manifest features of CF, but have infertility due to malformation of the vas deferens (19,20). Many of the infertile males carry one mutation that causes CF and an alteration in their second CFTR gene that has not been found in CF patients (20). Some of these individuals have elevated sweat chloride concentrations and chronic sinus disease, while others manifest no features of CF (21,22).
Sweat chloride concentrations >60 mmol/l have been used as a diagnostic standard for CF for >40 years (23). Only 1% of individuals with clinical features characteristic of CF have sweat chloride values in the normal range (23-25). Other disorders, such as pancreatitis, hypothyroidism and ectodermal dysplasia, have also been associated with elevated sweat chloride concentration (1). Interestingly, ~5% of apparently healthy individuals have increased sweat chloride concentrations (>50 mmol/l), but the underlying cause is unknown (26). The discovery of a family independently segregating CF and an isolated abnormality in sweat chloride presented a unique opportunity to investigate the molecular basis of sweat gland dysfunction. The family described here was studied originally by Feldman and colleagues (27) who had performed a prenatal diagnosis by linkage analysis and predicted an unaffected second child. However, this child had elevated sweat chloride values upon postnatal evaluation. Consequently, both parents were sweat tested, and the mother was found to have elevated sweat chloride values. We have identified mutations in each CFTR gene of the mother and child with isolated elevated sweat chloride concentrations. Extensive clinical evaluation of the mother revealed the absence of pulmonary or pancreatic disease characteristic of CF. Thus, defects in CFTR can give rise to isolated sweat gland dysfunction, in addition to the CF and male infertility phenotypes.
Clinical features of family members are summarized in Figure 1. The parents are first cousins once removed. The older child has elevated sweat chloride concentrations (62, 69 and 77 mmol/l), a decline in pulmonary function and non-mucoid Pseudomonas aeruginosa and Mycobacterium avium intracellulare in her sputum. She is pancreatic insufficient [fecal fat 51.4 g over 72 h (normal > 21) and absent stool trypsin] and takes 16 pancreas capsules five times a day. The younger child and mother are clinically well but have consistently elevated sweat chloride concentrations at levels comparable with those of the older child. Both have normal pulmonary function, normal sputum flora and no manifestations of exocrine pancreatic disease (steatorrhea, abdominal pain or vitamin deficiency). Serum immunoreactive trypsinogen has been assayed twice in the mother: the first test was found to be low (10.6 ng/ml; normal range: 13-36 ng/ml), but the repeat was normal (13 ng/ml). Tests to evaluate sweat duct function and CFTR function further ([beta]-adrenergic sweat stimulation and nasal potential difference) were not obtainable from the family. Although both parents have several siblings, only the mother's 32-year-old brother was available for clinical evaluation. He had a sweat chloride value of 22 mmol/l and normal pulmonary function tests and chest X-ray. His fertility status was not determined.
Screening for 16 CF mutations common among Caucasians by reverse dot-blot assay did not detect a mutation in any family member. Therefore, the entire coding region and flanking splice sites of each CFTR gene were screened by denaturing gradient gel electrophoresis (DGGE) or DNA sequencing (exons 9 and 23) to identify rare or novel mutations in this family. Exon 14a did not amplify by PCR from the older child's DNA sample. This result could have been due to a mutation in the region recognized by either PCR primer, or due to a genomic DNA deletion involving the entire region encompassed by the PCR primers. Genomic DNA from the older child and her family was digested with BglII and hybridized with a radiolabeled CFTR DNA probe specific for exon 14a. Southern blot analysis revealed an absence of the 4.2 kb fragment containing exon 14a in the child affected with CF (Fig. 2A). Mapping by a combination of BglII and HindIII digestions, Southern blotting and hybridization with CFTR cDNA probes indicated a genomic deletion of 6.8 kb (Fig. 2B) in one CFTR gene of each parent. The CFTR mRNA transcript from this gene is predicted to have an in-frame deletion of exon 14a that removes 43 amino acids from the second transmembrane domain of CFTR. The child with CF is homozygous for this mutation, while the child with elevated sweat chloride concentrations was heterozygous like her parents. Although we did not map the breakpoints of this mutation in each parent, it appears highly likely that the parents carry the same genomic deletion due to common ancestry.
RT-PCR was used to determine the consequences of the del14a and S1455X mutations upon transcript splicing and stability. cDNA reverse transcribed from nasal epithelial RNA obtained from both parents was amplified with primers corresponding to regions in CFTR exons 13 and 15. The paternal and maternal PCR products each had amplification of the size expected for the full-length CFTR mRNA transcript (463 bp) and smaller amplicons (334 bp) which corresponded to the size expected for omission of exon 14a from the CFTR transcripts (Fig. 3). Sequencing of the 334 bp amplicons confirmed that exon 14a was deleted. Hence, the 6.8 kb genomic DNA deletion causes an in-frame deletion of exon 14a such that the mRNA transcript is stable. Since nonsense mutations frequently cause severe reduction in mRNA levels (28), the stability of mRNA transcripts bearing the nonsense mutation S1455X was assessed as follows. Comparison of RT-PCR products from the father (del14a/normal) indicated that transcripts missing exon 14a were at levels comparable with normal CFTR mRNA transcripts (Fig. 3). Comparison of the RT-PCR products from the mother revealed that the intensity of the larger amplicon carrying the S1455X mutation was at similar levels to the smaller amplicon missing exon 14a (Fig. 3). Thus, normal CFTR mRNA transcripts and transcripts either bearing S1455X or missing exon 14a were present at similar levels, indicating that the S1455X transcripts were stable. To verify this result, we compared the levels of CFTR transcripts bearing S1455X with those missing exon 14a by RT-PCR amplification of exon 24 followed by hybridization with oligonucleotides specific for each transcript. Wild-type and mutant transcripts are predicted to amplify equally well from this region, such that within a linear range of amplification the relative amounts of RT-PCR products may be determined. Quantitation of the hybridization signals by phosphoimaging indicated that S1455X CFTR transcripts were stable, although less abundant (37.9 ± 1.99% SEM, n = 7) than del14a CFTR (62.1 ± 1.17% SEM). Since the S1455X CFTR mRNA was stable, we predicted that CFTR missing the last 26 amino acids was produced in vivo.
Processing of CFTRbearing S1455X was evaluated by immunoprecipitation using commercially available monoclonal antibodies directed against the R-domain or the last four residues at the C-terminus (Genzyme). CFTR was detected using protein kinase A (PKA)-mediated 32P-radiolabeling followed by denaturing gel electrophoresis and autoradiography. Both S1455X CFTR and wild-type CFTR products immunoprecipitated with the R-domain antibody have an apparent Mr of 175 kDa (Fig. 4). As expected, the S1455X CFTR mutant truncated at the C-terminus was not immunoprecipitated by the C-terminus antibody (Fig. 4). Wild-type CFTR protein (175 kDa) was immunoprecipitated with the C-terminus antibody from transfected HEK 293 cells, indicating that immunoprecipitation with this antibody was efficient.
Sweat chloride concentrations >60 mmol/l are associated with CF, malnutrition, hyperthyroidism and various inborn errors of metabolism (1). In CF patients, the abnormal sweat electrolytes are due to dysfunction of CFTR in the sweat duct. The presence of elevated sweat chloride concentrations in three members of a single family; one with CF and two in good health, raised the possibility that abnormal CFTR may cause sweat gland dysfunction in the absence of the CF phenotype. We have identified mutations in each CFTR gene of all three individuals. The mother and younger child with isolated elevated sweat chloride concentrations have the same genotype, while the child with CF has a different genotype. Since CF is a disorder of variable severity, it was important to exclude mild disease in the mother and younger child. Extensive evaluation at the Stanford CF Center indicated that neither had any clinical manifestation of CF. Other causes of a high concentration of sweat electrolytes, such as high sodium diet, hypothyroidism and skin disorders, were also excluded. Invasive studies that might have revealed subclinical evidence of CF, such as bronchoscopy or chest computed tomography, were felt to be clinically unjustified in these healthy individuals. The possibility that the mother or younger child will develop a CF phenotype in the future cannot be excluded. However, >90% of CF patients are diagnosed by 10 years of age, and <0.2% are identified after age 45 (25). The good health of the mother at 46 years of age suggested a very low likelihood that the S1455X/del14a genotype causes CF.
Over 700 mutations have been reported in the CFTR gene, but only a minor fraction (1%) are genomic deletions (CF Genetic Analysis Consortium, personal communication). The del14a mutation is particularly unique in that it is the only genomic deletion that encompasses a single exon (14a). Analysis of respiratory epithelial RNA from the mother and father who carried this mutation revealed that CFTR transcripts missing exon 14a were stable. Loss of exon 14a did not change the reading frame, indicating that the del14a allele encoded a CFTR protein missing 43 amino acids. The omitted region encompassed most of the seventh hydrophobic segment in the second membrane-associated domain of CFTR. This region appears critical for CFTR function since homozygosity for del14a produced a classic CF phenotype in the older child of the family studied here.
The S1455X mutation was implicated as the cause of the isolated elevated sweat chloride concentrations in the mother and younger daughter based on several lines of evidence. First, this mutation co-segregated with isolated sweat gland dysfunction in the family. Second, mutations in the extreme C-terminus of CFTR have not been identified in patients with CF. The most distal CF-associated mutation reported is a frameshift mutation predicted to introduce a termination codon at residue 1430 (31). Third, the extreme C-terminal region was believed to be irrelevant to CFTR function in the airways since synthetic C-terminus truncations distal to residue 1419 retain chloride channel function (32). Furthermore, studies have indicated that the C-terminal region of CFTR is not involved in the conduction or gating of chloride by the CFTR channel (33-35). Indeed, whole-cell patch-clamp data obtained from IB3-1 bronchial epithelial cells indicate that the chloride channel of S1455X CFTR functions similarly to wild-type CFTR. This probably explains why the mother and child carrying the S1455X mutation escaped the pulmonary and pancreatic manifestations of CF. However, the remote possibility exists that another undetected mutation on the S1455X allele may contribute to the phenotype. Moreover, since the functional data were derived from a heterologous airway epithelial expression system as patient samples were not available for patch-clamp studies, S1455X CFTR dysfunction in vivo may vary in a tissue-specific manner.
The S1455X mutation is predicted to eliminate a region containing two stretches of amino acids that are highly conserved among species (36-38). This region is not found in other proteins of the ATP-binding cassette family (39-42). The phenotype associated with the S1455X mutation indicates that the C-terminal sequences of CFTR have a functional role in the sweat gland. The gland is composed of two components, a coil that secretes isotonic fluid and a water-impermeable duct that absorbs sodium and chloride as the secreted fluid passes on its way to the surface of the skin. Loss of functional CFTR in CF patients leads to reduced chloride and sodium absorption in the duct and production of sweat with a high electrolyte content (43). Since the sweat duct is the only epithelial tissue in which CFTR is present at apical and basolateral membranes (12), one could speculate that CFTR localization, perhaps to the basolateral surface, may be dependent upon C-terminal sequences. Loss of these sequences due to the S1455X mutation prohibits CFTR trafficking to the basolateral surface, leading to reduced chloride permeability of the duct cells and elevated chloride concentrations in the sweat.
Although the mechanism of sweat gland dysfunction is enigmatic at present, identification of CFTR mutations in the mother and younger sister is of clinical relevance for evaluation of high sweat chloride values. Malformation of the vas deferens was considered to be a distinct autosomal recessive disorder until CFTR mutations were discovered in men with this disorder (19,44,45). This study indicates that CFTR mutations may be associated with elevated sweat chloride concentrations in otherwise healthy females.
The family was referred for mutation analysis to investigate the possible cause of isolated elevated sweat chloride concentrations (27). A detailed clinical history was taken by one of us (R.M.) for each subject, with questions regarding airway disease, bronchitis, pneumonia, sinusitis, asthma, failure to thrive, abdominal pain and stool frequency and consistency. Sweat chloride measurements were done by quantitative pilocarpine iontophoresis tests (24). Pulmonary function tests, chest X-rays and sputum cultures were performed to determine lung volume and spirometry. The body mass index (kg/m2) was calculated using weight (kg) and height (cm) measurements and expressed in tracking percentiles for age. Informed consent was obtained in accordance with institutional guidelines.
Total genomic DNA was assayed for 16 common CFTR mutations (R117H, 621+1G -> T, R334W, R349P, A455E, 1717-1G -> A, [Delta]I507, [Delta]F508, G542X, S549N, G551D, R553X, R560T, 3849+10 kb C -> T, W1282X, N1303K) by reverse dot-blot hybridization (46). Twenty five exons were screened by DGGE for mobility shifts (47), and, if necessary, dideoxy DNA sequencing was performed. Exons 9 and 23 were sequenced directly. Southern blot analysis was performed on 5 µg of genomic DNA digested with 40 U of BglII or HindIII (BRL), electrophoresed in 1% agarose, transferred to a nylon membrane (Hybondtm-N+; Amersham, Arlington Heights, IL) then hybridized for 12 h at 58°C with cDNA probes containing different exons of CFTR (6-10, 8-12, 13-16, 13-18, 14a, 14b-15, 15-18) that were random primer-labeled with 50 µCi of [[alpha]-32P]dCTP (Dupont NEN). The membranes were washed twice and exposed to X-ray film (Kodak, Rochester, NY) at -80°C for 2-10 days.
Total RNA was isolated from nasal epithelial scrapings and T84 cells using RNAzol B (Cinna/Biotecx, Friendswood, TX). First strand cDNA was synthesized from 3.5 µg of total RNA using Superscript Reverse Transcriptase (BRL, Gaithersburg, MD) and oligo(dT) 15mer (Boehringer Mannheim, Indianapolis, IN). The product was phenol/chloroform extracted prior to PCR, and one-quarter of the cDNA reaction product was used for all amplifications. PCR was performed for 30 cycles of denaturing at 94°C for 30 s, annealing at 56 or 58°C for 30 s, and extending at 72°C for 1 min using 2.5 U of Taq DNA polymerase (BRL), 0.4 mM ultrapure dNTPs (Pharmacia Biotech, Piscataway, NJ), Taq PCR buffer (50 mM KCl, 10 mM Tris, pH 8.3, 2.5 mM MgCl2) and 0.2 µM of a primer set. Four sets of oligonucleotide primers were used: 13e5'c (5'-TTCAGGCACGAAGGAGGC-3') and 15e3' (5'-CACATAATACGAACTGGTGC-3') annealing at 58°C; 23e5'a (5'-CACAGGATAGAAGCAATGC-3') and 24e3' (5'-CCATGAGCAAATGTCCCATG-3') annealing at 56°C; 24e5' (5'-ATTCCATCCAGAAACTGCTG-3') and 24e3' (5'-CCATGAGCAAATGTCCCATG-3') annealing at 56°C; and [beta]-actin-5' (5'-GCACTCTTCCAGCCTTCC-3') and [beta]-actin-3' (5'-GCGCTCAGGAGGAGCAAT-3') annealing at 58°C. The PCR products were either electrophoresed on a 2% agarose gel at 60 mV for 1 h and Southern blotted to a nylon membrane (Hybondtm-N+; Amersham, Arlington Heights, IL) (13e5'c/15e3' PCR products) or directly dot blotted on a nylon membrane (24e5'/24e3') for allele-specific oligonucleotide (ASO) hybridization. Oligonucleotide probes were end-labeled with 1 µCi of [[gamma]-32P]ATP (Dupont NEN, Boston, MA) using 10 U of T4 polynucleotide kinase according to the manufacturer's recommendations (New England Biolabs, Beverly, MA), and the labeled probe was collected by centrifugation through a SELECT spin column (5' -> 3' Inc., Boulder, CO) packed with 10% Sephadex G-25 (Pharmacia Biotech). Southern blots were hybridized at 37°C for 1 h with [gamma]-32P-labeled 13e5'd (5'-GCAAACTTGACTGAACTGG-3'), washed twice at 54°C and exposed to X-ray film for 20 min at -80°C. Dot-blots were hybridized at 42°C for 1 h with [gamma]-32P-labeled S1455X mutant probe (5'-CACTTGCTTCAGTTCCGG-3') or S1455 wild-type probe (5'-ACCGGAACTCAAGCAAGTG-3'), washed at room temperature and exposed to X-ray film for 10 min at -80°C. The signals were quantitated by phosphoimaging (Fugi BAS1000) with MacBas version 2.31 software (Koshin Graphics System Inc.).
The mutation S1455X was created in the CFTR-containing vector pBQ4.7 (gift from J. Rommens and L.-C. Tsui; The Hospital for Sick Children, Toronto) by single-strand mutagenesis (48) and then shuttled into the Rous sarcoma virus (RSV)-driven expression vector (pRSV-CFTR) using NcoI and SalI restriction sites common to both plasmids (30). Immunoprecipitation experiments were performed with either the CFTR R-domain-specific monoclonal antibody or CFTR C-terminus-specific monoclonal antibody upon lysates of transiently transfected human embryonic kidney (HEK) 293 cells (Genzyme, Cambridge, MA). HEK293 cells were grown in Eagle's minimal essential medium (EMEM) supplemented with 10% regular fetal bovine serum (FBS) (Biofluids Inc.) and 1% Pen/Strep/Neo (BRL) at 37°C, 5% CO2. Cells were grown in a 100 mm dish to 25% confluency, transfected with 10 µg of plasmid DNA, 50 µl of lipofectin (BRL) in 3.5 ml of Opti-MEM medium (BRL) for 6-8 h and lysed 72 h post-transfection for immunoprecipitation. Plasmid DNA not containing CFTR was used for mock transfections. Cells were lysed in 20 mM HEPES, pH 7.0, 150 mM NaCl, 1 mM EDTA, and 1% NP-40 supplemented with aprotinin and phenylmethylsulfonyl fluoride (PMSF) prior to use. Lysates were pre-cleared overnight at 4°C with protein G-Sepharose beads or protein A-Sepharose beads, and the protein concentration of the lysate was determined using a protein assay kit (Bio-Rad; Hercules, CA). Four mg (transiently transfected 293 cells) of protein was incubated in 1 ml of 1* RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS) with 1 µg of antibody for 90 min at 4°C. Then 20 µl of washed protein G-Sepharose (used with the R-domain Ab) or protein A-Sepharose (used with the C-terminus Ab) was added and incubated for 30 min at 4°C. Samples were centrifuged at 10 000 r.p.m.; the pellets were washed five times (10 min each at 4°C) with 1* RIPA buffer and once in Tris-buffered saline, pH 8.0. The precipitate was resuspended in 50 mM Tris, pH 7.5, 10 mM MgCl2, 0.1 mg/ml bovine serum albumin; 5 U of PKA (Sigma, St Louis, MO) and 10 µCi of [[gamma]-32P]ATP (Dupont NEN, Boston, MA) were added, and the volume was incubated at 30°C for 1 h. The beads were washed twice (10 min at 4°C) in 1× RIPA buffer, and the labeled proteins were eluted in standard electrophoresis buffer for 5 min at 37°C. The samples were electrophoresed on a 6% SDS-PAGE gel at 150 V for 1-2 h. The gels were then fixed, dried and auto- radiographed at -80°C for 1-6 h.
Whole-cell recordings were performed on IB3-1 cells transiently transfected with either pRSV-CFTR or pRSV-CFTR/S1455X using an inverted Nikon microscope (Nikon, Inc., Melville, NY), an Axopatch amplifier (Axon Instruments, Inc., Foster City, CA) and PCLAMP 6.0 software (Axon Instruments, Inc.). IB3-1 bronchial epithelial cells were derived from a CF patient and are deficient of functional CFTR (49). The cells were grown in LHC-8 medium (Biofluids, Inc., Rockville, MD) supplemented with 30 µg/ml ECGS (Collaborative Biomedical Products, Bedford, MA), 80 µg/ml tobramycin sulfate (Eli Lilly Co., Indianapolis, IN), 200 µg/ml imipenem (Merck & Co., Inc., West Point, PA), 1% fungizone and 5% prime FBS (Biofluids, Inc.). IB3-1 cells were transiently transfected at 30% confluency in a 35 mm dish for 6-8 h with either 3 µg of wild-type CFTR (pRSV-CFTR) or 3 µg of CFTR bearing the mutation S1455X (pRSV-CFTR/S1455X) using 15 µl of lipofectin and 1 ml of Opti-MEM. The cells were grown on collagen-coated glass coverslips (Bellco Glass, Inc., Vineland, NJ) for 72 h prior to whole-cell patch-clamp analysis. To facilitate detection of transfected cells, cells were co-transfected with the green fluorescent protein (GFP) reporter plasmid, pTR-UF5 at a 10:1 ratio (pRSV-CFTR:pTR-UF5). Cells expressing GFP were identified using a CF plan fluor 20× objective, a G-2A filter cube and an epifluorescence attachment (Nikon). Symmetrical Tris-HCl solutions were used in the bath (145 mM Tris-HCl pH 7.4, 1 mM CaCl2, 5 mM HEPES, 60 mM sucrose, 1 mM MgCl2) and pipet solutions (145 mM Tris-HCl pH 7.4, 5 mM HEPES, 5 mM ATP-Mg2+, 100 nM CaCl2, 2.5 mM EGTA) (50). The holding potential was set at -60 mV, and a voltage clamp protocol was followed stepping from -100 mV to +100 mV at 20 mV increments. Cells were pre-treated with CPT-cAMP (200 µM) at 37°C for at least 5 min before the Cl- channel blocker glybenclamide (50 µM) was added. Data points from steady-state levels were taken to generate current-voltage (I-V) plots. Plots were fitted using Origin 4.0 (Microcal, Northampton, MA). A unpaired Student's t-test was used to assess statistical significance of Cl- conductance between transfected and non-transfected IB3-1 cells; P < 0.05 was considered significant.
The authors thank Dr A. Beaudet for referring this family to us and Dr N. Muzyczka for the vector pTR-UF5. We are also grateful to Dr A. Mackova, Ms S. Allen and Mr R. Prasad for expert technical assistance. This study was supported by National Institute of Health grant DK44003 (G.R.C.) and HL47122 (W.B.G.), Specialized Center for Organized Research grant DK48977 (G.R.C. and W.B.G.), IGA MZ CR 2899-5, 3526-3, 2056-5, 4124-5 (M.M., Jr), GA CR 301/95/1606 (M.M., Jr) and the Cystic Fibrosis Foundation.
Human Molecular Genetics
Pages
Introduction
Results
Clinical evaluation of a family segregating CF and isolated elevated sweat chloride concentrations
Identification of nonsense mutation S1455X in mother and daughter with elevated sweat chloride concentrations
CFTR mRNA transcript bearing the S1455X mutation is stable
The S1455X CFTR truncated protein is processed to a mature state and functions similarly to wild-type CFTR
Discussion
Materials And Methods
Mutation analysis
Transcript analysis
Expression analysis
Whole-cell patch-clamp recording
Acknowledgements
References
REFERENCES
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