ABCMdb

ABCC7 p.Ser1455*

ClinVar:

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[hide] C-terminal truncations destabilize the cystic fibr... J Biol Chem. 1999 Jul 30;274(31):21873-7. Haardt M, Benharouga M, Lechardeur D, Kartner N, Lukacs GL
C-terminal truncations destabilize the cystic fibrosis transmembrane conductance regulator without impairing its biogenesis. A novel class of mutation.
J Biol Chem. 1999 Jul 30;274(31):21873-7., 1999-07-30 [PMID:10419506]

Abstract [show]
Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5- to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.

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19 Premature stop codons were as follows: Q1412X (A. Wallace and M. Tassabehji; ⌬70); S1455X and L1399X (⌬26 and ⌬82, respectively). Login to comment

[hide] A PDZ-interacting domain in CFTR is an apical memb... J Clin Invest. 1999 Nov;104(10):1353-61. Moyer BD, Denton J, Karlson KH, Reynolds D, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M, Stanton BA
A PDZ-interacting domain in CFTR is an apical membrane polarization signal.
J Clin Invest. 1999 Nov;104(10):1353-61., [PMID:10562297]

Abstract [show]
Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain-containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain-containing proteins play a key role in the apical polarization of ion channels in epithelial cells.

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21 In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. Login to comment
35 Toward these ends, we made chimeric constructs in which the green fluorescent protein (GFP) was linked to either wild-type (wt) CFTR or CFTR with truncations in the COOH-terminus (i.e., CFTR-∆TRL or CFTR-S1455X) and expressed these proteins in polarized kidney epithelial cells (MDCK) and human bronchial epithelial cells (16HBE14o-). Login to comment
37 In addition, the CFTR mutation S1455X, which lacks the 26 COOH-terminal amino acids including the PDZ-interacting domain, is mispolarized to the lateral membrane. Login to comment
52 pGFP-CFTR-S1455X encodes a GFP-CFTR fusion protein lacking the 26 CFTR COOH-terminal amino acids due to mutation of codon 1455 (which encodes serine) to a premature stop codon. Login to comment
86 In cells expressing GFP-CFTR-S1455X, the GFP mAb recognized 2 bands with relative molecular masses of approximately 210 and 240 kDa. Login to comment
113 Accordingly, we examined the cellular distribution of the COOH-terminal truncation mutant, S1455X, which lacks the COOH-terminal 26 amino acids including the PDZ-interacting domain (40). Login to comment
114 Patients with CFTR-S1455X have defective Cl-transport in sweat ducts (40). Login to comment
115 Examination of GFP-CFTR-S1455X localization by immunofluorescence confocal microscopy demonstrated that CFTR-S1455X was polarized to the lateral plasma 1356 The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10 Figure 1 The COOH-terminal PDZ-interacting domain (TRL) is required for polarization of CFTR to the apical membrane. Login to comment
130 GFP-CFTR-S1455X colocalized with the lateral membrane protein Na+-K+-ATPase (Figure 2d). Login to comment
131 Moreover, the ratio of GFP-CFTR-S1455X in the apical versus the basolateral plasma membrane was 0.2 ± 0.1 as determined by domain-selective cell-surface biotinylation (Table 1). Login to comment
133 That CFTR-S1455X was expressed primarily in the basolateral membrane, whereas CFTR-∆TRL was equally distributed in the apical and basolateral plasma membranes, suggests that some basolateral targeting information is suppressed or inactive in CFTR-∆TRL (see Discussion). Login to comment
136 To determine whether EPB50 interacts with and colocalizes with GFP-wt-CFTR in vivo, and whether the PDZ-interacting domain of CFTR is required for interaction and colocalization with EBP50, we coexpressed EBP50 with GFP-wt-CFTR, GFP-CFTR-∆TRL, or GFP-CFTR-S1455X in MDCK and COS cells. Examination of GFP-wt-CFTR and EBP50 by immunofluorescence confocal microscopy demonstrated that GFP-wt-CFTR and EBP50 colocalized to the apical plasma membrane of MDCK cells (Figure 3, a-c). Login to comment
139 By contrast, examination of GFP-CFTR-S1455X location by immunofluorescence confocal microscopy demonstrated that GFP-CFTR-S1455X did not colocalize with EBP50. Login to comment
140 GFP-CFTR-S1455X was polarized to the lateral plasma membrane, whereas EBP50 was polarized to the apical plasma membrane (Figure 3, g-i). Login to comment
141 To test the hypothesis that EBP50 and CFTR interact via the COOH-terminal PDZ-interacting domain of CFTR in vivo, we coexpressed EBP50 with GFP-wt-CFTR, GFP-CFTR-∆TRL, or GFP-CFTR-S1455X in COS cells and determined whether CFTR and EBP50 could be coimmunoprecipitated. Login to comment
142 EBP50 coimmunoprecipitated with GFP-wt-CFTR but not with GFP-CFTR-∆TRL (Figure 4) or GFP-S1455X. Login to comment
145 Discussion We have demonstrated that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. Our data also suggest that the polarization of CFTR to the apical plasma membrane involves interaction with an apical membrane PDZ The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10 1357 Table 1 Deletion of the PDZ-interacting domain of CFTR abrogates its apical polarization Cell line wt-CFTR CFTR-∆TRL CFTR-S1455X MDCK (Biotinylation) 7.5 ± 2.3 0.6A ± 0.3 0.2A ± 0.1 MDCK (Confocal) 3.0 ± 0.4 0.6A ± 0.1 0.1A ± 0.1 16HBE14o- (Confocal) 3.6 ± 0.6 0.7A ± 0.1 0.1A ± 0.1 Data are expressed as the ratio of CFTR expressed in the apical versus the basolateral plasma membrane. Login to comment
150 Figure 2 Confocal fluorescence micrographs (xz plane) of cells expressing GFP-wt-CFTR or GFP-CFTR-S1455X. Login to comment
153 (b) GFP-CFTR-S1455X is located in the lateral membrane of MDCK cells. Login to comment
154 (c) GFP-CFTR-S1455X is located in the lateral membrane of 16HBE14o-cells. Login to comment
155 (d) GFP-CFTR-S1455X (left panel in green) colocalizes with Na+-K+-ATPase (middle panel in red) in the lateral membrane (right panel is a merge of red and green channels, yellow-orange indicates colocalization). Login to comment
157 Note that some GFP-CFTR-S1455X is expressed in an intracellular compartment. Login to comment
180 Confocal fluorescence micrographs (xz plane) of MDCK cells coexpressing: (a-c) EBP50 and wt-CFTR, (d-e) EBP50 and CFTR-∆TRL, and (g-i) EBP50 and CFTR-S1455X. Login to comment
181 GFP-CFTR is green (a, d, g), EBP50 is red (b, e, h), and the merged red and green images are shown in c, f, and i. Colocalization of EBP50 and GFP-CFTR is orange-yellow in c and f. Note that some GFP-CFTR-S1455X and GFP-CFTR-∆TRL are expressed in an intracellular compartment (d and g). Login to comment
189 In the present study, we observed that approximately 30% of total cellular GFP-wt-CFTR was expressed in the plasma membrane, whereas only 1-3% of total cellular GFP-CFTR-∆TRL and GFP-S1455X was expressed in the plasma membrane. Login to comment
197 Although the polarization of CFTR-S1455X is dramatically different from wt-CFTR, this mutation does not cause CF, as pulmonary and pancreatic function appear to be normal; however, CFTR-S1455X results in elevated sweat chloride concentration (40). Login to comment
198 Because CFTR-S1455X is a functionally normal Cl-channel, we conclude that defective Cl-transport in sweat ducts is most likely due to the mispolarization of CFTR-S1455X to the lateral rather than the apical plasma membrane. Login to comment
199 We speculate that the relatively small fraction of CFTR-S1455X expressed at the apical membrane may be sufficient to sustain pulmonary and pancreatic function, but not sweat duct function. Login to comment
200 In addition, the fact that CFTR-S1455X was polarized to the lateral membrane, whereas CFTR-∆TRL was equally distributed between the apical and basolateral plasma membranes, suggests that some basolateral sorting information is suppressed in CFTR-∆TRL. Login to comment
202 Because sorting determinants are position dependent and hierarchical, deletion of the last 26 amino acids in CFTR may unmask the tyrosine and/or dileucine motifs, such that 1 or both of these motifs direct CFTR-S1455X to the lateral membrane (13, 14). Login to comment
216 Indeed, in MDCK cells stably expressing GFP-CFTR-∆TRL or GFP-CFTR-S1455X, we observed that CPT-cAMP-stimulated, transepithelial Cl-secretion was not different from parental, untransfected MDCK cells. Login to comment

[hide] Genotype and phenotype in cystic fibrosis. Respiration. 2000;67(2):117-33. Zielenski J
Genotype and phenotype in cystic fibrosis.
Respiration. 2000;67(2):117-33., [PMID:10773783]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene which encodes a protein expressed in the apical membrane of exocrine epithelial cells. CFTR functions principally as a cAMP-induced chloride channel and appears capable of regulating other ion channels. Besides the most common mutation, DeltaF508, accounting for about 70% of CF chromosomes worldwide, more than 850 mutant alleles have been reported to the CF Genetic Analysis Consortium. These mutations affect CFTR through a variety of molecular mechanisms which can produce little or no functional CFTR at the apical membrane. This genotypic variation provides a rationale for phenotypic effects of the specific mutations. The extent to which various CFTR alleles contribute to clinical variation in CF is evaluated by genotype-phenotype studies. These demonstrated that the degree of correlation between CFTR genotype and CF phenotype varies between its clinical components and is highest for the pancreatic status and lowest for pulmonary disease. The poor correlation between CFTR genotype and severity of lung disease strongly suggests an influence of environmental and secondary genetic factors (CF modifiers). Several candidate genes related to innate and adaptive immune response have been implicated as pulmonary CF modifiers. In addition, the presence of a genetic CF modifier for meconium ileus has been demonstrated on human chromosome 19q13.2. The phenotypic spectrum associated with mutations in the CFTR gene extends beyond the classically defined CF. Besides patients with atypical CF, there are large numbers of so-called monosymptomatic diseases such as various forms of obstructive azoospermia, idiopathic pancreatitis or disseminated bronchiectasis associated with CFTR mutations uncharacteristic for CF. The composition, frequency and type of CFTR mutations/variants parallel the spectrum of CFTR-associated phenotypes, from classic CF to mild monosymptomatic presentations. Expansion of the spectrum of disease associated with the CFTR mutant genes creates a need for revision of the diagnostic criteria for CF and a dilemma for setting nosologic boundaries between CF and other diseases with CFTR etiology.

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251 Sweat Ducts An interesting phenotype, presenting with elevated sweat Cl concentration in the absence of other CF symptoms, has been described in a patient with a nonsense mutation, S1455X. Login to comment
254 Detailed analyses of the S1455X CFTR variant have shown that the protein was normally processed and functional and therefore suggests that the truncated stretch of C-terminal amino acids plays some role in the sweat gland. Login to comment

[hide] The PDZ-interacting domain of cystic fibrosis tran... J Biol Chem. 2000 Sep 1;275(35):27069-74. Moyer BD, Duhaime M, Shaw C, Denton J, Reynolds D, Karlson KH, Pfeiffer J, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M, Stanton BA
The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane.
J Biol Chem. 2000 Sep 1;275(35):27069-74., 2000-09-01 [PMID:10852925]

Abstract [show]
Polarization of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel to the apical plasma membrane in epithelial cells is critical for vectorial chloride transport. Previously, we reported that the C terminus of CFTR constitutes a PDZ-interacting domain that is required for CFTR polarization to the apical plasma membrane and interaction with the PDZ domain-containing protein EBP50 (NHERF). PDZ-interacting domains are typically composed of the C-terminal three to five amino acids, which in CFTR are QDTRL. Our goal was to identify the key amino acid(s) in the PDZ-interacting domain of CFTR with regard to its apical polarization, interaction with EBP50, and ability to mediate transepithelial chloride secretion. Point substitution of the C-terminal leucine (Leu at position 0) with alanine abrogated apical polarization of CFTR, interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane, and chloride secretion. Point substitution of the threonine (Thr at position -2) with alanine or valine had no effect on the apical polarization of CFTR, but reduced interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane as well as chloride secretion. By contrast, individual point substitution of the other C-terminal amino acids (Gln at position -4, Asp at position -3 and Arg at position -1) with alanine had no effect on measured parameters. We conclude that the PDZ-interacting domain, in particular the leucine (position 0) and threonine (position -2) residues, are required for the efficient, polarized expression of CFTR in the apical plasma membrane, interaction of CFTR with EBP50, and for the ability of CFTR to mediate chloride secretion. Mutations that delete the C terminus of CFTR may cause cystic fibrosis because CFTR is not polarized, complexed with EBP50, or efficiently expressed in the apical membrane of epithelial cells.

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36 EXPERIMENTAL PROCEDURES Expression Vectors-pGFP-CFTR, pGFP-CFTR-⌬TRL, and pGFP-CFTR-S1455X, encoding enhanced GFP fused to the N terminus of wt-CFTR, CFTR lacking the 3 C-terminal amino acids, and CFTR lacking the 26 C-terminal amino acids, respectively, were constructed as described previously (23, 29). Login to comment
81 By contrast, EBP50 could not be co-immunoprecipitated with CFTR-QDTRA or CFTR-⌬TRL, which are nonpolarized, and, as shown previously, CFTR-S1455X (23) (Fig. 2 and Table I). Login to comment
82 CFTR-S1455X, a naturally occurring mutation that lacks the 26 C-terminal amino acids, is polarized to the lateral membrane (23). Login to comment
92 By contrast, in cells stably expressing mutant CFTRs that were not polarized to the apical plasma membrane, including CFTR-⌬TRL, CFTR-S1455X and CFTR-QDTRA, FIG. 1. Login to comment
104 CFTR AP/BL ratio Co-IP EBP50 Isc % ␮A/cm2 wt-CFTR 10.4 Ϯ 2.0 100 8.3 Ϯ 0.8 ⌬TRL 0.6 Ϯ 0.3a 0a 2.6 Ϯ 0.4a QDTRA 0.7 Ϯ 0.2a 0a 2.0 Ϯ 0.3a QDTAL 10.8 Ϯ 1.8 40a - QDARL 10.9 Ϯ 2.0 10a 3.6 Ϯ 0.6a QDVRL 11.2 Ϯ 1.9 16a 4.8 Ϯ 0.5a QATRL 15.2 Ϯ 2.0 74 8.6 Ϯ 0.9 ADTRL 11.4 Ϯ 2.0 78 7.6 Ϯ 1.5 S1455X 0.2 Ϯ 0.1a 0a 1.7 Ϯ 0.2a a Indicates cAMP-stimulated chloride secretion was not significantly different from parental, untransfected cells (Fig. 3). Login to comment
114 Similar inefficient expression was observed for CFTR-S1455X, CFTR-QDTRA, CFTR-QDVRL and CFTR-QDARL (2-5% of total CFTR was expressed in the plasma membranes). Login to comment
129 By contrast, CFTR proteins that did not associate with EBP50, including QDTRA (* indicates protein could not be detected) and, as shown previously, S1455X and ⌬TRL (23), were not polarized to the apical membrane (Fig. 1). Login to comment
141 By contrast, CFTR mutants that were not polarized to the apical membrane, including ⌬TRL, S1455X, and QDTRA, did not produce cAMP-stimulated chloride currents significantly different from cAMP-stimulated currents in untransfected, parental cells. Login to comment
144 Number of monolayers studied was: 14 for parental cells, 22 for wt-CFTR, 14 for ⌬TRL, 8 for S1455X, 21 for QDTRA, 14 for QDVRL, 17 for QDARL, 5 for QATRL, and 4 for ADTRL. Login to comment

[hide] E3KARP mediates the association of ezrin and prote... J Biol Chem. 2000 Sep 22;275(38):29539-46. Sun F, Hug MJ, Lewarchik CM, Yun CH, Bradbury NA, Frizzell RA
E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells.
J Biol Chem. 2000 Sep 22;275(38):29539-46., 2000-09-22 [PMID:10893422]

Abstract [show]
Although it is generally recognized that cystic fibrosis transmembrane conductance regulator (CFTR) contains a PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at its COOH terminus, the identity of the PDZ domain protein(s) that interact with CFTR is uncertain, and the functional impact of this interaction is not fully understood. By using human airway epithelial cells, we show that CFTR associates with Na(+)/H(+) exchanger (NHE) type 3 kinase A regulatory protein (E3KARP), an EBP50/NHE regulatory factor (NHERF)-related PDZ domain protein. The PDZ binding motif located at the COOH terminus of CFTR interacts preferentially with the second PDZ domain of E3KARP, with nanomolar affinity. In contrast to EBP50/NHERF, E3KARP is predominantly localized (>95%) in the membrane fractions of Calu-3 and T84 cells, where CFTR is located. Moreover, confocal immunofluorescence microscopy of polarized Calu-3 monolayers shows that E3KARP and CFTR are co-localized at the apical membrane domain. We also found that ezrin associates with E3KARP in vivo. Co-expression of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimulated CFTR Cl(-) currents. These results support the concept that E3KARP functions as a scaffold protein that links CFTR to ezrin. Since ezrin has been shown previously to function as a protein kinase A anchoring protein, we suggest that one function served by the interaction of E3KARP with both ezrin and CFTR is to localize protein kinase A in the vicinity of the R-domain of CFTR. Since ezrin is also an actin-binding protein, the formation of a CFTR.E3KARP.ezrin complex may be important also in stabilizing CFTR at the apical membrane domain of airway cells.

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301 Support for this concept is provided by the naturally occurring CFTR mutation, S1455X, which deletes part of the CFTR COOH terminus (33). Login to comment

[hide] Localization of sequences within the C-terminal do... J Biol Chem. 2001 Jan 12;276(2):1291-8. Gentzsch M, Riordan JR
Localization of sequences within the C-terminal domain of the cystic fibrosis transmembrane conductance regulator which impact maturation and stability.
J Biol Chem. 2001 Jan 12;276(2):1291-8., 2001-01-12 [PMID:11022033]

Abstract [show]
Some disease-associated truncations within the 100-residue domain C-terminal of the second nucleotide-binding domain destabilize the mature protein (Haardt, M., Benharouga, M., Lechardeur, D., Kartner, N., and Lukacs, G. L. (1999) J. Biol. Chem. 274, 21873-21877). We now have identified three short oligopeptide regions in the C-terminal domain which impact cystic fibrosis transmembrane conductance regulator (CFTR) maturation and stability in different ways. A highly conserved hydrophobic patch (region I) formed by residues 1413-1416 (FLVI) was found to be crucial for the stability of the mature protein. Nascent chain stability was severely decreased by shortening the protein by 81 amino acids (1400X). This accelerated degradation was sensitive to proteasome inhibitors but not influenced by brefeldin A, indicating that it occurred at the endoplasmic reticulum. The five residues at positions 1400 to 1404 (region II) normally maintain nascent CFTR stability in a positional rather than a sequence-specific manner. A third modulating region (III) constituted by residues 1390 to 1394 destabilizes the protein. Hence the nascent form regains stability on further truncation back to residues 1390 or 1380, permitting some degree of maturation and a low level of cyclic AMP-stimulated chloride channel activity at the cell surface. Thus while not absolutely essential, the C-terminal domain strongly modulates the biogenesis and maturation of CFTR.

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235 Another truncation detected in a family where the sweat gland may be the sole tissue effected, S1455X (42), would not influence the turnover of either form of CFTR. Login to comment

[hide] Molecular screening of the CFTR gene in men with a... Mol Hum Reprod. 2000 Dec;6(12):1063-7. Jezequel P, Dubourg C, Le Lannou D, Odent S, Le Gall JY, Blayau M, Le Treut A, David V
Molecular screening of the CFTR gene in men with anomalies of the vas deferens: identification of three novel mutations.
Mol Hum Reprod. 2000 Dec;6(12):1063-7., [PMID:11101688]

Abstract [show]
Many studies have shown that congenital absence of the vas deferens (CAVD) is a genital cystic fibrosis transmembrane conductance regulator (CFTR)-mediated phenotype, with a broad spectrum of abnormalities causing male infertility. The genotype of these patients includes mutations in the CFTR gene, e.g. DeltaDeltaF508, R117H and the T5 allele; all of which are commonly found in CAVD. In this study we have screened the entirety of CFTR gene in 47 males with anomalies of the vas deferens: 37 cases of congenital bilateral absence of the vas deferens, three cases of congenital unilateral absence of the vas deferens and seven cases of obstructive azoospermia with hypoplastic vas deferens. Among the 94 chromosomes studied, 65 mutations, of which three are novel (2789+2insA, L1227S, 4428insGA), were identified. The majority of patients (63.8%) had two detectable CFTR gene mutations. Furthermore, high frequencies of the DeltaDeltaF508 mutation (44.7%), the T5 allele (36.2%) and R117H mutation (19.1%) were observed.

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74 Other authors (Mickle et al., 1998; Moyer et al., 1999) One mutation detected and one T5 allele (15/47 ϭ 31.9%) have demonstrated that a nonsense S1455X mutation in exon 1 ∆F508/- (TG)10T9/(TG)12T5 24 encoded a truncated version of the CFTR protein which 3 ∆F508/- (TG)10T9/(TG)12T5 missed the last 26 amino acids and mispolarized to the lateral5 ∆F508/- (TG)10T9/(TG)12T5 6 ∆F508/- (TG)10T9/(TG)12T5 membrane of epithelia. Login to comment
75 The child and mother bearing the 10 ∆F508/- (TG)10T9/(TG)12T5 del14a/S1455X genotype were clinically well but had consist-13 ∆F508/- (TG)10T9/(TG)13T5 ently elevated sweat chloride concentrations (74 mmol/l). Login to comment

[hide] Comprehensive mutation screening in a cystic fibro... Pediatrics. 2001 Feb;107(2):280-6. Wine JJ, Kuo E, Hurlock G, Moss RB
Comprehensive mutation screening in a cystic fibrosis center.
Pediatrics. 2001 Feb;107(2):280-6., [PMID:11158459]

Abstract [show]
OBJECTIVES AND BACKGROUND: The identities of a cystic fibrosis (CF) patient's CFTR mutations can influence therapeutic strategies, but because >800 CFTR mutations exist, cost-effective, comprehensive screening requires a multistage approach. Single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA) can be an important part of mutation detection, but must be calibrated within each laboratory. The sensitivity of a combined commercial-SSCP/HA approach to genotyping in a large, ethnically diverse US center CF population has not been established. STUDY DESIGN: We screened all 27 CFTR exons in 10 human participants who had an unequivocal CF diagnosis including a positive sweat chloride test and at least 1 unknown allele after commercial testing for the 70 most common mutations by SSCP/HA. These participants were compared with 7 participants who had negative sweat tests but at least 1 other CF-like symptom meriting complete genotyping. RESULTS: For the 10 CF participants, we detected 11 of 16 unknown alleles (69%) and all 4 of the known alleles (100%), for an overall rate of 75% inpatients not fully genotyped by conventional 70 mutation screen. For 7 participants with negative sweat tests, we confirmed 1 identified mutation in 14 alleles and detected 3 additional mutations. Mutations detected in both groups included 7 missense mutations (S13F, P67L, G98R, S492F, G970D, L1093P, N1303K) and 9 deletion, frameshift, nonsense or splicing mutations (R75X, G542X, DeltaF508, 451-458Delta8 bp, 5T, 663DeltaT, exon 13 frameshift, 1261+1G-->A and 3272-26A-->G). Three of these mutations were novel (G970D, L1093P, and 451-458Delta8 bp(1)). Thirteen other changes were detected, including the novel changes 1812-3 ins T, 4096-278 ins T, 4096-265 ins TG, and 4096-180 T-->G. CONCLUSION: When combined with the 70 mutation Genzyme test, SSCP/HA analysis allows for detection of >95% of the mutations in an ethnically heterogeneous CF center population. We discuss 5 possible explanations that could account for the few remaining undetected mutations.

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86 Mutations in the Stanford CF Mutation Database After Screening With the Genzyme70 Assay Mutation n % n % ⌬F508 353 67.11% 353 67.11% Splice mutations 16 3.04% 621ϩ1 G3T 5 0.95% 1717-1 G3A 5 0.95% 2789ϩ5 G3A 1 0.19% 1898ϩ1 G3A 1 0.19% 3849ϩ10 kb C3T 4 0.76% Stop mutations 31 5.89% Q493X 1 0.19% G542X 13 2.47% R553X 4 0.76% R1162X 1 0.19% W1282X 10 1.90% S1455X 2 0.38% Insertions/deletions 9 1.71% 681 del C 1 0.19% 2184 del A 2 0.38% 3859 del C 5 0.95% 3905 ins T 1 0.19% Missense mutations 33 6.27% G85E 4 0.76% R117H 3 0.57% R334W 6 1.14% G551D 14 2.66% R560T 3 0.57% N1303K 3 0.57% Unknown mutations 84 15.97% 84 15.97% Total 526 100.00% 526 100.00% ARTICLES tients with positive sweat tests were selected for SSCP/HA analysis based on clinical status, ethnicity, and previous screening with the Genzyme70 assay. Login to comment

[hide] A PDZ-binding motif is essential but not sufficien... J Cell Sci. 2001 Feb;114(Pt 4):719-26. Milewski MI, Mickle JE, Forrest JK, Stafford DM, Moyer BD, Cheng J, Guggino WB, Stanton BA, Cutting GR
A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane.
J Cell Sci. 2001 Feb;114(Pt 4):719-26., [PMID:11171377]

Abstract [show]
Localization of ion channels and transporters to the correct membrane of polarized epithelia is important for vectorial ion movement. Prior studies have shown that the cytoplasmic carboxyl terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the apical localization of this protein. Here we show that the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains. Co-expression of the C terminus with full-length CFTR results in redistribution of CFTR from apical to basolateral membranes, indicating that both proteins interact with the same target at the apical membrane. Amino acid substitution and deletion analysis confirms the importance of a PDZ-binding motif D-T-R-L> for apical localization. However, two other C-terminal regions, encompassing amino acids 1370-1394 and 1404-1425 of human CFTR, are also required for localizing to the apical plasma membrane. Based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif.

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114 This truncation corresponds to the naturally occurring CFTR mutation S1455X that was associated with sweat gland dysfunction (Mickle et al., 1998). Login to comment
144 (a) Loss of apical localization of GFP-CFTR 1370-1480 ∆ag construct (green) after truncation of last 26 amino acids (S1455X), truncation of last three amino acids (∆TRL), or substitution of last three amino acids with alanines (TRL→AAA). Login to comment

[hide] Regulatory interaction between the cystic fibrosis... J Biol Chem. 2001 May 18;276(20):17236-43. Epub 2001 Feb 22. Ahn W, Kim KH, Lee JA, Kim JY, Choi JY, Moe OW, Milgram SL, Muallem S, Lee MG
Regulatory interaction between the cystic fibrosis transmembrane conductance regulator and HCO3- salvage mechanisms in model systems and the mouse pancreatic duct.
J Biol Chem. 2001 May 18;276(20):17236-43. Epub 2001 Feb 22., 2001-05-18 [PMID:11278980]

Abstract [show]
The pancreatic duct expresses cystic fibrosis transmembrane conductance regulator (CFTR) and HCO3- secretory and salvage mechanisms in the luminal membrane. Although CFTR plays a prominent role in HCO3- secretion, the role of CFTR in HCO3- salvage is not known. In the present work, we used molecular, biochemical, and functional approaches to study the regulatory interaction between CFTR and the HCO3- salvage mechanism Na+/H+ exchanger isoform 3 (NHE3) in heterologous expression systems and in the native pancreatic duct. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. In the pancreatic duct, CFTR increases expression of NHE3 in the luminal membrane. Thus, luminal expression of NHE3 was reduced by 53% in ducts of homozygote DeltaF508 mice. Accordingly, luminal Na+-dependent and HOE694- sensitive recovery from an acid load was reduced by 60% in ducts of DeltaF508 mice. CFTR and NHE3 were co-immunoprecipitated from PS120 cells expressing both proteins and the pancreatic duct of wild type mice but not from PS120 cells lacking CFTR or the pancreas of DeltaF508 mice. The interaction between CFTR and NHE3 required the COOH-terminal PDZ binding motif of CFTR, and mutant CFTR proteins lacking the C terminus were not co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120 cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ binding motif, augmented cAMP-dependent inhibition of NHE3 activity by 31%. These findings reveal that CFTR controls overall HCO3- homeostasis by regulating both pancreatic ductal HCO3- secretory and salvage mechanisms.

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60 Site-directed Mutagenesis-Oligonucleotide-directed mutagenesis using the GeneEditor mutagenesis kit (Promega, Madison, WI) was performed in the CFTR expression vector pCMVNot6.2 to delete the C-terminal 4 (⌬DTRL) or 26 (S1455X) amino acids. Login to comment
63 The mutagenesis primers were as follows: ⌬DTRL, 5Ј-GGA GAC AGA AGA AGA GGT GTA AGA TAC AAG GCT TTA GAG AG-3Ј; S1455X, 5Ј-GCT CTT TCC CCA CCG GAA CTG AAG CAA GTG CAA GTC TAA GCC-3Ј. Login to comment
174 Another finding of note in Fig. 5 is the behavior of a mutant CFTR protein lacking the final C-terminal 26 amino acids (S1455X). Login to comment
176 Similar to the findings with the CFTR-⌬DTRL, the CFTR-S1455X did not associate with NHE3 (Fig. 5B) and had no effect on NHE3 activity (Fig. 5C). Login to comment
208 PS120/NHE3 cells were transfected with mammalian expressing vectors for WT-CFTR, CFTR-⌬DTRL, and CFTR-S1455X. Login to comment

[hide] A combined analysis of the cystic fibrosis transme... Mol Biol Evol. 2001 Sep;18(9):1771-88. Chen JM, Cutler C, Jacques C, Boeuf G, Denamur E, Lecointre G, Mercier B, Cramb G, Ferec C
A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models.
Mol Biol Evol. 2001 Sep;18(9):1771-88., [PMID:11504857]

Abstract [show]
Over the past decade, nearly 1,000 variants have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in classic and atypical cystic fibrosis (CF) patients worldwide, and an enormous wealth of information concerning the structure and function of the protein has also been accumulated. These data, if evaluated together in a sequence comparison of all currently available CFTR homologs, are likely to refine the global structure-function relationship of the protein, which will, in turn, facilitate interpretation of the identified mutations in the gene. Based on such a combined analysis, we had recently defined a "functional R domain" of the CFTR protein. First, presenting two full-length cDNA sequences (termed sCFTR-I and sCFTR-II) from the Atlantic salmon (Salmo salar) and an additional partial coding sequence from the eastern gray kangaroo (Macropus giganteus), this study went further to refine the boundaries of the two nucleotide-binding domains (NBDs) and the COOH-terminal tail (C-tail), wherein NBD1 was defined as going from P439 to G646, NBD2 as going from A1225 to E1417, and the C-tail as going from E1418 to L1480. This approach also provided further insights into the differential roles of the two halves of CFTR and highlighted several well-conserved motifs that may be involved in inter- or intramolecular interactions. Moreover, a serious concern that a certain fraction of missense mutations identified in the CFTR gene may not have functional consequences was raised. Finally, phylogenetic analysis of all the full-length CFTR amino acid sequences and an extended set of exon 13--coding nucleotide sequences reinforced the idea that the rabbit may represent a better CF model than the mouse and strengthened the assertion that a long-branch attraction artifact separates the murine rodents from the rabbit and the guinea pig, the other Glires.

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542 Indeed, neither S1455X (Mickle et al. 1998) nor Q1476X (http:// www.genet.sickkids.on.ca/cftr) caused a severe CF phenotype. Login to comment

[hide] Aggregation of misfolded proteins can be a selecti... J Biol Chem. 2002 Sep 13;277(37):34462-70. Epub 2002 Jun 25. Milewski MI, Mickle JE, Forrest JK, Stanton BA, Cutting GR
Aggregation of misfolded proteins can be a selective process dependent upon peptide composition.
J Biol Chem. 2002 Sep 13;277(37):34462-70. Epub 2002 Jun 25., 2002-09-13 [PMID:12084728]

Abstract [show]
Intracellular aggregation of misfolded proteins is observed in a number of human diseases, in particular, neurologic disorders in which expanded tracts of polyglutamine residues play a central role. A variety of other proteins are prone to aggregation when mutated, indicating that this process is a common pathologic mechanism for inherited disorders. However, little is known about the relationship between the sequence of aggregating peptides and the specificity of intracellular accumulation. Here we demonstrate that substitution of two residues eliminates aggregation of a 111-amino acid peptide derived from the C-terminal portion of the cystic fibrosis transmembrane conductance regulator (CFTR). We also show that fusion to a reporter protein considerably alters the subcellular distribution of aggregating peptide. When fused to green fluorescent protein, the peptide containing amino acids 1370-1480 of CFTR accumulates in large perinuclear or nuclear aggregates. The same CFTR fragment devoid of green fluorescent protein localizes predominantly to discrete accumulations associated with mitochondria. Importantly, both types of accumulation are dependent on the presence of the same two amino acids within the CFTR sequence. Co-expression studies show that both CFTR-derived proteins can co-localize in large cytoplasmic/nuclear aggregates. However, neither CFTR construct accumulates in intracellular inclusions formed by N-terminal fragment of huntingtin. In addition to unique accumulation patterns, each aggregating peptide shows differences in association with chaperone proteins. Thus, our results indicate that the process of intracellular aggregation can be a selective process determined by the composition of the aggregating peptides.

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125 To examine whether this C-terminal region contributed to the unusual accumulation pattern of HA-CFTR 1370-1480, we deleted the last 26 amino acids in this construct by introducing the naturally occurring CFTR mutation S1455X (33), associated with abnormal localization of the full-length protein (31). Login to comment

[hide] Effects of C-terminal deletions on cystic fibrosis... Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1937-42. Epub 2003 Feb 10. Ostedgaard LS, Randak C, Rokhlina T, Karp P, Vermeer D, Ashbourne Excoffon KJ, Welsh MJ
Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia.
Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1937-42. Epub 2003 Feb 10., 2003-02-18 [PMID:12578973]

Abstract [show]
To better understand the function of the conserved C terminus of the cystic fibrosis (CF) transmembrane conductance regulator, we studied constructs containing deletions in the C-terminal tail. When expressed in well differentiated CF airway epithelia, each construct localized predominantly to the apical membrane and generated transepithelial Cl(-) current. The results suggested that neither the C-terminal PSD-95/Discs-large/ZO-1 (PDZ)-interacting motif nor other C-terminal sequences were absolutely required for apical expression in airway epithelia. Surprisingly, deleting an acidic cluster near the C terminus reduced both channel opening rate and transepithelial Cl(-) transport, indicating that it influences channel gating. These results may help explain the relative paucity of CF-associated mutations in the C terminus.

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7 Deletion of 26 C-terminal residues by the S1455X mutation was associated with elevated sweat Cl- concentrations, but not other manifestations of CF (4), whereas deletion of the last 70 residues by the Q1412X mutation caused CF (C. J. Taylor, personal communication). Login to comment
117 In addition, whole-cell patch-clamp studies of BHK-21 and IB3-1 cells reported that the S1455X mutation had no effect on current, but the Q1412X mutation reduced current by 90% (4, 19). Login to comment
184 These findings are consistent with previous reports that maturation and degradation of C-terminally truncated CFTR (S1455X and D1425X) is similar to WT in COS and BHK cells (19-21). Login to comment
248 The S1455X was associated with an elevated sweat Cl-concentration, but not airway or pancreatic disease (4), and the 1476X deletion was found in a person with congenital bilateral absence of the vas deferens (personal communication, M. Claustres, Montpellier, France). Login to comment

[hide] The role of the C terminus and Na+/H+ exchanger re... J Biol Chem. 2003 Jun 13;278(24):22079-89. Epub 2003 Mar 21. Benharouga M, Sharma M, So J, Haardt M, Drzymala L, Popov M, Schwapach B, Grinstein S, Du K, Lukacs GL
The role of the C terminus and Na+/H+ exchanger regulatory factor in the functional expression of cystic fibrosis transmembrane conductance regulator in nonpolarized cells and epithelia.
J Biol Chem. 2003 Jun 13;278(24):22079-89. Epub 2003 Mar 21., 2003-06-13 [PMID:12651858]

Abstract [show]
The conserved C-terminal peptide motif (1476DTRL) of the cystic fibrosis transmembrane conductance regulator (CFTR) ensures high affinity binding to different PSD-95/Disc-large/zonula occludens-1 (PDZ) domain-containing molecules, including the Na+/H+ exchanger regulatory factor (NHERF)/ezrin-radixin-moesin-binding phosphoprotein of 50 kDa. The physiological relevance of NHERF binding to CFTR is not fully understood. Individuals with mutations resulting in premature termination of CFTR (S1455X or Delta26 CFTR) have moderately elevated sweat Cl- concentration, without an obvious lung and pancreatic phenotype, implying that the CFTR function is largely preserved. Surprisingly, when expressed heterologously, the Delta26 mutation was reported to abrogate channel activity by destabilizing the protein at the apical domain and inducing its accumulation at the basolateral membrane (Moyer, B., Denton, J., Karlson, K., Reynolds, D., Wang, S., Mickle, J., Milewski, M., Cutting, G., Guggino, W., Li, M., and Stanton, B. (1999) J. Clin. Invest. 104, 1353-1361). The goals of this study were to resolve the contrasting clinical and cellular phenotype of the Delta26 CFTR mutation and evaluate the role of NHERF in the functional expression of CFTR at the plasma membrane. Complex formation between CFTR and NHERF was disrupted by C-terminal deletions, C-terminal epitope tag attachments, or overexpression of a dominant negative NHERF mutant. These perturbations did not alter CFTR expression, metabolic stability, or function in nonpolarized cells. Likewise, inhibition of NHERF binding had no discernible effect on the apical localization of CFTR in polarized tracheal, pancreatic, intestinal, and kidney epithelia and did not influence the metabolic stability or the cAMP-dependent protein kinase-activated chloride channel conductance in polarized pancreatic epithelia. On the other hand, electrophysiological studies demonstrated that NHERF is able to stimulate the cAMP-dependent protein kinase-phosphorylated CFTR channel activity in intact cells. These results help to reconcile the discordant genotype-phenotype relationship in individuals with C-terminal truncations and indicate that apical localization of CFTR involves sorting signals other than the C-terminal 26 amino acid residues and the PDZ-binding motif in differentiated epithelia.

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2 Individuals with mutations resulting in premature termination of CFTR (S1455X or ⌬26 CFTR) have moderately elevated sweat Cl-concentration, without an obvious lung and pancreatic phenotype, implying that the CFTR function is largely preserved. Login to comment

[hide] Mutations located in exon 24 of the CFTR gene are ... Clin Genet. 2003 Sep;64(3):266-8. Bienvenu T, Viel M, Leroy C, Van Esch H, Fajac I, Dusser D, Hubert D
Mutations located in exon 24 of the CFTR gene are associated with a mild cystic fibrosis phenotype.
Clin Genet. 2003 Sep;64(3):266-8., [PMID:12919146]

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3 However, Mickle et al. described a nonsense mutation, S1455X, located in exon 24, which was associated with elevated sweat chloride concentrations in the absence of the CF phenotype in female compound heterozygotes (2). Login to comment

[hide] Emerging drug treatments for cystic fibrosis. Expert Opin Emerg Drugs. 2003 Nov;8(2):523-35. Zeitlin PL
Emerging drug treatments for cystic fibrosis.
Expert Opin Emerg Drugs. 2003 Nov;8(2):523-35., [PMID:14662004]

Abstract [show]
Cystic fibrosis (CF) is one of the most common life-shortening inherited disorders. Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene disrupt the localisation and function of the cAMP-mediated chloride channel. Most of the morbidity and mortality arise from the lung disease which is characterised by excessive inflammation and chronic infection. Research into the mechanisms of wild-type and mutant CFTR biogenesis suggest that multiple drug targets can be identified. This review explores the current understanding of the nature of the different mutant CFTR forms and the potential for repair of the chloride channel defect. High-throughput screening, pharmacogenomics and proteomics bring recent technological advances to the field.

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88 Class of mutation Molecular mechanism Pancreatic status (if known) Examples 1 No CFTR protein synthesis PI W1282X, G542X, R553X, 621 + 1 G→T, 1717-1 G→A, 3905insT, 394delTT 2 Abnormal CFTR processing and trafficking PI ∆F508, N1303K, P574H 3 Defective CFTR regulation (normal trafficking) PI G551D, G551S, G1349D, S1255P 4 Decreased CFTR chloride conductance PS R117H, R334W, R347P, P547H 5 Reduced synthesis and trafficking of normal CFTR PS A455E, 3849 + 10kb C→T, (5T) 6A Reduced apical stability PI S1455X, Q1412S, 4326delTC, 4279insA 6B Defective regulation of other ion channels PI G551D Note that the G551D is placed in Class 3 for defective regulation and Class 6B for defective regulation of the outwardly rectifying chloride channel. Login to comment

[hide] Isolated elevated sweat chloride concentrations in... Am J Med Genet A. 2005 Mar 1;133A(2):207-8. Salvatore D, Tomaiuolo R, Vanacore B, Elce A, Castaldo G, Salvatore F
Isolated elevated sweat chloride concentrations in the presence of the rare mutation S1455X: an extremely mild form of CFTR dysfunction.
Am J Med Genet A. 2005 Mar 1;133A(2):207-8., 2005-03-01 [PMID:15666307]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been shown to cause typical cystic fibrosis (CF) and several milder phenotypes. We report on two asymptomatic sisters who had isolated increased sweat chloride concentrations, and in whom systematic scanning of the whole coding region of the CFTR gene revealed the F508del/S1455X genotype.

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0 American Journal of Medical Genetics 133A:-208 (2005) Clinical Report Isolated Elevated Sweat Chloride Concentrations in the Presence of the Rare Mutation S1455X: An Extremely Mild Form of CFTR Dysfunction Donatello Salvatore,1 * Rossella Tomaiuolo,2 Borghina Vanacore,2 Ausilia Elce,2 Giuseppe Castaldo,2,3 and Francesco Salvatore2 1 Pediatric Division, Cystic Fibrosis Center, San Carlo Hospital, Potenza, Italy 2 CEINGE-Advanced Biotechnologies and Department of Biochemistry and Medical Biotechnologies, University of Naples ''Federico II``, Naples, Italy 3 Faculty of Sciences, University of Molise, Isernia, Italy Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been shown to cause typical cystic fibrosis (CF) and several milder phenotypes. Login to comment
1 We report on two asymptomatic sisters who had isolated increased sweat chloride concentrations, and in whom systematic scanning of the whole coding region of the CFTR generevealedtheF508del/S1455X genotype. Login to comment
9 Mickle et al. [1998] described a mother and a daughter who had isolated elevated sweat chloride concentrations, and the non-sense mutation S1455X in compound heterozygosis with the del14a deletion [Mickle et al., 1998]. Login to comment
10 We report on two asymptomatic sisters who had increased sweat chloride concentrations, and in whom systematic scanning of the whole coding region of the CFTR gene revealed the F508del/S1455X genotype. Login to comment
27 The analysis revealed the non-sense mutation S1455X, in compound heterozygosity with F508del. Login to comment
35 E-mail: saverdon@tiscali.it Received 31 March 2004; Accepted 21 October 2004 DOI 10.1002/ajmg.a.30518 ß 2005 Wiley-Liss, Inc. and gene scanning confirmed the presence of the F508del/ S1455X genotype (Table I). Login to comment
37 The analysis revealed the presence of the mutation F508del in heterozygosis in the mother and of the mutation S1455X in heterozygosis in the father. Login to comment
38 DISCUSSION Our case report confirms that S1455X is a rare mutation associated with isolated elevated sweat chloride concentrations in the absence of the CF phenotype. Login to comment
39 Although, we cannot exclude changes in the patients` clinical conditions in the future, the long follow-up (8 years) of these two sisters and the normal clinical picture of the previously reported patients, indicate that S1455X is related to the isolated sweat gland dysfunction [Moyer et al., 1999]. Login to comment
40 The possible explanation of this phenotype is that the CFTR mRNA transcript bearing the S1455X mutation is stable, and encodes a version of CFTR that lacks the last 26 amino acids, which is processed to a mature state and functions similarly to wild-type CFTR [Mickle et al., 1998]. Login to comment
41 In addition, the S1455X CFTR mutation is mispolarized to the lateral membrane of epithelial cells [Moyer et al., 1999]. Login to comment
42 The transport of the electrolytes across the respiratory epithelia should be studied in vivo by means of measurement of nasal potential difference (NPD) in patients carrying the S1455X mutation, in order to evaluate the organ specific expression of the mutated gene, the hypothesis being that sodium and chloride transport might be normal or near normal at the level of airway epithelial cells despite the abnormal sweat electrolytes. Login to comment
45 The most distal CF-associated mutation reported is a missense mutation predicted to replace aspartic acid for asparagine at amino acid 1445 [Cystic Fibrosis Genetic Analysis Consortium, 2004], whereas mutations more distal with respect to S1455X have been associated to the congenital bilateral absence of vasa deferentes. Login to comment
46 All compound heterozygotes with the S1455X mutation so far reported are female, so we cannot exclude the possibility that this mutation in males would be related to isolated elevated sweat chloride and infertility. Login to comment
47 The fact that a very mild phenotype is associated with S1455X is important for genetic counseling. Login to comment
49 In the case of our adult patient, we calculated the risk of having a CF child of 1:334, but the risk could be further ''halved`` based on the hypothesis that a S1455X compound heterozygote would be asymptomatic. Login to comment

[hide] Gene transfer of CFTR to airway epithelia: low lev... Am J Physiol Lung Cell Mol Physiol. 2005 Dec;289(6):L1123-30. Epub 2005 Aug 5. Farmen SL, Karp PH, Ng P, Palmer DJ, Koehler DR, Hu J, Beaudet AL, Zabner J, Welsh MJ
Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR.
Am J Physiol Lung Cell Mol Physiol. 2005 Dec;289(6):L1123-30. Epub 2005 Aug 5., [PMID:16085675]

Abstract [show]
Gene transfer of CFTR cDNA to airway epithelia is a promising approach to treat cystic fibrosis (CF). Most gene transfer vectors use strong viral promoters even though the endogenous CFTR promoter is very weak. To learn whether expressing CFTR at a low level in a fraction of cells would correct Cl(-) transport, we mixed freshly isolated wild-type and CF airway epithelial cells in varying proportions and generated differentiated epithelia. Epithelia with approximately 20% wild-type cells generated approximately 70% the transepithelial Cl(-) current of epithelia containing 100% wild-type cells. These data were nearly identical to those previously obtained with CFTR expressed under control of a strong promoter in a CF epithelial cell line. We also tested high level CFTR expression using the very strong cytomegalovirus (CMV) promoter as well as the cytokeratin-18 (K18) promoter. In differentiated airway epithelia, the CMV promoter generated 50-fold more transgene expression than the K18 promoter, but the K18 promoter generated more transepithelial Cl(-) current at high vector doses. Using functional studies, we found that with marked overexpression, some CFTR channels were present in the basolateral membrane where they shunted Cl(-) flow, thereby reducing net transepithelial Cl(-) transport. These results suggest that very little CFTR is required in a fraction of CF epithelial cells to complement Cl(-) transport because transepithelial Cl(-) flow is limited at the basolateral membrane. Thus they suggest a broad leeway in promoter strength for correcting the CF gene transfer, although at very high expression levels CFTR may be mislocalized to the basolateral membrane.

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275 Third, evidence that mislocalization of CFTR might have a physiological consequence comes from the study of the S1455X mutation, which was located in the basolateral membrane in airway epithelial cells (27). Login to comment
272 Third, evidence that mislocalization of CFTR might have a physiological consequence comes from the study of the S1455X mutation, which was located in the basolateral membrane in airway epithelial cells (27). Login to comment

[hide] The cystic fibrosis transmembrane regulator forms ... Proc Am Thorac Soc. 2004;1(1):28-32. Guggino WB
The cystic fibrosis transmembrane regulator forms macromolecular complexes with PDZ domain scaffold proteins.
Proc Am Thorac Soc. 2004;1(1):28-32., [PMID:16113408]

Abstract [show]
Cystic fibrosis transmembrane regulator (CFTR), an epithelial Cl- channel defective in cystic fibrosis, is localized at the apical membrane of epithelial cells. CFTR interacts with a number of ion channels (outwardly rectifying chloride channels, epithelial sodium channels, and inwardly rectifying potassium channels), protein kinases A and C, and putative protein phosphatases and ATP transporters. These data suggest that CFTR may exist in macromolecular complexes with these and other scaffolding proteins at the apical membrane. PDZ domain proteins have been shown to localize and cluster CFTR in the Golgi and at the plasma membrane. This review highlights the role of PDZ domain proteins in regulating the trafficking and processing of CFTR.

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130 Several years ago, our group reported 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 but with no evidence of pulmonary or pancreatic disease characteristic of cystic fibrosis. Login to comment
131 Further study showed that the S1455X-CFTR mutant generated cAMP-activated whole-cell chloride currents similar to wild-type CFTR. These data are consistent with the relatively normal lung and pancreatic function observed in the mother and her daughter (32). Login to comment
135 When the S1455X mutant is expressed in polarized, heterologous epithelial cells, a significant portion accumulates at the lateral membrane. Login to comment
146 There are limited data on a small number of individuals with the S1455X mutation, which makes it difficult to generalize on the role of the C terminus in CFTR biology. Login to comment

[hide] Modifier genetics: cystic fibrosis. Annu Rev Genomics Hum Genet. 2005;6:237-60. Cutting GR
Modifier genetics: cystic fibrosis.
Annu Rev Genomics Hum Genet. 2005;6:237-60., [PMID:16124861]

Abstract [show]
Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder in the Caucasian population, affecting about 30,000 individuals in the United States. The gene responsible for CF, the CF transmembrane conductance regulator (CFTR), was identified 15 years ago. Substantial variation in the many aspects of the CF phenotype among individuals with the same CFTR genotype demonstrates that factors independent of CFTR exert considerable influence on outcome in CF. To date, the majority of published studies investigating the cause of disease variability in CF report associations between candidate genes and some aspect of the CF phenotype. However, a definitive modifier gene for CF remains to be identified. Despite the challenges posed by searches for modifier effects, studies of affected twins and siblings indicate that genetic factors play a substantial role in intestinal manifestations. Identifying the factors contributing to variation in pulmonary disease, the primary cause of mortality, remains a challenge for CF research.

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193 This point may be best illustrated by the identification of four individuals in two unrelated families who carry a severe CF mutation paired with a rare nonsense mutation in CFTR (S1455X). Login to comment
557 Isolated elevated sweat chloride concentrations in the presence of the rare mutation S1455X: an extremely mild form of CFTR dysfunction. Login to comment

[hide] Establishing a diagnosis of cystic fibrosis. Chron Respir Dis. 2004;1(4):205-10. Southern KW, Peckham D
Establishing a diagnosis of cystic fibrosis.
Chron Respir Dis. 2004;1(4):205-10., [PMID:16281647]

Abstract [show]
Cystic fibrosis (CF) is a recessively inherited condition caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Characterization of the genetic defect has improved understanding of the condition and, in the majority of cases, diagnosis is straightforward. However, in a significant number, diagnosis remains a challenge. This paper will discuss the management of these issues and reflect on atypical presentations. In addition we will discuss situations in which genetic variations of the CFTR gene are not associated with a classical CF phenotype and the implications for practice in both paediatric and adult clinics.

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54 One mutation of the CFTR gene (S1455X) results in an abnormal sweat test but no other phenotypic evidence of CF.10 This is a nonsense mutation that results in a truncated CFTR protein with the final 26 amino acids missing. Login to comment

[hide] Mild cystic fibrosis revealed by persistent hypona... Clin Genet. 2005 Dec;68(6):552-3. Epaud R, Girodon E, Corvol H, Niel F, Guigonis V, Clement A, Feldmann D, Bensman A, Ulinski T
Mild cystic fibrosis revealed by persistent hyponatremia during the French 2003 heat wave, associated with the S1455X C-terminus CFTR mutation.
Clin Genet. 2005 Dec;68(6):552-3., [PMID:16283887]

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0 Letter to the Editor Mild cystic fibrosis revealed by persistent hyponatremia during the French 2003 heat wave, associated with the S1455X C-terminus CFTR mutation To the Editor: Cystic fibrosis (CF) - an autosomal recessive disorder with widely variable presentation - is caused by a dysfunction of the CF transmembrane conductance regulator (CFTR) protein. Login to comment
13 The patient was a compound heterozygote for the F508del (maternally inherited) and the S1455X mutation located in the last exon 24 (paternally inherited). Login to comment
17 The S1455X mutation, encoding a truncated CFTR protein missing the last 26 amino acids, was originally described in the sib and the mother of a CF patient, who were compound heterozygotes for the S1455X mutation and a genomic 8.6 kb deletion removing exon 14a (3). Login to comment
19 A recent report documented two sisters with the [F508del] þ [S1455X] genotype and elevated sweat chloride values. Login to comment
21 Functional studies failed to elucidate the association of S1455X with elevated sweat chloride values (5-8). Login to comment
25 All rights reserved CLINICAL GENETICS doi: 10.1111/j.1399-0004.2005.00525.x 552 vas deferens (CBAVD) (4428insAG), apparently isolated CBAVD (E1473X and E1476X), and isolated positive sweat test (S1455X) (3, 4). Login to comment
26 Contrary to the previous S1455X-associated cases, our compound heterozygous [F508del] þ [S1455X] patient is a boy, and CBAVD-associated infertility may possibly occur. Login to comment
28 S1455X might not have a specific effect on CFTR function, but belong to a group of mild C-terminus CFTR truncation mutations. Login to comment
42 Isolated elevated sweat chloride concentrations in the presence of the rare mutation S1455X: an extremely mild form of CFTR dysfunction. Login to comment

[hide] The relevance of sweat testing for the diagnosis o... Clin Biochem Rev. 2005 Nov;26(4):135-53. Mishra A, Greaves R, Massie J
The relevance of sweat testing for the diagnosis of cystic fibrosis in the genomic era.
Clin Biochem Rev. 2005 Nov;26(4):135-53., [PMID:16648884]

Abstract [show]
Cystic fibrosis (CF) is the most common inherited disorder of childhood. The diagnosis of CF has traditionally been based on clinical features with confirmatory evidence by sweat electrolyte analysis. Since 1989 it has been possible to also use gene mutation analysis to aid the diagnosis. Cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has advanced our understanding of CF, in particular the molecular basis of an expanded CF phenotype. However, because there are over 1000 mutations and 200 polymorphisms, many without recognised effects on CFTR, the molecular diagnosis can be troublesome. This has necessitated measurement of CFTR function with renewed interest in the sweat test. This review provides an overview of the clinical features of CF, the diagnosis and complex genetics. We provide a detailed discussion of the structure and function of CFTR and the classification of CFTR mutations. Sweat electrolyte analysis is discussed, from the physiology of sweating to the rigours of a properly performed sweat test and its interpretation. With this information it is possible to understand the relevance of the sweat test in the genomic era.

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244 Highsmith and colleagues (1994) studied 23 patients with pulmonary disease characteristic of CF but with a normal sweat test and identified a point mutation in intron 19 of the CFTR gene, termed 3849+10kb C-T.15 This mutation produces an alternative splicing site and decreased amounts of CFTR mRNA can be detected.16 Thus, according to the classification of the CFTR mutations, this mutation falls into Class V.16,67 Other mutations associated with normal or borderline sweat electrolytes are R117H, D1152H, A455E, G551S and 2789+5G - A.9,24,78 An interesting phenotype, presenting with elevated sweat chloride concentration in the absence of other CF symptoms, has been described in a patient with a nonsense mutation, S1455X.105 This mutation truncates 26 amino acids from the C-terminus of the protein product. Login to comment
246 CFTR mRNA transcripts bearing the S1455X mutation were normally processed and functional, which therefore suggests that the truncated stretch C-terminal amino acid plays some role in the sweat gland only. Login to comment

[hide] Cystic fibrosis and formes frustes of CFTR-related... Respiration. 2007;74(3):241-51. Southern KW
Cystic fibrosis and formes frustes of CFTR-related disease.
Respiration. 2007;74(3):241-51., [PMID:17534127]

Abstract [show]
Cystic fibrosis (CF) is the commonest genetic cause of bronchiectasis in the Caucasian population. Since identification of the putative gene in 1989, the molecular basis of the condition has become clearer with characterisation of the unique pathophysiology. The small airways are the primary site of lung disease, with an intense but localised inflammatory picture, dominated by neutrophils. The clinical heterogeneity is explained to some degree by the distinct molecular consequences of the many mutations that have been recognised to affect the CF transmembrane conductance regulator (CFTR) gene; however other genes appear to modify the phenotype as well as environmental exposure. It has become increasingly apparent that certain conditions may result from CFTR dysfunction without fulfilling diagnostic criteria for CF. In some cases this may result in single organ disease for which the term CF (or CFTR)-related disease has been advocated. Congenital bilateral absence of the vas deferens is the most clearly characterised of these. In other cases where a mild CF phenotype is apparent, atypical CF is probably a better term. It remains unclear whether carrier status predisposes to certain conditions such as chronic rhinosinusitis or pancreatitis.

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175 In contrast, a nonsense mutation (S1455X) results in truncation of the final 26 amino acids in CFTR and people with this mutation have a positive sweat test with raised sweat chloride levels, but no evidence of lung disease [107]. Login to comment

[hide] Restoration of W1282X CFTR activity by enhanced ex... Am J Respir Cell Mol Biol. 2007 Sep;37(3):347-56. Epub 2007 May 31. Rowe SM, Varga K, Rab A, Bebok Z, Byram K, Li Y, Sorscher EJ, Clancy JP
Restoration of W1282X CFTR activity by enhanced expression.
Am J Respir Cell Mol Biol. 2007 Sep;37(3):347-56. Epub 2007 May 31., [PMID:17541014]

Abstract [show]
Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Premature termination codons represent a common minority of CFTR mutations, and are caused by base pair substitutions that produce abnormal stop codons in the coding sequence. Select aminoglycosides induce "translational readthrough" of premature stop codons and have been shown to restore full-length functional protein in a number of preclinical and clinical settings. We studied two well-described premature termination codons found in the distal open reading frame of CFTR, W1282X and R1162X, expressed in polarizing and nonpolarizing cells. Our findings indicate that W1282X CFTR-expressing cells demonstrate significantly greater CFTR activity when overexpressed compared with R1162X CFTR cells, even when truncated protein is the predominant form. In addition, our results show that the combination of stimulated expression and stop codon suppression produces additive effects on CFTR-mediated ion transport. These findings provide evidence that W1282X CFTR exhibits membrane localization and retained chloride channel function after enhanced expression, and suggest that patients harboring this mutation may be more susceptible to CFTR rescue.

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208 Supporting this hypothesis, Mickle and coworkers reported that a disease-causing CFTR mutation produced by a PTC located 26 amino acids from the C-terminus (S1455X CFTR) retains partial function and demonstrates a mild pulmonary phenotype (but abnormal ion transport in the sweat gland [42]). Login to comment

[hide] Atypical cystic fibrosis and CFTR-related diseases... Clin Rev Allergy Immunol. 2008 Dec;35(3):116-23. Paranjape SM, Zeitlin PL
Atypical cystic fibrosis and CFTR-related diseases.
Clin Rev Allergy Immunol. 2008 Dec;35(3):116-23., [PMID:18493878]

Abstract [show]
Cystic fibrosis (CF), which is among the most common life-shortening recessive illnesses, is caused by mutations of the CF transmembrane conductance regulator (CFTR) and typically involves chronic infection and progressive obstruction of the respiratory tract as well as pancreatic exocrine insufficiency. Disease severity, to some extent, correlates with organ sensitivity to CFTR dysfunction and to the amount of functional protein, which is influenced by the type of mutation. Atypical CF represents approximately 2% of affected individuals, and includes cases presenting in adolescence or adulthood with pancreatic exocrine sufficiency, normal or borderline sweat chloride concentrations, or with a single predominant clinical feature. This review briefly describes diagnostic methods and phenotypic characteristics of classic and atypical CF, as well as CFTR-related diseases, conditions in which mutated CFTR may contribute to the pathogenesis but do not strictly fit established diagnostic criteria.

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64 Determination of the transepithelial nasal potential difference has been beneficial in establishing a CF Table 1 Mutations, sites, and molecular consequences associated with either an atypical presentation of CF respiratory disease or pancreatic sufficiency or late-onset pancreatic insufficiency (http:// www.genet.sickkids.on.ca) Mutation Site Consequence Atypical presentation M1210I Exon 19 Met to Ile at 1210 S1455X Exon 24 Ser to Stop at 1455 1811+18G→A Intron 11 mRNA splicing defect L346P Exon 7 Leu to Pro at 346 Y161D Exon 4 Tyr to Asp at 161 R31C Exon 2 Arg to Cys at 31 I752S Exon 13 Ile to Ser at 752 2811G/T Exon 15 Sequence variation Pancreatic sufficiency or late-onset pancreatic insufficiency R600G Exon 13 Arg to Gly at 600 D1152H Exon 18 Asp to His at 1152 Y89C Exon 3 Tyr to Cys at 89 R117H Exon 4 Arg to His at 117 D110E Exon 4 Asp to Glu at 110 296 + 3insT Intron 2 mRNA splicing defect E217G Exon 6a Glu to Gly at 217 V392G Exon 8 Val to Gly at 392 N1088D Exon 17b Asn to Asp at 1088 S737F Exon 13 Missense 1716+1G→A Intron 10 mRNA splicing defect R334W Exon 7 Arg to Trp at 334 R347P Exon 7 Arg to Pro at 347 A455E Exon 9 Ala to Glu at 455 P574H Exon 12 Pro to His at 574 3850-3T→G Intron 19 mRNA splicing defect diagnosis in many atypical cases. Login to comment

[hide] A C-terminal motif found in the beta2-adrenergic r... Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8496-501. Hall RA, Ostedgaard LS, Premont RT, Blitzer JT, Rahman N, Welsh MJ, Lefkowitz RJ
A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.
Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8496-501., 1998-07-21 [PMID:9671706]

Abstract [show]
The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.

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149 Interestingly, a naturally occurring truncation of the tail of CFTR (S1455X) has been recently reported (24). Login to comment

[hide] Molecular aspects of renal anionic drug transport. Annu Rev Physiol. 2002;64:563-94. Russel FG, Masereeuw R, van Aubel RA
Molecular aspects of renal anionic drug transport.
Annu Rev Physiol. 2002;64:563-94., [PMID:11826280]

Abstract [show]
Multiple organic anion transporters in the proximal tubule of the kidney are involved in the secretion of drugs, toxic compounds, and their metabolites. Many of these compounds are potentially hazardous on accumulation, and it is therefore not surprising that the proximal tubule is also an important target for toxicity. In the past few years, considerable progress has been made in the cloning of these transporters and their functional characterization following heterologous expression. Members of the organic anion transporter (OAT), organic anion transporting polypeptide (OATP), multidrug resistance protein (MRP), sodium-phosphate transporter (NPT), and peptide transporter (PEPT) families have been identified in the kidney. In this review, we summarize our current knowledge on their localization, molecular and functional characteristics, and substrate and inhibitor specificity. A major challenge for the future will be to understand how these transporters work in concert to accomplish the renal secretion of specific anionic substrates.

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224 The CFTR mutant S1455X, which lacks the26C-terminalaminoacids,nolongerbindstoNHERF-1andismislocatedtothe lateral membrane upon expression in kidney (MDCK) and airway (16HBE14o-) epithelial cells (125). Login to comment
254 The CFTR mutant S1455X, which lacks the26C-terminalaminoacids,nolongerbindstoNHERF-1andismislocatedtothe lateral membrane upon expression in kidney (MDCK) and airway (16HBE14o-) epithelial cells (125). Login to comment

[hide] Keratin K18 increases CFTR surface expression by b... J Biol Chem. 2012 Oct 8. Duan Y, Sun Y, Zhang F, Zhang WK, Wang D, Wang Y, Cao X, Hu W, Xie C, Cuppoletti J, Magin TM, Wang H, Wu Z, Li N, Huang P
Keratin K18 increases CFTR surface expression by binding to its C-terminal hydrophobic patch.
J Biol Chem. 2012 Oct 8., [PMID:23045527]

Abstract [show]
Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to cystic fibrosis, but the regulation of CFTR is not fully understood. Here, we identified the intermediate filament protein keratin K18 (K18) as a CFTR binding protein by various approaches. We mapped a highly conserved hydrophobic patch (F1413LVI) in the CFTR carboxy-terminus, known to determine plasmalemmal CFTR stability, as the K18 binding site. On the other hand, the C-terminal tail of K18 was found to be a critical determinant for binding CFTR. Overexpression of K18 in cells robustly increased the surface expression of wild-type CFTR, whereas depletion of K18 through RNA interference specifically diminished it. K18 binding increased the surface expression of CFTR by accelerating its apical recycling rate without altering CFTR's biosynthesis, maturation or internalization. Importantly, CFTR surface expression was markedly reduced in duodenal and gallbladder epithelia of K18-/- mice. Taken together, our results suggest that K18 increases the cell surface expression of CFTR by interacting with CFTR's C-terminal hydrophobic patch. These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner K8 may be modifier genes in cystic fibrosis.

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23 Interestingly, clinical studies suggest that the deletion of CFTR`s C-terminal 26 residues by the S1455X mutation elevates the chloride concentration in sweat without producing any other CF symptoms, while the deletion of the C-terminal 69 residues by Q1412X mutation results in severe CF (8-10). Login to comment
238 It is possible that mutation Q1412X in CFTR, but not mutation S1455X, disrupts the K18 binding site and thus leads to the loss of plasmalemmal CFTR in epithelia and causes severe CF in patients. Login to comment
17 Interestingly, clinical studies suggest that the deletion of the CFTR C-terminal 26 residues by the S1455X mutation elevates the chloride concentration in sweat without producing any other CF symptoms, whereas the deletion of the C-terminal 69 residues by the Q1412X mutation results in severe CF (8-10). Login to comment
291 It is possible that mutation Q1412X in CFTR, but not mutation S1455X, disrupts the K18-binding site and thus leads to the loss of plasmalemmal CFTR in epithelia and causes severe CF in patients. Login to comment

[hide] Analysis of the CFTR gene in Iranian cystic fibros... J Cyst Fibros. 2008 Mar;7(2):102-9. Epub 2007 Jul 27. Alibakhshi R, Kianishirazi R, Cassiman JJ, Zamani M, Cuppens H
Analysis of the CFTR gene in Iranian cystic fibrosis patients: identification of eight novel mutations.
J Cyst Fibros. 2008 Mar;7(2):102-9. Epub 2007 Jul 27., [PMID:17662673]

Abstract [show]
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.

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38 1 p.N1303K E21 C to G at 4041 Asn to Lys at 1303 6 p.S1455X E24 C to G at 4496 Ser to stop at 1455 1 c.186-?_296+?del E2 Large in frame deletion starting in intron 1, ending in intron 2 1 c.1342-?_1524+?del E9 Large in frame deletion starting in intron 8, ending in intron 9 1 c.406-?_1716+?del E4-E10 Large in frame deletion starting in intron 3, ending in intron 10 2 Mutations described for the first time appear in bold. Login to comment
66 Results A total of 69 unrelated CF patients (38 male and 31 female; aged between 2 months and 15 years) of Iranian Table 2 Genotype of CFTR genes in 53 Iranian patients Genotype Exon/intron Number of patients p.F508del/p.F508del E10/E10 10 p.F508del/p.R1162X E10/E19 2 p.F508del/p.T1036I E10/E17a 1 p.F508del/p.R1066C E10/E17b 1 p.F508del/c.1342-?_1524+?del E10/E9 1 p.S466X/p.S466X E10/E10 4 c.2183_2184delAAinsG/ c.2183_2184delAAinsG E13/E13 4 c.2183_2184delAAinsG/c.186- ?_296+?del E13/E2 1 p.N1303K/p.N1303K E21/E21 2 p.N1303K/p.S945L E21/E15 1 p.N1303K/c.1677delTA E21/E10 1 p.G542X/p.G542X E11/E11 2 p.G542X/c.2789+5GNA E11/I14b 1 c.3120+1GNA/c.3120+1GNA I16/I16 2 c.3120+1GNA/c.3121-1GNA I16 1 c.3121-1GNA/p.T1086I I16/E17b 1 c.3130delA/c.3130delA E17a/E17a 2 p.D192G/p.D192G E5/E5 1 p.R334W/p.R334W E7/E7 1 p.R334W/p.S945L E7/E15 1 p.R334W/p.L1077P E7/E17b 1 c.1525-1GNA/c.1525-1GNA I9/I9 1 p.S549R/p.S549R E11/E11 1 p.A566D/p.A566D E12/E12 1 c.1898+1GNT/c.1898+1GNT I12/I12 1 c.2576delA/p.S1455X/ E13/E24 1 c.2184insA/c.1677delTA E10/E13 1 p.R785X/p.R785X E13/E13 1 c.2752-1_2756delGGTGGCinsTTG/ c.2752-1_2756delGGTGGCinsTTG I14a/E14b 1 c.2789+5GNA/c.2789+5GNA I14b/I14b 1 p.K1177X/p.K1177X E19/E19 1 c.406-?_1716+?del/c.406-?_1716+?del E4-E10/E4-E10 1 Total 53 origin were extensively studied for the presence of mutations in the CFTR gene, for the presence of the deep intronic 3849+10 kbC→T mutation, and large deletions/ duplications. Login to comment
96 It was found once in a patient that carried S1455X in compound heterozygosity. Login to comment
65 Results A total of 69 unrelated CF patients (38 male and 31 female; aged between 2 months and 15 years) of Iranian Table 2 Genotype of CFTR genes in 53 Iranian patients Genotype Exon/intron Number of patients p.F508del/p.F508del E10/E10 10 p.F508del/p.R1162X E10/E19 2 p.F508del/p.T1036I E10/E17a 1 p.F508del/p.R1066C E10/E17b 1 p.F508del/c.1342-?_1524+?del E10/E9 1 p.S466X/p.S466X E10/E10 4 c.2183_2184delAAinsG/ c.2183_2184delAAinsG E13/E13 4 c.2183_2184delAAinsG/c.186- ?_296+?del E13/E2 1 p.N1303K/p.N1303K E21/E21 2 p.N1303K/p.S945L E21/E15 1 p.N1303K/c.1677delTA E21/E10 1 p.G542X/p.G542X E11/E11 2 p.G542X/c.2789+5GNA E11/I14b 1 c.3120+1GNA/c.3120+1GNA I16/I16 2 c.3120+1GNA/c.3121-1GNA I16 1 c.3121-1GNA/p.T1086I I16/E17b 1 c.3130delA/c.3130delA E17a/E17a 2 p.D192G/p.D192G E5/E5 1 p.R334W/p.R334W E7/E7 1 p.R334W/p.S945L E7/E15 1 p.R334W/p.L1077P E7/E17b 1 c.1525-1GNA/c.1525-1GNA I9/I9 1 p.S549R/p.S549R E11/E11 1 p.A566D/p.A566D E12/E12 1 c.1898+1GNT/c.1898+1GNT I12/I12 1 c.2576delA/p.S1455X/ E13/E24 1 c.2184insA/c.1677delTA E10/E13 1 p.R785X/p.R785X E13/E13 1 c.2752-1_2756delGGTGGCinsTTG/ c.2752-1_2756delGGTGGCinsTTG I14a/E14b 1 c.2789+5GNA/c.2789+5GNA I14b/I14b 1 p.K1177X/p.K1177X E19/E19 1 c.406-?_1716+?del/c.406-?_1716+?del E4-E10/E4-E10 1 Total 53 origin were extensively studied for the presence of mutations in the CFTR gene, for the presence of the deep intronic 3849+10 kbC࢐T mutation, and large deletions/ duplications. Login to comment
95 It was found once in a patient that carried S1455X in compound heterozygosity. Login to comment

[hide] Purinergic signaling underlies CFTR control of hum... J Cyst Fibros. 2004 Jun;3(2):99-117. Braunstein GM, Zsembery A, Tucker TA, Schwiebert EM
Purinergic signaling underlies CFTR control of human airway epithelial cell volume.
J Cyst Fibros. 2004 Jun;3(2):99-117., [PMID:15463893]

Abstract [show]
BACKGROUND: Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function in cystic fibrosis (CF) causes dysregulation of multiple ion channels, water channels, and acid-base transporters in epithelia. As such, we hypothesized that dysregulation of many critical ion channels and transporters may cause defects in human airway epithelial cell volume regulation. METHODS: Cell volume, regulatory volume decrease, and its regulation was assessed in real-time via Coulter Counter Multisizer III-driven electronic cell sizing in non-CF, CF, and CFTR-complemented CF human airway epithelial cells. SPQ halide fluorescence assay of hypotonicity-induced chloride efflux provided indirect validation of the cell volume assays. RESULTS: CFTR, via autocrine ATP signaling, governs human airway epithelial cell volume regulation. Non-CF cells and wild-type (WT)-CFTR-transfected CF cells had normal regulatory volume decrease (RVD) responses that were attenuated by blockade of autocrine and paracrine purinergic signaling. In contrast, parental IB3-1 CF cells or IB3-1 cells expressing CFTR mutants (DeltaF508, G551D, and S1455X) failed to RVD. CF cell RVD was rescued by agonists to P2Y G protein-coupled receptors and, more robustly, by agonists to P2X purinergic receptor channels. CONCLUSIONS: Loss of CFTR and CFTR-driven autocrine ATP signaling may underlie defective cell volume regulation and dysregulated ion, water, and acid-base transport in CF airway epithelia.

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6 In contrast, parental IB3-1 CF cells or IB3-1 cells expressing CFTR mutants (DF508, G551D, and S1455X) failed to RVD. Login to comment
119 IB3-1 cells express the DF508-CFTR and W1282X-CFTR mutations endogenously [35,49]. Login to comment
121 No CFTR protein was detectable in ''mock controls``, while band B ''core glycosylated`` (130-140 kDa) and band C ''maturely glycosylated`` (160-180 kDa) forms of CFTR were found for WT-CFTR, T1478X (also DTRL)-CFTR, S1455X-CFTR, and G551D-CFTR [9,51,52]. Login to comment
122 S1455X-CFTR showed a similar profile to T1478X-CFTR (data not shown). Login to comment
163 In similar experiments, DF508-CFTR and G551D-CFTR required at least 40 min to return to this threshold, and S1455X-CFTR required 30 min (data not shown). Login to comment
185 Cells expressing the more severe mutations, DF508-CFTR and G551D-CFTR, or the more severe C-terminal truncation mutant, S1455X-CFTR, were unable to fully recover their cell volume after hypotonic cell swelling. Login to comment
205 However, the cells transiently transfected with DF508-CFTR, G551D-CFTR, and S1455X-CFTR did not show a response (Fig. 5B). Login to comment
118 No CFTR protein was detectable in ''mock controls``, while band B ''core glycosylated`` (130-140 kDa) and band C ''maturely glycosylated`` (160-180 kDa) forms of CFTR were found for WT-CFTR, T1478X (also DTRL)-CFTR, S1455X-CFTR, and G551D-CFTR [9,51,52]. Login to comment
158 In similar experiments, DF508-CFTR and G551D-CFTR required at least 40 min to return to this threshold, and S1455X-CFTR required 30 min (data not shown). Login to comment
180 Cells expressing the more severe mutations, DF508-CFTR and G551D-CFTR, or the more severe C-terminal truncation mutant, S1455X-CFTR, were unable to fully recover their cell volume after hypotonic cell swelling. Login to comment
200 However, the cells transiently transfected with DF508-CFTR, G551D-CFTR, and S1455X-CFTR did not show a response (Fig. 5B). Login to comment

[hide] Cystic fibrosis: a multiple exocrinopathy caused b... Am J Med. 1998 Jun;104(6):576-90. Schwiebert EM, Benos DJ, Fuller CM
Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein.
Am J Med. 1998 Jun;104(6):576-90., [PMID:9674722]

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299 Examples of such nonsense mutations are G542X in NBF1, W1282X in NBF2, and S1455X in the C-terminus of the CFTR protein. Login to comment

[hide] A mutation in the cystic fibrosis transmembrane co... Hum Mol Genet. 1998 Apr;7(4):729-35. Mickle JE, Macek M Jr, Fulmer-Smentek SB, Egan MM, Schwiebert E, Guggino W, Moss R, Cutting GR
A mutation in the cystic fibrosis transmembrane conductance regulator gene associated with elevated sweat chloride concentrations in the absence of cystic fibrosis.
Hum Mol Genet. 1998 Apr;7(4):729-35., [PMID:9499426]

Abstract [show]
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 in vivo , 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.

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1 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. Login to comment
4 CFTR mRNA transcripts bearing the S1455X mutation were stable in vivo, implying that this allele encoded a truncated version of CFTR missing the last 26 amino acids. Login to comment
5 Loss of this region did not affect processing of transiently expressed S1455X-CFTR compared with wild-type CFTR. Login to comment
7 Preservation of chloride channel function of S1455X-CFTR was consistent with normal lung and pancreatic function in the mother and her daughter. Login to comment
28 Shading indicates CFTR alleles: del14a alleles are solid, S1455X alleles are hatched and the wild-type allele is unshaded. Login to comment
54 Identification of nonsense mutation S1455X in mother and daughter with elevated sweat chloride concentrations Screening for 16 CF mutations common among Caucasians by reverse dot-blot assay did not detect a mutation in any family member. Login to comment
71 This novel mutation is predicted to change the codon for serine at residue 1455 to an opal termination codon, and is designated S1455X.Both theproband`s mother and sister are compound heterozygotes for deletion 14a and S1455X (Fig. 1). Login to comment
73 Since the del14a mutation caused the CF phenotype in the homozygous state, we predicted that the S1455X mutation was the cause of the isolated elevated sweat chloride concentrations in the sister and mother. Login to comment
74 CFTR mRNA transcript bearing the S1455X mutation is stable RT-PCR was used to determine the consequences of the del14a and S1455X mutations upon transcript splicing and stability. Login to comment
78 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. Login to comment
81 Immunoprecipitation of wild-type and S1455X CFTR transiently expressed in HEK 293 cells. Login to comment
82 Wild-type CFTR and S1455X CFTR proteins immunoprecipitated with the R-domain-specific monoclonal antibody are indicated by the arrow.Both migrateat ~175kDa.TheS1455XCFTRtruncated protein was not detected by immunoprecipitation with the C-terminus-specific antibody. Login to comment
85 the larger amplicon carrying the S1455X mutation was at similar levels to the smaller amplicon missing exon 14a (Fig. 3). Login to comment
86 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. Login to comment
87 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. Login to comment
89 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). Login to comment
90 Since the S1455X CFTR mRNA was stable, we predicted that CFTR missing the last 26 amino acids was produced in vivo. Login to comment
91 The S1455X CFTR truncated protein is processed to a mature state and functions similarly to wild-type CFTR Processing of CFTR bearing 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). Login to comment
93 Both S1455X CFTR and wild-type CFTR products immunoprecipitated with the R-domain antibody have an apparent Mr of 175 kDa (Fig. 4). Login to comment
94 As expected, the S1455X CFTR mutant truncated at the C-terminus was not immunoprecipitated by the C-terminus antibody (Fig. 4). Login to comment
96 Since the S1455X mutant appears to undergo post-translational modifications similar to wild-type CFTR, we expected that the mutant protein would be properly folded and inserted into the cell membrane. Login to comment
97 To assay function, wild-type and S1455X CFTRcDNAs were transiently transfected into CF airway epithelial cells (IB3-1) devoid of functional CFTR. Login to comment
99 Whole-cell chloride current activated by cAMP in IB3-1 cells transfected with wild-type CFTR or S1455X CFTR.Current-voltage plots for wild-type CFTR (left) and S1455X CFTR (right) in the presence of CPT-cAMP (200 µM) are shown as filled symbols. Login to comment
103 Incubation with a non-hydrolyzable form of cAMP (CPT-cAMP; 200 µM) evoked chloride currents of similar magnitude in wild-type CFTR (+100 mV: 1090.9 ± 84.2 SEM pA; n = 12) and S1455X CFTR (+100 mV: 938.9± 114.4 pA; n = 20) transfected cells that were significantly higher (P < 0.05) than non-transfected cells (+100 mV: 89.3 ± 34.0 pA; n = 5). Login to comment
106 Whole-cell patch-clamp analysis of IB3-1 cells transfected with S1455X CFTR displayed cAMP-activated currents that were outwardly rectified and inhibited by glybenclamide (+100 mV: 302.7 ± 127.5 pA; n = 7). Login to comment
107 Thus, S1455X CFTR generates robust cAMP-activated chloride currents similarly to wild-type CFTR. Login to comment
119 The good health of the mother at 46 years of age suggested a very low likelihood that the S1455X/ del14a genotype causes CF. Login to comment
126 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. Login to comment
131 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. Login to comment
132 This probably explains why the mother and child carrying the S1455X mutation escaped the pulmonary and pancreatic manifestations of CF. Login to comment
133 However, the remote possibility exists that another undetected mutation on the S1455X allele may contribute to the phenotype. Login to comment
134 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. Login to comment
135 The S1455X mutation is predicted to eliminate a region containing two stretches of amino acids that are highly conserved among species (36-38). Login to comment
137 The phenotype associated with the S1455X mutation indicates that the C-terminal sequences of CFTR have a functional role in the sweat gland. Login to comment
141 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. Login to comment
164 Dot-blots were hybridized at 42_C for 1 h with γ-32P-labeled S1455X mutant probe (5'-CACTTGCTTCAGTT- CCGG-3') or S1455 wild-type probe (5'-ACCGGAACTCAAG- CAAGTG-3'), washed at room temperature and exposed to X-ray film for 10 min at -80_C. Login to comment
166 Expression analysis 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). Login to comment
180 Whole-cell patch-clamp recording 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.). Login to comment
183 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. Login to comment
2 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. Login to comment
6 Loss of this region did not affect processing of transiently expressed S1455X-CFTR compared with wild-type CFTR. Login to comment
8 Preservation of chloride channel function of S1455X-CFTR was consistent with normal lung and pancreatic function in the mother and her daughter. Login to comment
29 Shading indicates CFTR alleles: del14a alleles are solid, S1455X alleles are hatched and the wild-type allele is unshaded. Login to comment
55 Identification of nonsense mutation S1455X in mother and daughter with elevated sweat chloride concentrations Screening for 16 CF mutations common among Caucasians by reverse dot-blot assay did not detect a mutation in any family member. Login to comment
72 This novel mutation is predicted to change the codon for serine at residue 1455 to an opal termination codon, and is designated S1455X.Both theproband`s mother and sister are compound heterozygotes for deletion 14a and S1455X (Fig. 1). Login to comment
75 CFTR mRNA transcript bearing the S1455X mutation is stable RT-PCR was used to determine the consequences of the del14a and S1455X mutations upon transcript splicing and stability. Login to comment
79 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. Login to comment
83 Wild-type CFTR and S1455X CFTR proteins immunoprecipitated with the R-domain-specific monoclonal antibody are indicated by the arrow.Both migrateat ~175kDa.TheS1455XCFTRtruncated protein was not detected by immunoprecipitation with the C-terminus-specific antibody. Login to comment
88 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. Login to comment
92 The S1455X CFTR truncated protein is processed to a mature state and functions similarly to wild-type CFTR Processing of CFTR bearing 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). Login to comment
95 As expected, the S1455X CFTR mutant truncated at the C-terminus was not immunoprecipitated by the C-terminus antibody (Fig. 4). Login to comment
98 To assay function, wild-type and S1455X CFTRcDNAs were transiently transfected into CF airway epithelial cells (IB3-1) devoid of functional CFTR. Login to comment
100 Whole-cell chloride current activated by cAMP in IB3-1 cells transfected with wild-type CFTR or S1455X CFTR.Current-voltage plots for wild-type CFTR (left) and S1455X CFTR (right) in the presence of CPT-cAMP (200 &#b5;M) are shown as filled symbols. Login to comment
104 Incubation with a non-hydrolyzable form of cAMP (CPT-cAMP; 200 &#b5;M) evoked chloride currents of similar magnitude in wild-type CFTR (+100 mV: 1090.9 &#b1; 84.2 SEM pA; n = 12) and S1455X CFTR (+100 mV: 938.9&#b1; 114.4 pA; n = 20) transfected cells that were significantly higher (P < 0.05) than non-transfected cells (+100 mV: 89.3 &#b1; 34.0 pA; n = 5). Login to comment
108 Thus, S1455X CFTR generates robust cAMP-activated chloride currents similarly to wild-type CFTR. Login to comment
120 The good health of the mother at 46 years of age suggested a very low likelihood that the S1455X/ del14a genotype causes CF. Login to comment
127 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. Login to comment
136 The S1455X mutation is predicted to eliminate a region containing two stretches of amino acids that are highly conserved among species (36-38). Login to comment
138 The phenotype associated with the S1455X mutation indicates that the C-terminal sequences of CFTR have a functional role in the sweat gland. Login to comment
142 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. Login to comment
165 Dot-blots were hybridized at 42C for 1 h with b3;-32P-labeled S1455X mutant probe (5'-CACTTGCTTCAGTT- CCGG-3') or S1455 wild-type probe (5'-ACCGGAACTCAAG- CAAGTG-3'), washed at room temperature and exposed to X-ray film for 10 min at -80C. Login to comment
167 Expression analysis 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). Login to comment
181 Whole-cell patch-clamp recording 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.). Login to comment
184 IB3-1 cells were transiently transfected at 30% confluency in a 35 mm dish for 6-8 h with either 3 &#b5;g of wild-type CFTR (pRSV-CFTR) or 3 &#b5;g of CFTR bearing the mutation S1455X (pRSV-CFTR/S1455X) using 15 &#b5;l of lipofectin and 1 ml of Opti-MEM. Login to comment

[hide] Regulated trafficking of the CFTR chloride channel... Eur J Cell Biol. 2000 Aug;79(8):544-56. Kleizen B, Braakman I, de Jonge HR
Regulated trafficking of the CFTR chloride channel.
Eur J Cell Biol. 2000 Aug;79(8):544-56., [PMID:11001491]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR), the ABC transporter encoded by the cystic fibrosis gene, is localized in the apical membrane of epithelial cells where it functions as a cyclic AMP-regulated chloride channel and as a regulator of other ion channels and transporters. Whereas a key role of cAMP-dependent phosphorylation in CFTR-channel gating has been firmly established, more recent studies have provided clear evidence for the existence of a second level of cAMP regulation, i.e. the exocytotic recruitment of CFFR to the plasma membrane and its endocytotic retrieval. Regulated trafficking of the CFTR Cl- channel has sofar been demonstrated only in a subset of CFTR-expressing cell types. However, with the introduction of more sensitive methods to measure CFTR cycling and submembrane localization, it might turn out to be a more general phenomenon that could contribute importantly to both the regulation of CFTR-mediated chloride transport itself and to the regulation of other transporters and CFTR-modulated cellular functions. This review aims to summarize the present state of knowledge regarding polarized and regulated CFTR trafficking and endosomal recycling in epithelial cells, to discuss present gaps in our understanding of these processes at the cellular and molecular level, and to consider its possible implications for cystic fibrosis.

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47 However, expressing the C-terminally truncated green fluorescent protein (GFP)- CFTR-S1455X construct shifted the CFTR localization to the basolateral membrane, suggesting that the deleted 26 amino acids somehow mask interaction of basolateral targeting signals in the CFTR deletion mutant and thereby prevent basolateral localization (Moyer et al., 1999). Login to comment
49 In light of these studies it would be of interest to assess whether CFTR-S1455X (but not WT-CFTR) has acquired the capacity to interact with the AP-1 complex in cells expressing the novel m1B adaptor subunit (Folsch et al., 1999). Login to comment

[hide] Distribution of CFTR mutations in the Czech popula... J Cyst Fibros. 2013 Sep;12(5):532-7. doi: 10.1016/j.jcf.2012.12.002. Epub 2012 Dec 29. Krenkova P, Piskackova T, Holubova A, Balascakova M, Krulisova V, Camajova J, Turnovec M, Libik M, Norambuena P, Stambergova A, Dvorakova L, Skalicka V, Bartosova J, Kucerova T, Fila L, Zemkova D, Vavrova V, Koudova M, Macek M, Krebsova A, Macek M Jr
Distribution of CFTR mutations in the Czech population: positive impact of integrated clinical and laboratory expertise, detection of novel/de novo alleles and relevance for related/derived populations.
J Cyst Fibros. 2013 Sep;12(5):532-7. doi: 10.1016/j.jcf.2012.12.002. Epub 2012 Dec 29., [PMID:23276700]

Abstract [show]
BACKGROUND: This two decade long study presents a comprehensive overview of the CFTR mutation distribution in a representative cohort of 600 Czech CF patients derived from all regions of the Czech Republic. METHODS: We examined the most common CF-causing mutations using the Elucigene CF-EU2v1 assay, followed by MLPA, mutation scanning and/or sequencing of the entire CFTR coding region and splice site junctions. RESULTS: We identified 99.5% of all mutations (1194/1200 CFTR alleles) in the Czech CF population. Altogether 91 different CFTR mutations, of which 20 were novel, were detected. One case of de novo mutation and a novel polymorphism was revealed. CONCLUSION: The commercial assay achieved 90.7%, the MLPA added 1.0% and sequencing increased the detection rate by 7.8%. These comprehensive data provide a basis for the improvement of CF DNA diagnostics and/or newborn screening in our country. In addition, they are relevant to related Central European populations with lower mutation detection rates, as well as to the sizeable North American "Bohemian diaspora".

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56 The S1455X mutation was observed in compound heterozygosity with the F508del in a male patient who was clinically diagnosed at age 7 years, in which "repeated bronchitis" led to sweat testing (mean concentration 70 mmol/l). Login to comment
57 Interestingly, the patient's asymptomatic father bears mutation S1455X in trans to a novel variant S1456N and the patient's apparently healthy brother has the maternal-F508del/S1456N genotype. Login to comment
76 We have not included the male with the S1455X/F508del in our cohort, since he only suffers from "isolated elevated sweat chloride concentrations" [20] and does not meet the diagnostic criteria for CF [10]. Login to comment

[hide] Analysis of cystic fibrosis gene mutations in chil... J Med Case Rep. 2014 Oct 10;8:339. doi: 10.1186/1752-1947-8-339. Dell'Edera D, Benedetto M, Gadaleta G, Carone D, Salvatore D, Angione A, Gallo M, Milo M, Pisaturo ML, Di Pierro G, Mazzone E, Epifania AA
Analysis of cystic fibrosis gene mutations in children with cystic fibrosis and in 964 infertile couples within the region of Basilicata, Italy: a research study.
J Med Case Rep. 2014 Oct 10;8:339. doi: 10.1186/1752-1947-8-339., [PMID:25304080]

Abstract [show]
INTRODUCTION: Cystic fibrosis is the most common autosomal recessive genetic disease in the Caucasian population. Extending knowledge about the molecular pathology on the one hand allows better delineation of the mutations in the CFTR gene and the other to dramatically increase the predictive power of molecular testing. METHODS: This study reports the results of a molecular screening of cystic fibrosis using DNA samples of patients enrolled from January 2009 to December 2013. Patients were referred to our laboratory for cystic fibrosis screening for infertile couples. In addition, we identified the gene mutations present in 76 patients affected by cystic fibrosis in the pediatric population of Basilicata. RESULTS: In the 964 infertile couples examined, 132 subjects (69 women and 63 men) resulted heterozygous for one of the CFTR mutations, with a recurrence of carriers of 6.85%. The recurrence of carriers in infertile couples is significantly higher from the hypothetical value of the general population (4%). CONCLUSIONS: This study shows that in the Basilicata region of Italy the CFTR phenotype is caused by a small number of mutations. Our aim is to develop a kit able to detect not less than 96% of CTFR gene mutations so that the relative risk for screened couples is superimposable with respect to the general population.

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59 As mentioned before, molecular screening Table 2 Comparison between the results obtained in this study and those obtained in a previous study Castaldo et al. [14] Mutations observed in the present study F508del 55.8% (29) 48.62% (141) N1303K 3.8% (2) 9.31% (27) G542X 3.8% (2) 8.96% (26) W1282X 3.8% (2) 1.03% (3) 2183AA>G 5.8% (3) 2.76% (8) R1162X 0 0 1717-1G>A 1.9% (1) 0 T338I 0 0 R347P 0 0.69% (2) 711+5G>A 0 0 852del22 5.8% (3) 1.03% (3) 4382delA 0 0.69% (2) 1259insA 0 0.34% (1) 4016insT 0 0.34% (1) R553X 0 0.34% (1) R1158X 0 0 L1077P 0 1.03% (3) I502T 0 0 3849+10kbC>T 1.9% (1) 0.34% (1) D579G 0 0.69% (2) G1244E 3.8% (2) 0 G1349D 0 0.34% (1) 2789+5G>A 0 1.03% (3) 711+1G>T 0 0 L1065P 0 0 2522insC 0 0 E585X 0 0 G85E 0 0 G178R 0 0 D1152H 0 3.10% (9) I148T-3195del6 0 0 I148T (alone) 0 4.48% (13) R334W 0 0 DI507 0 0.69% (2) I1005R 0 0 3272-26A>G 0 0 2711delT 0 0 L558S 1.9% (1) 0.34% (1) W1063X 0 0 D110H 0 0 S549R (A>C) 1.9% (1) 0.69% (2) 2184insA 0 0 3131del22 0 0 Table 2 Comparison between the results obtained in this study and those obtained in a previous study (Continued) R709N 0 0 A349V 0 0 4015insA 0 0 Y849X 1.9% (1) 0.34% (1) G551D 0 1.03% (3) 621+3A>G 0 0.34% (1) E831X 0 0 I507del 0 0.69% (2) IVS8 TG12/t5 0 1.03% (3) H139R (A->G) 0 0.34% (1) 1248+1G>A 0 0.34% (1) R74W;V201M;D1270N 0 0.69% (2) S1455X 0 0.34% (1) dele 2,3 (21kb) 0 0.34% (1) 991del5 0 0.34% (1) UNKNOWN 7 %(4) 4.83% (14) F508C 0 0.69% (2) TOTAL 52 290 of CF is highly recommended in the USA by the National Institutes of Health Consensus Development Conference Statement on genetic testing for cystic fibrosis [17]. Login to comment

[hide] A Genotypic-Oriented View of CFTR Genetics Highlig... Mol Med. 2015 Apr 21;21:257-75. doi: 10.2119/molmed.2014.00229. Lucarelli M, Bruno SM, Pierandrei S, Ferraguti G, Stamato A, Narzi F, Amato A, Cimino G, Bertasi S, Quattrucci S, Strom R
A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis.
Mol Med. 2015 Apr 21;21:257-75. doi: 10.2119/molmed.2014.00229., [PMID:25910067]

Abstract [show]
Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype-phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype-phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype-phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway.

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390 L1077P c.3230T>C CF-PI CF-causing p.Leu1077Pro Y1092X(C>A) c.3276C>A CF-PI CF-causing p.Tyr1092* M1137V c.3409A>G CFTR-RD nd p.Met1137Val D1152H c.3454G>C CF-PI,CF-PS,CFTR-RD varying clinical consequence p.Asp1152His R1162X c.3484C>T CF-PI CF-causing p.Arg1162* D1168G c.3503A>G CFTR-RD nd p.Asp1168Gly 3667ins4 c.3535_3536insTCAA CF-PI CF-causing p.Thr1179IlefsX17 S1206X c.3617C>A uncertain: CF-PI and/or CF-PS nd p.Ser1206* I1234V c.3700A>G CF-PI,CF-PS CF-causing p.Ile1234Val S1235R c.3705T>G CFTR-RD non CF-causing p.Ser1235Arg 3849+10kbC>T c.3717+12191C>T CF-PI,CF-PS CF-causing V1240G c.3719T>G CFTR-RD nd p.Val1240Gly G1244R c.3730G>A uncertain: CF-PI and/or CF-PS nd p.Gly1244Arg G1244E c.3731G>A CF-PI,CF-PS CF-causing p.Gly1244Glu G1247R(G>C) c.3739G>C CF-PS nd p.Gly1247Arg W1282X c.3846G>A CF-PI CF-causing p.Trp1282* Q1291R c.3872A>G CF-PI,CF-PS,CFTR-RD nd p.Gln1291Arg 4016insT c.3884_3885insT CF-PI CF-causing p.Ser1297PhefsX5 4040delA c.3908delA CF-PI nd p.Asn1303ThrfsX25 N1303K c.3909C>G CF-PI CF-causing p.Asn1303Lys ex22-24del c.3964-3890_4443+3143del9454ins5 CF-PI nd ex22,23del c.3964-78_4242+577del1532 CF-PI CF-causing 4168delCTAAGCC c.4036_4042del CF-PI nd p.Leu1346MetfsX6 G1349D c.4046G>A CF-PI CF-causing p.Gly1349Asp H1375P c.4124A>C uncertain: CF-PI and/or CF-PS nd p.His1375Pro S1455X c.4364C>G CF-PS,CFTR-RD nd p.Ser1455* Q1476X c.4426C>T CFTR-RD nd p.Gln1476* nd,Not determined.According to the three rules described (see Materials and Methods),each mutated allele was classified according to its clinical outcome.It was impossible to univocally assign 16 of the 125 different mutated alleles to one or more macrocategories.A comparison with the CFTR2 project (11) (http://www.cftr2.org) is shown.The alleles are ordered according to their nucleotidic position. Login to comment

[hide] Inconclusive diagnosis of cystic fibrosis after ne... Pediatrics. 2015 Jun;135(6):e1377-85. doi: 10.1542/peds.2014-2081. Epub 2015 May 11. Ooi CY, Castellani C, Keenan K, Avolio J, Volpi S, Boland M, Kovesi T, Bjornson C, Chilvers MA, Morgan L, van Wylick R, Kent S, Price A, Solomon M, Tam K, Taylor L, Malitt KA, Ratjen F, Durie PR, Gonska T
Inconclusive diagnosis of cystic fibrosis after newborn screening.
Pediatrics. 2015 Jun;135(6):e1377-85. doi: 10.1542/peds.2014-2081. Epub 2015 May 11., [PMID:25963003]

Abstract [show]
OBJECTIVES: To prospectively study infants with an inconclusive diagnosis of cystic fibrosis (CF) identified by newborn screening (NBS; "CF screen positive, inconclusive diagnosis" [CFSPID]) for disease manifestations. METHODS: Infants with CFSPID and CF based on NBS from 8 CF centers were prospectively evaluated and monitored. Genotype, phenotype, repeat sweat test, serum trypsinogen, and microbiology data were compared between subjects with CF and CFSPID and between subjects with CFSPID who did (CFSPID-->CF) and did not (CFSPID-->CFSPID) fulfill the criteria for CF during the first 3 years of life. RESULTS: Eighty-two subjects with CFSPID and 80 subjects with CF were enrolled. The ratio of CFSPID to CF ranged from 1:1.4 to 1:2.9 in different centers. CFTR mutation rates did not differ between groups; 96% of subjects with CFSPID and 93% of subjects with CF had 2 mutations. Subjects with CFSPID had significantly lower NBS immunoreactive trypsinogen (median [interquartile range]:77 [61-106] vs 144 [105-199] mug/L; P < .0001) than did subjects with CF. Pseudomonas aeruginosa and Stenotrophomonas maltophilia were isolated in 12% and 5%, respectively, of subjects with CFSPID. CF was diagnosed in 9 of 82 (11%) subjects with CFSPID (genotype and abnormal sweat chloride = 3; genotype alone = 4; abnormal sweat chloride only = 2). Sweat chloride was abnormal in CFSPID-->CF patients at a mean (SD) age of 21.3 (13.8) months. CFSPID-->CF patients had significantly higher serial sweat chloride (P < .0001) and serum trypsinogen (P = .009) levels than did CFSPID-->CFSPID patients. CONCLUSIONS: A proportion of infants with CFSPID will be diagnosed with CF within the first 3 years. These findings underscore the need for clinical monitoring, repeat sweat testing at age 2 to 3 years, and extensive genotyping.

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103 In combination with a disease-causing mutation, R117H-7T has been associated with diagnostic uncertainties in CF, TABLE 2 Genotypes of Subjects With CFSPID According to Initial Sweat Chloride Measurements Sweat Chloride ,30 mmol/L Sweat Chloride 30-59 mmol/L Allele 1 Allele 2 n Allele 1 Allele 2 n F508dela R117H (7T)b 9 F508dela R117Cd 2c F508dela 5Tb 2 F508dela L206Wd 2c F508dela D1152Hb 2 F508dela P67Ld 1c F508dela R117Hb 1 F508dela 5Tb 8 F508dela D1270Nb 1 F508dela R117H (7T)b 3 F508dela L997F 3 F508dela R117Hb 3 F508dela 1716G.A 1 F508dela S1455X 1c F508dela 621+3G.A 1 F508dela R170H 1 F508dela I1328T 1 F508dela I148T 1 F508dela L967S 1 F508dela L997F 1 F508dela M1137T 1 F508dela Q1476X 1 F508dela Y301C 1 F508dela S1235R 1 1717-1G.Aa D1152Hb 1 F508dela T1299I 1 2183AA.Ga 5Tb 1 2183AA.Ga R117Cd 1 2183AA.Ga S431G 1 2789+5G.Aa R117H (7T)b 1 3849+10kbC.Ta 3041-15T.G 1 3849+10kbC.Ta 3041-15T.G 1 621+1G.Ta R117H (7T)b 1 621+1G.Ta G1069Rb 1 711+1G.Ta D1152Hb 1 G542Xa L206Wd 1c G542Xa R117H (7T)b 1 G542Xa C1410T 1 G542Xa D1152Hb 1 G551Da 5Tb 1 G551Da D1152Hb 1 N1303Ka 5Tb 1 N1303Ka D1152Hb 1 R1162Xa R117H (7T)b 1c N1303Ka E527G 1 R553Xa 5Tb 1 R117H (5T)a 5Tb 1 R553Xa L997F 1 R117H (7T)b R117H (7T)b 1 R560Ta G576A 1 R117H (7T)b 3041_71G.C 1 W1282Xa 5Tb 2 R117Hb Q1476X 1 F508dela - 2 R117H (5T)a - 1 -, no mutation identified on the second allele. Login to comment
108 TABLE 3 Characteristics of Subjects With CFSPID Who Later Met Diagnostic Criteria of CF Subject Number Allele 1 Allele 2 Ethnicity NBS IRT, mg/L Initial Sweat Chloride, mmol/L Highest Sweat Chloride, mmol/L Country 1 F508del R117C White 105.8 36 61 Canada 2 F508del S1455X White 66.6 46 74 Canada 3 F508del P67L White 151.2 38 38 Canada 4 F508del L206W White 83.8 58 64 Canada 5 G542X L206W White 67 49 66 Canada 6 F508del L206W White 59.9 45 45 Canada 7 R1162X R117H-7T White 126 36 70 Italy 8 2183AA.G R117C White 129 32 32 Italy 9 F508del R117C White 80.4 48 56 Canada e OOI et al including in newborn-screened infants with equivocal CF diagnosis and in older individuals with single-organ manifestations of CF.17,18,20-22 As in the case of the 7 subjects who were initially classified as CFSPID but who were subsequently recognized to carry 2 disease-causing mutations on the basis of the CFTR2 project, the diagnostic consequences (benign versus disease-causing) of the CFTR mutations identified in all of the other subjects with CFSPID may not be apparent until later on, when new genetic information becomes available and classification of CFTR mutations currently considered to be of "unknown" consequences is updated. Login to comment