ABCMdb

PMID: 17541014

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., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Trp1282* Restoration of W1282X CFTR Activity by Enhanced Expression Steven M. Rowe, Karoly Varga, Andras Rab, Zsuzsa Bebok, Kevin Byram, Yao Li, Eric J. Sorscher, and John P. Clancy Departments of Medicine, Pediatrics, and Physiology and Biophysics, the Gregory Fleming James Cystic Fibrosis Research Center; and Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Login to comment 3 ABCC7 p.Trp1282* ABCC7 p.Arg1162* 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. Login to comment 4 ABCC7 p.Trp1282* ABCC7 p.Arg1162* 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. Login to comment 6 ABCC7 p.Trp1282* 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. Login to comment 16 ABCC7 p.Trp1282* Importantly, all subjects with nonsense mutations in both of these studies carried at least one copy of W1282X CFTR, a PTC that is particularly common in this population (found in up to 40% of Israeli patients with CF) (5, 16, 17). Login to comment 21 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg1162* Based on the promising clinical findings in patients possessing W1282X CFTR, coupled with variable in vivo responses to premature stop suppression, we designed experiments to examine the effects of stop codon site on two common PTCs found in CFTR (R1162X and W1282X). Login to comment 22 ABCC7 p.Trp1282* ABCC7 p.Trp1282* Our results indicate CLINICAL RELEVANCE Evidence that W1282X cystic fibrosis transmembrane conductance regulator (CFTR) retains chloride channel function improves our understanding of CFTR biology, and may explain why subjects harboring W1282X CFTR appear more susceptible to the strategy of premature termination codon suppression. Login to comment 24 ABCC7 p.Trp1282* ABCC7 p.Arg1162* E-mail: smrowe@ uab.edu Am J Respir Cell Mol Biol Vol 37. pp 347-356, 2007 Originally Published in Press as DOI: 10.1165/rcmb.2006-0176OC on May 31, 2007 Internet address: www.atsjournals.org that enhanced expression is sufficient to restore measurable activity to W1282X CFTR but not R1162X CFTR. Login to comment 26 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg1162* ABCC7 p.Arg1162* MATERIALS AND METHODS Development of Stable Cell Lines Expressing W1282X and R1162X CFTR W1282X and R1162X CFTR cDNA were stably introduced into HeLa and CFBE41o-cells using TranzVector (Tranzyme, Inc., Birmingham, AL). Login to comment 50 ABCC7 p.Trp1282* ABCC7 p.Arg1162* SPQ Studies of Halide Efflux in HeLa Cells HeLa cells stably expressing W1282X, R1162X, or wild type CFTR were studied with the halide quenched dye 6-methoxy-N-(3-sulfopropyl)- quinolinium (SPQ; Molecular Probes, Inc., Eugene, OR) as previously described (27, 29). Login to comment 63 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Briefly, CFBE41o2 cells expressing W1282X or R1162X CFTR were seeded on Costar 0.4-mm permeable supports (5 3 105 cells/filter, 6.5 mm diameter; Bethesda, MD) after coating with fibronectin as previously described (32). Login to comment 72 ABCC7 p.Trp1282* ABCC7 p.Arg1162* The CFBE41o2 W1282X and CFBE41o2 R1162X cells were handled in exactly the same fashion during our studies. Login to comment 85 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Arg1162* RESULTS Preferential Enhancement of CFTR Activity in W1282X CFTR Expressing Cells by Sodium Butyrate Previous studies have examined the function of several CFTR molecules containing clinically relevant premature stop codons in transient, high-level expression systems using nonpolarizing cell types (including G542X, R553X, R1162X, and W1282X CFTR), with variable levels of constitutive and regulated CFTR activity described (7, 8, 33). Login to comment 88 ABCC7 p.Trp1282* ABCC7 p.Arg1162* We limited our comparisons to R1162X and W1282X CFTR in readily comparable model systems widely used in CF research. Login to comment 89 ABCC7 p.Trp1282* ABCC7 p.Arg1162* These two mutants occur in the distal portion of CFTR (immediately after TMD-2 for R1162X CFTR, and approximately one third through NBD-2 for W1282X CFTR). Login to comment 91 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Figure 1 summarizes functional studies in HeLa cells expressing R1162X CFTR and W1282X CFTR. Login to comment 94 ABCC7 p.Trp1282* In Figure 1A, increasing concentrations of NaBu exposure led to dramatic increases in halide transport in W1282X CFTR-transduced cells. Login to comment 95 ABCC7 p.Arg1162* By contrast, NaBu had little effect on halide efflux in R1162X CFTR (Figure 1B). Login to comment 99 ABCC7 p.Trp1282* ABCC7 p.Arg1162* W1282X CFTR-expressing cells had significantly increased agonist-stimulated Isc relative to R1162X CFTR-expressing cells at baseline (no NaBu treatment). Login to comment 102 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Halide transport in W1282X and R1162X CFTR transduced HeLa cells. Login to comment 103 ABCC7 p.Trp1282* (A) Fluorescence-based halide efflux (SPQ) activity of W1282X CFTR HeLa cells after treatment with increasing concentrations of NaBu or control (vehicle media) for 24 h. Closed arrowhead represents basal CFTR activity after switch to dequenched buffer, and the open arrowhead signifies fluorescence after the addition of CFTR agonists forskolin (20 mM) and genistein (50 mM). Login to comment 104 ABCC7 p.Trp1282* ABCC7 p.Arg1162* * Halide efflux of W1282X compared with R1162X CFTR transduced cells was significantly increased after treatment with NaBu (P , 0.001, 6 SEM). Login to comment 105 ABCC7 p.Arg1162* (B) Parallel experiments performed in R1162X CFTR HeLa cells showing no activation of halide efflux after CFTR stimulation. Login to comment 107 ABCC7 p.Trp1282* ABCC7 p.Arg1162* with NaBu, further enhancement of agonist-stimulated Isc was demonstrated in W1282X CFTR-transduced cells relative to R1162X CFTR. Login to comment 108 ABCC7 p.Arg1162* ABCC7 p.Arg1162* The negative results seen in the R1162X CFTR cells demonstrate that NaBu has no detectable functional effects on either the R1162X CFTR transgene or endogenous DF508 CFTR expressed in these cells (albeit at extremely low levels [32]). Login to comment 109 ABCC7 p.Trp1282* Together, the studies in both HeLa and CFBE41o2 cell lines demonstrate similar results: NaBu treatment has unique effects on halide transport only in W1282X CFTR-expressing cells. Login to comment 111 ABCC7 p.Trp1282* NaBu treatment results in increased Isc in W1282X expressing cells. Login to comment 114 ABCC7 p.Trp1282* ABCC7 p.Arg1162* (A) Representative tracings in CFBE41o2 cells expressing W1282X CFTR (left) and R1162X CFTR (right) after treatment with NaBu (500 mM) or control conditions for 24 h. Login to comment 115 ABCC7 p.Trp1282* Experiments show increased Isc in W1282X CFTR-transduced CFBE41o2 cells pretreated with NaBu after forskolin (forsk, 20 mM) and genistein (gen, 50 mM) stimulation. Login to comment 118 ABCC7 p.Trp1282* ABCC7 p.Arg1162* (B) Summary data of CFBE41o-cells expressing W1282X (solid bars) or R1162X CFTR (open bars) exposed to various concentrations of NaBu versus control (vehicle media) for 24 h, then studied in modified Ussing chambers under voltage clamp conditions. Login to comment 119 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Short-circuit currents were greater in W1282X compared with R1162X CFTR-transduced cells under control and NaBu-treated conditions. Login to comment 121 ABCC7 p.Trp1282* ABCC7 p.Arg1162* ABCC7 p.Arg1162* *P , 0.05 compared with W1282X control, ‡P , 0.05 compared with R1162X at same condition, ‡‡P , 0.01 compared with R1162X at same condition, (6 SEM). Login to comment 122 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Enhancement of R1162X and W1282X CFTR Expression by Sodium Butyrate NaBu has been shown to deacetylate the CMV promoter and enhance expression of transgenes in other model systems (34-36). Login to comment 123 ABCC7 p.Trp1282* To better understand the mechanism of the activating effects of NaBu in HeLa and CFBE41o2 cells expressing W1282X CFTR, we measured mutant CFTR expression at the mRNA and protein level. Login to comment 124 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Figure 3 demonstrates that under basal conditions, W1282X and R1162X CFTR-transduced cells exhibit slightly greater CFTR mRNA levels relative to endogenous CFTR in Calu-3 cells (Figure 3A); however, protein expression across the three cell lines are qualitatively similar, probably due to differences in processing efficiency between the native and transduced genes (32). Login to comment 125 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Figure 4 summarizes real-time PCR studies comparing R1162X and W1282X CFTR expression in HeLa and CFBE41o2 cells. Login to comment 126 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Figure 4A shows dose-response studies with NaBu in transduced HeLa cells, and Figure 4B shows similar studies in CFBE41o2 cells expressing R1162X or W1282X CFTR. Login to comment 127 ABCC7 p.Trp1282* ABCC7 p.Arg1162* NaBu led to dose-dependent increases in both R1162X and W1282X CFTR mRNA levels in HeLa cells, with similar enhancing effects seen in CFBE41o2 cells. Login to comment 132 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Relative expression of R1162X and W1282X CFTR in transduced HeLa cells is similar to the Calu-3 type. Login to comment 133 ABCC7 p.Trp1282* ABCC7 p.Arg1162* (A) R1162X and W1282X CFTR transduced HeLa cells grown to confluence and then assayed by RT-PCR after RNA isolation showed mRNA expression within 3-fold compared with endogenously expressing Calu-3 cells. Login to comment 138 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Relative transcript levels of R1162X and W1282X CFTR in transduced HeLa (A) and CFBE41o2 (B) cells. Login to comment 141 ABCC7 p.Trp1282* ABCC7 p.Arg1162* W1282X CFTR mRNA levels under control conditions were 72% of R1162X CFTR expression. Login to comment 146 ABCC7 p.Trp1282* ABCC7 p.Arg1162* R1162X CFTR mRNA levels under control conditions were 83% of W1282X expression levels. Login to comment 149 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg1162* Together, the results from Figures 1-6 provide evidence that NaBu enhanced the expression of truncated CFTR (without significantly increasing the level of full-length protein) in both W1282X and R1162X CFTR-expressing cells; moreover only W1282X CFTR-expressing cells demonstrated increased agonist stimulated ion transport after NaBu treatment. Login to comment 150 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Synergistic Effects of Stop Codon Suppression and Enhanced Expression in R1162X and W1282X CFTR-Expressing Cells We next examined the effects of NaBu combined with G418 (an aminoglycoside and previously described potent suppressor of premature stop mutations) on full-length CFTR production in HeLa and CFBE41o2 cells transduced with either of the two CFTR premature stop codons of interest. Login to comment 152 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Treatment with the combination of NaBu and G418 for 16 hours led to increased full-length core glycosylated (band B) and fully glycosylated (band C) CFTR production in HeLa (Figure 6A) and CFBE41o2 (Figure 6B) cells transduced with R1162X or W1282X CFTR. Login to comment 156 ABCC7 p.Trp1282* ABCC7 p.Arg1162* To confirm the presence of truncated W1282X CFTR at the cell surface compared with R1162X-expressing cells, immunofluorescence studies were performed. Login to comment 158 ABCC7 p.Trp1282* A high proportion of cells positive for surface-localized truncated W1282X CFTR was detectable after treatment with NaBu alone (55% of cells, P , 0.001 versus control, probed with anti-NBD-1 antibody). Login to comment 160 ABCC7 p.Trp1282* ABCC7 p.Arg1162* In contrast, R1162X CFTR-transduced cells demonstrated overall lower surface expression of both full-length and truncated protein (controlled for treatment condition), reflecting less efficient surface localization of truncated protein relative to W1282X CFTR (P , 0.001 by logistic regression analysis). Login to comment 162 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Full-length CFTR expression after NaBu, G418, and combination treatment in W1282X or R1162X CFTR-transduced HeLa (A) and CFBE41o2 (B) cells. Login to comment 163 ABCC7 p.Trp1282* ABCC7 p.Arg1162* (A) Immunoprecipitation with C-terminus-specific antibody (24-1) followed by in vitro phosphorylation of W1282X and R1162X CFTR-transduced HeLa cells is shown. Login to comment 169 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Detection of truncated R1162X and W1282X CFTR in HeLa (A) and CFBE41o2 (B) cells. Login to comment 170 ABCC7 p.Trp1282* ABCC7 p.Arg1162* (A) HeLa Cells were selectively probed for truncated CFTR using an antibody directed against NBD-1 in W1282X and R1162X CFTR-transduced cells. Login to comment 174 ABCC7 p.Trp1282* ABCC7 p.Arg1162* NaBu treatment significantly enhanced W1282X (z 140 kD, left panel) or R1162X (z 110 kD, right panel) CFTR protein expression. Login to comment 177 ABCC7 p.Trp1282* ABCC7 p.Arg1162* The synergistic effects of G418 and NaBu on W1282X and R1162X CFTR activity were also measured by SPQ (Figure 8). Login to comment 178 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg1162* The top portion shows examples of experiments in W1282X CFTR-expressing cells, and the bottom portion summarizes results from multiple paired studies comparing R1162X and W1282X CFTR-expressing cells. Login to comment 180 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg1162* ABCC7 p.Arg1162* G418 treatment had positive effects on halide transport in both R1162X and W1282X CFTR-expressing cells (reflecting readthrough of PTCs), and NaBu treatment had significantly stronger effects on agonist stimulated halide transport in the W1282X CFTR-expressing cells relative to cells transduced with R1162X CFTR. Login to comment 181 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Combination treatment with G418 and NaBu dramatically increased halide efflux in cells expressing W1282X CFTR, with smaller synergistic effects seen in the R1162X CFTR-transduced cells. Login to comment 182 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Together, the results from Figures 6-8 provide evidence for synergistic effects on CFTR activity and surface localization produced by enhanced expression (NaBu treatment) coupled with stop codon suppression (G418 treatment) in W1282X and R1162X CFTR-transduced cells, and further emphasize apparent differences in the relative susceptibility of these CFTR mutations to functional rescue. Login to comment 184 ABCC7 p.Trp1282* ABCC7 p.Arg1162* We identified distinct differences in the responses of R1162X and W1282X CFTR to treatment designed to enhance transgene expression (NaBu). Login to comment 186 ABCC7 p.Trp1282* However immunohistochemical detection using anti-NBD-1 antibody revealed significantly greater cell surface localization of W1282X CFTR Figure 7. Login to comment 187 ABCC7 p.Trp1282* NaBu increases surface-localized truncated CFTR expression in W1282X CFTR-expressing cells as detected by immunofluorescence. Login to comment 188 ABCC7 p.Trp1282* ABCC7 p.Arg1162* (A) Immunofluorescence staining of W1282X (left) and R1162X CFTR (right)-expressing HeLa cells after 24 h exposure to NaBu (2.5mM), NaBu 1 G418 (500 mg/ml), or untreated (vehicle control) conditions. Login to comment 190 ABCC7 p.Trp1282* ABCC7 p.Arg1162* W1282X CFTR-transduced cells demonstrate significantly greater number of cells with surface localized truncated CFTR after 24 h treatment with NaBu compared with R1162X cells (55.2% versus 10.4% of cells with surface staining, respectively; P , 0.001). Login to comment 191 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg1162* A significant number of cells with surface localized full-length CFTR is detected in both cell lines after the addition of G418, although a greater percentage was seen in W1282X-transduced cells (29.1% of W1282X cells exhibit surface staining after G418 1 NaBu versus 0.5% under control conditions, P , 0.001; 5.4% and 0.06% surface staining was seen in R1162X cells, respectively, P , 0.01). Login to comment 195 ABCC7 p.Trp1282* ABCC7 p.Arg1162* *P , 0.05 versus control condition for each cell type; †P , 0.001 comparing W1282X versus R1162X cells at exact same treatment condition. Login to comment 197 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Furthermore, W1282X (but not R1162X) CFTR activity was partially restored in both cell lines after treatment with NaBu alone (Figures 1, 2, and 8). Login to comment 199 ABCC7 p.Trp1282* ABCC7 p.Trp1282* Together, our results suggest that increasing the expression of W1282X CFTR was sufficient to restore partial CFTR activity, and this effect was likely due to retained function of truncated W1282X CFTR that localizes to the plasma membrane. Login to comment 200 ABCC7 p.Arg1162* In contrast, increasing expression of the truncated R1162X CFTR did not restore functional activity. Login to comment 203 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Because mRNA levels were similar after NaBu treatment for both mutations, enhanced efficiency of cellular processing and localization of W1282X CFTR relative to R1162X likely account for the observed differences. Login to comment 207 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg1162* ABCC7 p.Arg1162* As R1162X CFTR is truncated immediately after the TMD-2 domain and W1282X CFTR is truncated within the NBD-2 domain, our results suggest that critical sequences between R1162X and W1282X may be responsible for functional differences between the mutations. Login to comment 208 ABCC7 p.Ser1455* 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 209 ABCC7 p.Trp1282* Our findings are also in agreement with single channel studies recently reported by Wang and colleagues, in which low-level activity was retained in W1282X CFTR expressing HeLa cells without treatments to enhance protein expression (43). Login to comment 210 ABCC7 p.Trp1282* Moreover, surface localization of W1282X CFTR in HeLa cells was confirmed using a biotinylation assay. Login to comment 213 ABCC7 p.Trp1282* ABCC7 p.Arg1162* CFTR-mediated halide transport in W1282X and R1162X CFTR-transduced HeLa cells studied by SPQ. Login to comment 214 ABCC7 p.Trp1282* Top: representative experiments in W1282X CFTR-transduced cells at various conditions. Login to comment 215 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Bottom: change in halide transport after stimulation with forskolin (20 mM) and genistein (50 mM) in W1282X or R1162X CFTR-transduced HeLa cells. Login to comment 217 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Results demonstrate CFTR activity in W1282X CFTR-transduced cells after treatment with NaBu that is not present in R1162X CFTR-transduced cells. Login to comment 218 ABCC7 p.Trp1282* ABCC7 p.Arg1162* *P , 0.001 for treated conditions compared with untreated controls, †P , 0.001 for W1282X versus R1162X under identical conditions, n 5 30 cells/condition, 6 SEM. Login to comment 221 ABCC7 p.Arg553* ABCC7 p.Gly542* In previously published studies, our laboratory has reported that high-level expression of the two most common CFTR premature stop mutations G542X and R553X CFTR (using transient vaccinia-based expression in non-polarizing cells) led to constitutive halide transport function lacking cAMP-dependent regulation (8, 27). Login to comment 224 ABCC7 p.Trp1282* ABCC7 p.Arg1162* The similar findings between the two cell types studied here (airway and nonairway) suggest that fundamental differences in cellular processing of R1162X and W1282X CFTR are retained across both model systems. Login to comment 225 ABCC7 p.Trp1282* Studies examining PKA-dependent and independent regulation of the two mutations will be needed to determine if cellular differences are also seen in regards to cAMP activation of truncated W1282X CFTR (as is seen with DF508 CFTR expressed in these two cell types) (32). Login to comment 230 ABCC7 p.Trp1282* In the context of adequate expression, W1282X CFTR function could be demonstrated at the cell membrane of both polarizing and nonpolarizing cell types. Login to comment 231 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg1162* ABCC7 p.Arg1162* While our studies demonstrate distinct differences in truncated protein activity between R1162X and W1282X CFTR, they do not rule out the possibility that NaBu treatment leads to enhanced levels of background translational readthrough of W1282X CFTR relative to R1162X CFTR. Login to comment 232 ABCC7 p.Trp1282* In fact, this is suggested by a higher proportion of W1282X cells with detectable full-length CFTR at the cell surface of NaBu-treated HeLa cells (13.6 versus 3.7% of cells, respectively; Figure 7). Login to comment 233 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg1162* Background readthrough is not likely to account for all the findings presented here, as combination treatment with NaBu and G418 led to similar levels of total (surface and nonsurface) full-length CFTR in cell lines expressing either R1162X or W1282X CFTR (Figures 6A and 6B), but NaBu alone led to much larger increases in truncated W1282X CFTR activity (Figures 1 and 2). Login to comment 234 ABCC7 p.Trp1282* Further studies directly comparing protein expression and function from CFTR cDNAs containing premature stop codons with and without distal CFTR open reading frames will be necessary to definitively determine the contribution of low-level background readthrough to the activity of W1282X CFTR. Login to comment 237 ABCC7 p.Trp1282* To date, the strongest suppressive and corrective effects have been seen in studies completed within the Israeli population (16, 17, 20), which is enriched with patients possessing a limited repertoire of premature stop mutations (particularly W1282X CFTR). Login to comment 238 ABCC7 p.Trp1282* ABCC7 p.Trp1282* At least three interpretations may be responsible for the treatment effects seen in these studies relative to others in patients with CF, including (1) enhancement of truncated but functionally active W1282X CFTR expression through inhibition of mRNA degradation by aminoglycoside-induced suppression of nonsense-mediated decay; (2) inherent, CFTR-independent population differences influencing CFTR mRNA stability among different study populations; or (3) differences in the relative suppressibility of Israeli subjects (particularly those subjects harboring W1282X CFTR). Login to comment 239 ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Arg1162* ABCC7 p.Arg1162* For example, although both W1282X and R1162X CFTR are caused by an abnormal UGA stop codon, W1282X is flanked by an adenine (as opposed to guanine in R1162X), a factor that enhances susceptibility to translational readthrough (8). Login to comment 240 ABCC7 p.Trp1282* ABCC7 p.Arg1162* Regardless of the contributor(s), the differences between R1162X and W1282X CFTR activity after enhancement of expression should be considered when interpreting the results of related clinical trials designed to restore CFTR function through suppression of premature stop mutations. Login to comment 241 ABCC7 p.Trp1282* ABCC7 p.Arg1162* In summary, the results reported here demonstrate that W1282X CFTR function can be partially restored after enhancement of transcription relative to R1162X CFTR. Login to comment 243 ABCC7 p.Trp1282* The results suggest W1282X may be more susceptible to corrective therapies than other mutations, and should be considered in the interpretation of clinical trials examining agents to suppress premature stop codons as a treatment strategy for genetic diseases. Login to comment