ABCMdb

PMID: 25799511

Rapino D, Sabirzhanova I, Lopes-Pacheco M, Grover R, Guggino WB, Cebotaru L
Rescue of NBD2 mutants N1303K and S1235R of CFTR by small-molecule correctors and transcomplementation.
PLoS One. 2015 Mar 23;10(3):e0119796. doi: 10.1371/journal.pone.0119796. eCollection 2015., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg RESEARCH ARTICLE Rescue of NBD2 Mutants N1303K and S1235R of CFTR by Small-Molecule Correctors and Transcomplementation Daniele Rapino1,2 , Inna Sabirzhanova1,2 , Miqu&#e9;ias Lopes-Pacheco1,2 , Rahul Grover1,2 , William B. Guggino2 , Liudmila Cebotaru1 * 1 Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America, 2 Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America * lcebotaru@jhmi.edu. Login to comment 3 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg To provide information on potential therapeutic strategies for mutations in NBD2, we studied, using a combination of biochemical approaches and newly created cell lines, two disease-causing NBD2 mutants, N1303K and S1235R. Login to comment 5 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Inhibition of proteasomes with MG132 or aggresomes with tubacin rescued the immature B and mature C bands of N1303K and S1235R, indicating that degradation occurs via proteasomes and aggresomes. Login to comment 12 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Our results show that the N1303K mutation has a more profound effect on NBD2 processing than S1235R and that small-molecule correctors increase the maturation of bands B and C in NBD2 mutants. Login to comment 15 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Finally our results are important because they suggest that gene or corrector molecule therapies either alone or in combination individualized for NBD2 mutants may be beneficial for patients bearing N1303K or S1235R mutations. Login to comment 16 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg PLOS ONE | DOI:10.1371/journal.pone.0119796 March 23, 2015 1 / 17 a11111 OPEN ACCESS Citation: Rapino D, Sabirzhanova I, Lopes-Pacheco M, Grover R, Guggino WB, Cebotaru L (2015) Rescue of NBD2 Mutants N1303K and S1235R of CFTR by Small-Molecule Correctors and Transcomplementation. Login to comment 31 ABCC7 p.Gly551Asp Recently, the FDA has approved Kalydeco (VX-770) for patients with the G551D mutation, which is also associated with severe CF [7]. Login to comment 32 ABCC7 p.Gly551Asp Unlike ƊF508-CFTR, G551D is a gating mutant that traffics to the plasma membrane but requires potentiation of its defective channel activity [8]. Login to comment 33 ABCC7 p.Gly551Asp Patients taking Kalydeco, which was derived from a basic understanding of the need of potentiation for the G551D mutant channel function [9], have shown remarkable improvement in most of the clinical manifestations of their disease [7]. Login to comment 37 ABCC7 p.Gly551Asp There has been much interest in the gating and trafficking mutations in NBD1, such as G551D and ƊF508-CFTR. Login to comment 38 ABCC7 p.Ala455Glu We have recently shown that another NBD1 mutant, A455E, can be rescued [12]. Login to comment 44 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg To address this question, we have now focused on two NBD2 mutations, N1303K and S1235R. Login to comment 45 ABCC7 p.Asn1303Lys N1303K has a high incidence in Europe, and it has been reported to be the second most common mutation in Italy (4%). Login to comment 47 ABCC7 p.Asn1303Lys N1303K is classified as a severe mutation with respect to the pancreas (causing pancreatic insufficiency and diabetes mellitus) [18]; it is also associated with precocious and severe lung symptoms such as newborn pneumothorax [19]. Login to comment 48 ABCC7 p.Ser1235Arg The incidence of S1235R is approximately 1% (3.3% in Eastern Europe), significantly higher than other rare mutations. Login to comment 49 ABCC7 p.Asn1303Lys Phenotypic manifestations appear to be milder and more variable than those associated with N1303K and are mainly related to chronic pancreatitis, associated with sweat chloride test results [20-23] that are on the borderline of what is considered to be typical of CF. Login to comment 60 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg The human embryonic kidney Flp-In-293 cell line was used to generate two stable cell lines expressing the PBI CMV2 N1303K CFTR and PBI CMV2 S1235R CFTR plasmids (provided by Phil Thomas (U. Texas, Southwestern). Login to comment 63 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg PBI CMV2 N1303K CFTR, pc-DNA Ɗ27-264, PBI CMV2 S1235R CFTR, PBI CMV2 CFTR and PBI CMV2 ƊF508 plasmids were used to perform transient transfections using Lipofectamine 2000. Login to comment 93 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg The effect of proteasome, aggresome, and lysosome inhibitors (MG132, tubacin, and E64, respectively) on the N1303K and S1235R mutations was also studied. Login to comment 98 ABCC7 p.Asn1303Lys Dieter Gruenert, University of California, San Francisco) were stably transfected with N1303K using Flp-In technology. Login to comment 99 ABCC7 p.Asn1303Lys Once the cell line was established and verified to contain N1303K by western blotting, the cells were grown on Snapwell filters (Corning Costar, Acton, MA; 3407). Login to comment 109 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg ABCC7 p.Ser1235Arg SPSS (version 17.0 SPSS, Inc, Chicago, III) was used for data analysis Results Characterization of N1303K and S1235 mutations cDNA of N1303K and S1235R, wild-type CFTR, and ƊF508 was transfected into the Cos-7 cell line, and the protein expression of both mutations, N1303K and S1235R, was compared with wt CFTR and ƊF508, the most common CF mutation. Login to comment 110 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Densitometry analysis showed that N1303K expressed a level of immature band B similar to that of ƊF508 and a barely detectable mature band C. S1235R expressed both band B and C again at lower levels compared to wt CFTR (Fig. 1). Login to comment 113 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg In sharp contrast, the results differed for the NBD2 mutants: Temperature rescue had no effect on the maturation of band C of either N1303K or S1235R (Fig. 1). Login to comment 114 ABCC7 p.Ser1235Arg The only significant effect was an increase in the mature band B of S1235R when the cells were grown at the reduced temperature. Login to comment 115 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Next, we applied inhibitors of proteasomes, aggresomes, and lysosomes in order to investigate the degradation pathway of the N1303K and S1235R mutants (Figs. 2 and 3). Login to comment 116 ABCC7 p.Asn1303Lys When we applied MG132, a non-specific inhibitor of proteasome degradation [12], the expression of band B and C in N1303K CFTR was significantly increased, indicating that this mutation is degraded in the proteasome. Login to comment 118 ABCC7 p.Asn1303Lys On the other hand, when we applied E64, an inhibitor of cysteine proteinases [31], to cells expressing N1303K, we saw no significant change in either the B or C band, indicating that this mutant is not degraded in lysosomes (Fig. 2). Login to comment 119 ABCC7 p.Asn1303Lys We saw an even greater increase in both the immature B and mature C bands of N1303K when tubacin was applied, this strong effect, especially on the immature B band, indicates a major role for aggresome formation [32] in the degradation of this mutant. Login to comment 120 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Like N1303K, the S1235R mutation was not affected by E64 inhibition. Login to comment 121 ABCC7 p.Ser1235Arg Interestingly, when we applied MG132 to cells expressing the S1235R mutant, we saw a 6-fold increase in the immature B band (Fig. 3). Login to comment 126 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Characterization and temperature correction of N1303K and S1235R. Login to comment 127 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg (A) Cos7 cells were transfected with wild type (wt) CFTR, ƊF508, N1303K, or S1235R and then grown either continuously at 37&#b0;C or at 25&#b0;C for 24 h. Login to comment 130 ABCC7 p.Asn1303Lys Neither the B nor the C band of N1303K is increased by 25&#b0;C treatment. Login to comment 131 ABCC7 p.Ser1235Arg For S1235R, some increase in band B is seen, but no significant effect on band C. Login to comment 135 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Degradation pathways for the N1303K mutation. An HEK 293 cell line stably expressing N1303K was treated with (A) the lysosome inhibitor E64, (B) proteasome inhibitor MG132, or (C) aggresome inhibitor tubacin. Login to comment 136 ABCC7 p.Asn1303Lys (D) The graph shows that the N1303K mutant is mainly degraded by the proteasome and aggresome; no effect was detected after lysosome treatment. Login to comment 139 ABCC7 p.Ser1235Arg ABCC7 p.Ser1235Arg Degradation pathways for the S1235R mutation. An HEK 293 cell line stably expressing S1235R was treated with (A) the lysosome inhibitor E64, (B) proteasome inhibitor MG132, or (C) aggresome inhibitor tubacin. Login to comment 140 ABCC7 p.Ser1235Arg (D) The graph shows that the S1235R mutant is mainly degraded by proteasomes and aggresomes; no effect was detected after lysosome treatment. Login to comment 144 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Rescue of N1303K and S1235R by small-molecule correctors. Login to comment 145 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg HEK 293 cell lines stably expressing either (A) N1303K or (B) S1235R were treated with compound alone (C3, C4, C18, CFFT 002, CFFT 003) or the combination of two compounds (C3+C4, C4+C18, or C3+C18). Login to comment 146 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg (C and D) Graphs show that all compounds are able to significantly increase bands B and C of N1303K and S1235R. Login to comment 150 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg doi:10.1371/journal.pone.0119796.g004 Application of small-molecule correctors has a significant effect in rescuing the protein expression levels of N1303K and S1235R A number of corrector molecules have been shown to rescue ƊF508-CFTR [33], and the question we asked here is whether they would rescue mutants in NBD2. Login to comment 153 ABCC7 p.Asn1303Lys In the case of the N1303K mutation, most of the correctors increased both the B and C bands. Login to comment 155 ABCC7 p.Asn1303Lys Both combinations significantly increased the levels of B and C bands of N1303K, by at least two to three folds. Login to comment 156 ABCC7 p.Ser1235Arg In the case of S1235R, all correctors appeared to have a similar effect either alone or in combination, giving an increase of approximately two fold in both the B and C bands (Fig. Login to comment 158 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Stability of both the B and C bands of N1303K and S1235R is significantly higher than that of the control, after treatment with corrector compounds Cells were treated with cycloheximide to inhibit protein synthesis [34] and then harvested at time 0 or after 1, 2, 4, 8, or 24 h. Login to comment 159 ABCC7 p.Asn1303Lys Western blot analysis showed that the half-life of the C band of N1303K was approximately 4-8 h (Fig. Login to comment 161 ABCC7 p.Asn1303Lys Interestingly, the B band of N1303K could be detected at 24 h. Login to comment 163 ABCC7 p.Asn1303Lys The mature C band of N1303K had a relatively short half-life when compared to that been reported for wt-CFTR, indicating that although some of the C band was processed, it was less stable than the mature C band of wt-CFTR. Login to comment 165 ABCC7 p.Asn1303Lys Unlike ƊF508-CFTR, whose immature B band has a very short half-life, the immature B band of N1303K was still detectable after 24 h. Login to comment 166 ABCC7 p.Asn1303Lys This stability may indicate that the processing of the N1303K mutant is arrested in the ER and is amenable to rescue by an appropriate combination of correctors such as C3+C4 or C4+C18 (Fig. 4). Login to comment 167 ABCC7 p.Ser1235Arg On the other hand, the half-life of the S1235R mutation exceeded 24 h (Fig. Login to comment 170 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Thus, it appears that corrector compounds exert their effect by increasing the amount of C and B bands in S1235R, whereas in the case of the N1303K mutant, the half-life is also increased. Login to comment 171 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Small-molecule compounds increase the amount of mature CFTR localized to the plasma membrane Biotinylation was performed to determine whether the treatment with small-molecule correctors of N1303K and S1235R could increase the amount of band C in these CFTR mutants that was present at the plasma membrane. When compounds C3 + C4 at 5 bc;M were applied, they were able to increase by more than two fold the amount of mature CFTR capable of reaching the cell surface, as compared with the controls for both mutants (Fig. 7). Login to comment 172 ABCC7 p.Asn1303Lys Fig 5. Half-live of N1303K with and without treatment with compounds. HEK 293 cell lines stably expressing either mutant were treated with cycloheximide (25bc;g/ml) and harvested after 1, 2, 4, 8, or 24 h. Login to comment 174 ABCC7 p.Asn1303Lys When C3+C4 5 (bc;M) was applied, the levels of both the B and C bands were higher at each time point for (B, D) N1303K. Login to comment 175 ABCC7 p.Asn1303Lys The major effect of the corrector combination was a prolongation of the half-life of the C band of N1303K. Login to comment 178 ABCC7 p.Ser1235Arg doi:10.1371/journal.pone.0119796.g005 Fig 6. Half-live of S1235R with and without treatment with compounds. HEK 293 cell lines stably expressing either mutant were treated with cycloheximide (25bc;g/ml) and harvested after 1, 2, 4, 8, or 24 h. Login to comment 179 ABCC7 p.Ser1235Arg (A, C) The half-life of the C band of the S1235R mutant exceeds 24 h. Login to comment 180 ABCC7 p.Ser1235Arg When C3+C4 5 (bc;M) was applied, the levels of both the B and C bands were higher at each time point for S1235R (B, D). Login to comment 186 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Compounds C3+C4 (5 bc;M) demonstrate the ability to significantly increase the delivery of the fully glycosylated protein to the plasma membrane, in the case of the N1303K (A and C) and S1235R (B and D) mutations. Login to comment 189 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Co-immunoprecipitation of CFTR, HSPs, VCP, and HDAC6 in the N1303K and S1235R cell lines treated or untreated with the compounds. HEK 293 cell lines stably expressing either mutant were treated with C3+C4 (5bc;M), and CFTR was immunoprecipitated with anti-CFTR antibody M3A7. Login to comment 198 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg As shown in Figs. 8 and 9, a number of chaperones, including heat shock proteins (HSP) 27, 40, 70, and 90, bind to the N1303K and S1235R mutants. Login to comment 204 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg It is also noteworthy that the reduction in binding was greater with the N1303K mutant than with the S1235R mutant (Fig. 8B). Login to comment 205 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Given that N1303K causes a more severe form of CF than does S1235R, these results may indicate a more avid engagement of N1303K with the degradative process. Login to comment 206 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg The binding data precisely paralleled the increase in the rescue of mature C band of N1303K, which was increased by more than 2 folds by the combined treatment with C3+C4, as compared with the rescue of S1235R (Fig. 8B). Login to comment 208 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg The main difference in the total lysate is a significant increase in the total amount of HSP27 for S1235R and a significant but slightly decreased amount of HSP27 for N1303K when compared to the control (HEK 293 cell lines without any CFTR mutation) (Fig. 9). Login to comment 209 ABCC7 p.Ala455Glu Transcomplementation by Ɗ27-264 is able to rescue NBD2 mutants We have previously shown that truncated forms of CFTR such as Ɗ264 and Ɗ27-264-CFTR can rescue the NBD1 mutants ƊF508-CFTR and A455E CFTR [27,42], but it was still not clear whether the NBD2 mutants could also be transcomplemented. Login to comment 210 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Transient transfection of the Ɗ27-264 plasmid into the N1303K and S1235R HEK Flp-In-293 stable cell lines was able to rescue the N1303K mutation, increasing the band C expression by 3-fold when compared to the control (Fig. Login to comment 212 ABCC7 p.Ser1235Arg Treatment with the CFTR truncated form Ɗ27-264 was also able to rescue band C of S1235R, increasing it by more than 2-fold (Fig. Login to comment 214 ABCC7 p.Asn1303Lys Importantly, when the chloride transport ability of the N1303K mutant was measured in CFBE stably expressing the mutant, we saw a dramatic increase in Isc in the cells infected with AAV1 containing Ɗ264-CFTR. Login to comment 217 ABCC7 p.Asn1303Lys 10A, B) and chloride transport of the N1303K mutant (Fig. 11). Login to comment 218 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Discussion Here we have studied two mutations in NBD2, S1235R and N1303K, and showed that both can be rescued by small-molecule correctors, used in combination, and transcomplementation using a truncated version of CFTR. Login to comment 219 ABCC7 p.Asn1303Lys The N1303K mutation is associated with severe CF and is a processing mutant in NBD2, with minimal chloride conductance and mature C band at the cell surface when assessed in rat Fischer thyroid cells (FRT) and HeLa cells [43]. Login to comment 222 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Total lysate levels of CFTR, HSPs, VCP and HDAC6 in the N1303K and S1235R cell lines. Login to comment 224 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg (B) The main difference in the total lysate is a significant increase in the total amount of HSP27 for S1235R and a significant but slightly decreased binding of HSP27 to N1303K when compared with the control (HEK 293 cell lines without any CFTR mutation). Login to comment 225 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg The graphs show that HSP27 in the cell line bearing the S1235R mutant, whether treated or untreated with the compounds, is four times higher than in the control and the N1303K cell line. Login to comment 230 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Effect of transcomplementation with Ɗ27-264 on N1303K and S1235R mutants. Login to comment 232 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Ɗ27-264-is able to increase significantly band C of (A) N1303K and (B) S1235R. Login to comment 233 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Graphs show that the fully glycosylated protein is increased by 3 times for (C) N1303K and more than 2 times for (D) S1235R. Login to comment 238 ABCC7 p.Asn1303Lys The lysine residue, on the other hand, is disruptive, as suggested by the poor function and trafficking of N1303K; it could also potentially serve as a site for abnormal ubiquitination, further disrupting the normal trafficking of CFTR. Login to comment 239 ABCC7 p.Ser1235Arg S1235R, in contrast, is associated with mild or minimal disease [43]. Login to comment 241 ABCC7 p.Gly551Asp ABCC7 p.Ser1235Arg In NBD1, the equivalent amino acid is glutamic acid, and thus the arginine replacement in S1235R seems to be mildly disruptive; the conductance measured in FRT cells is approximately 79% of wt-CFTR, and the protein processing is nearly equivalent to that measured for the G551D mutant [43]. Login to comment 244 ABCC7 p.Ala455Glu ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Unlike ƊF508-CFTR and A455E, both of which undergo significant trafficking of immature band B to mature band C [12], the mature bands of N1303K and S1235R are not enhanced when cells are grown at low temperature. Login to comment 250 ABCC7 p.Ser1235Arg S1235R is particularly sensitive to tubacin, the aggresome inhibitor. Login to comment 251 ABCC7 p.Ala455Glu ABCC7 p.Ser1235Arg This response of S1235R is in sharp contrast to that of A455E, which is not sensitive to tubacin. Login to comment 252 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg There was surprisingly little effect of lysosomal inhibition, especially on the S1235R mutant, even though this mutant does have significant quantities of mature C band at the plasma membrane. When protein translation is inhibited by cycloheximide, the immature B band of N1303K is more stable than ƊF508-CFTR, which is rapidly degraded and disappears rather quickly (see Fig 11. Login to comment 253 ABCC7 p.Asn1303Lys Transcomplementation rescues N1303K CFTR currents. Login to comment 254 ABCC7 p.Asn1303Lys CFBE cells stably expressing N1303K-CFTR were grown on Transwell supports and treated with either 10 bc;l of AAV1 containing Ɗ264 CFTR as described in Materials and Methods. Login to comment 259 ABCC7 p.Ala455Glu These results point to differences in the processing of these NBD2 mutants when compared to two of the mutations in NBD1, A455E and ƊF508-CFTR. Login to comment 263 ABCC7 p.Asn1303Lys Bands B and C of N1303K were significantly increased when small-molecule correctors were applied, with an additive effect when they were administered together. Login to comment 264 ABCC7 p.Ser1235Arg Similar results were found for S1235R; all compounds were effective to some extent when administered individually. Login to comment 265 ABCC7 p.Asn1303Lys However, when they were administered together, we saw a less remarkable effect than we had observed for N1303K. Login to comment 267 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg The results showed a significant increase in the presence of the fully glycosylated bands of both N1303K and S1235R at the cell surface when C3+C4 was applied. Login to comment 273 ABCC7 p.Asn1303Lys The most profound effect was noted for N1303K, when we combined a Class I corrector with one from Class II, indicating that mutations in NBD2 may also have effects on the other parts of CFTR, which themselves will have to be corrected. Login to comment 274 ABCC7 p.Asn1303Lys Interesting, the combination of C3+C4 had a large impact on the interaction of N1303K with proteins associated with ERAD, another indication of their combined effectiveness. Login to comment 280 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg We show here that transfection of the Ɗ27-264 plasmid does indeed lead to rescue of the fully glycosylated band of N1303K and S1235R. Login to comment 284 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg ABCC7 p.Ser1235Arg In summary Our results suggest that the N1303K mutation has a more profound effect on CFTR processing than does S1235R, consistent with the observation that N1303K causes severe CF and S1235R only mild-to-borderline disease [19,23,46]. Login to comment 287 ABCC7 p.Asn1303Lys ABCC7 p.Ser1235Arg Finally, our results suggest that patients with N1303K or S1235R mutations may be good candidates for therapy with small-molecule correctors, combined with transcomplementation with the truncated form of CFTR, Ɗ27-264. Login to comment 310 ABCC7 p.Gly551Asp Accurso FJ, Rowe SM, Clancy JP, Boyle MP, Dunitz JM, Durie PR, et al. Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation. Login to comment 322 ABCC7 p.Ala455Glu 2012; 67: 12-18. doi: 10.1136/thoraxjnl-2011-200393 PMID: 21825083 12. Cebotaru L, Rapino D, Cebotaru V, Guggino WB Correcting the cystic fibrosis disease mutant, A455E CFTR. Login to comment 335 ABCC7 p.Asn1303Lys Federici S, Iron A, Reboul MP, Desgeorges M, Claustres M, Bremont F, et al. [CFTR gene analyis in 207 patients with cystic fibrosis in southwest France: high frequency of N1303K and 1811+1.6bA>G mutations]. Login to comment 337 ABCC7 p.Asn1303Lys Cotellessa M, Mazzella M, Bruno C, Buzzanca C, Minicucci L, Gandino M, et al. N1303K mutation and diabetes mellitus in cystic fibrosis. Login to comment 339 ABCC7 p.Asn1303Lys Ataseven F, Ozer S, Yilmaz R, Senayli A (2010) Bilateral spontaneous pneumothorax in a newborn with N1303K mutation of cystic fibrosis (CFTR) gene. Login to comment 402 ABCC7 p.Asn1303Lys Osborne L, Santis G, Schwarz M, Klinger K, Dork T, McIntosh I, et al. Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene. Login to comment