ABCMdb

PMID: 24520399

Char JE, Wolfe MH, Cho HJ, Park IH, Jeong JH, Frisbee E, Dunn C, Davies Z, Milla C, Moss RB, Thomas EA, Wine JJ
A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor.
PLoS One. 2014 Feb 10;9(2):e88564. doi: 10.1371/journal.pone.0088564. eCollection 2014., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly551Asp ABCC7 p.Arg117His A Little CFTR Goes a Long Way: CFTR-Dependent Sweat Secretion from G551D and R117H-5T Cystic Fibrosis Subjects Taking Ivacaftor Jessica E. Char1 , Marlene H. Wolfe1 , Hyung-ju Cho1 , Il-Ho Park1 , Jin Hyeok Jeong1 , Eric Frisbee1 , Colleen Dunn2 , Zoe Davies2 , Carlos Milla2 , Richard B. Moss2 , Ewart A. Login to comment 1 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His C. Thomas3 , Jeffrey J. Wine1,2,3 * 1 Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California, United States of America, 2 Department of Pediatrics, Stanford University School of Medicine, Stanford, California, United States of America, 3 Department of Psychology, Stanford University, Stanford, California, United States of America Abstract To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Login to comment 12 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Citation: Char JE, Wolfe MH, Cho H-j, Park I-H, Jeong JH, et al. (2014) A Little CFTR Goes a Long Way: CFTR-Dependent Sweat Secretion from G551D and R117H-5T Cystic Fibrosis Subjects Taking Ivacaftor. Login to comment 24 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp VX-770 was shown to improve the function of G551D in cultured cells [3], and patients having at least one G551D mutation treated with oral ivacaftor showed marked clinical improvement [4,5], leading to FDA approval of ivacaftor for use in G551D patients. Login to comment 25 ABCC7 p.Arg117His ABCC7 p.Arg117His Recently, ivacaftor was shown to improve CFTR-dependent ion transport in human airway epithelial cells carrying an R117H mutation [6], and trials are now underway to determine if ivacaftor will be clinically beneficial for patients with R117H-5T mutations. Login to comment 35 ABCC7 p.Gly551Asp Given the assay`s present inability to discriminate between PI and PS subjects, it was important to determine if it could detect functional efficacy of ivacaftor in CF subjects carrying the G551D mutation, where its clinical efficacy has already been demonstrated [4,5]. Login to comment 36 ABCC7 p.Gly551Asp Therefore, in the present study we measured the efficacy of ivacaftor in 4 individuals of G551D/F508del genotype. Login to comment 38 ABCC7 p.Arg117His Therefore, we also used the assay to measure the efficacy of ivacaftor in two CF subjects with R117H-5T mutations. Login to comment 40 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117His After written informed consent, six adults with genotypes G551D/F508del, one with G551D/R117H-5T and one with I507del/R117H-5T were enrolled, along with a healthy control. Login to comment 46 ABCC7 p.Gly551Asp One G551D subject who was not enrolled in an ivacaftor trial was studied off drug prior to FDA approval and then on-drug after starting ivacaftor as prescribed by his physician. Login to comment 60 ABCC7 p.Gly551Asp CFTR independent (M-sweat) and CFTR dependent (C-sweat) secretion in G551D CF subjects. Login to comment 78 ABCC7 p.Arg117His ABCC7 p.Arg117His doi:10.1371/journal.pone.0088564.g001 measures of PO and c for R117H [12] and mRNA transcript measurements of n [13] to provide an independent estimate of unpotentiated R117H-5T channel function. Login to comment 80 ABCC7 p.Gly551Asp For G551D, our corrected C/M values were compared with single channel measures made (6) ivacaftor [14,15]. Login to comment 88 ABCC7 p.Gly551Asp G551D/F508del subjects. Login to comment 90 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp (nl/gland) MCh Rate Estimate nl/gl/min Cktl Avg Final Vol (pl/gl) Cktl Rate (pl/gl/min) C mean/ M mean % WT % WT (Highest 12 M-sweat) % WT (Hi-12, Loss-Corrected) S1 G551D/F508del (2) ivacaftor S1 M 10/20/2011 71 0 0% 75.83 5.06 0 0 0.00% 0.00% - - S1 M 1/13/2012 85 0 0% 64.58 4.31 0 0 0.00% 0.00% - - S1 M 2/4/2012 75 0 0% 71.34 4.76 0 0 0.00% 0.00% - - S1 (2) Averages 77 0 0% 70.58 4.71 0 0 0.00% 0.00% 0.00% 0.00% S1 G551D/F508del (+) ivacaftor S1 M 3/23/2012 82 13 16% 70.99 4.73 152 5.06 0.11% 0.42% - - S1 M 4/10/2012 71 15 21% 91.91 6.13 105 3.52 0.06% 0.22% - - S1 M 4/17/2012 66 11 17% 97.02 6.47 110 3.66 0.06% 0.22% - - S1 (+) A xverages 73 13 18% 86.64 5.78 122 4.08 0.07% 0.29% 0.64% 1.59% S2 G551D/F508del (2) ivacaftor S2 F 2/2/2012 99 0 0% 28.61 1.91 0 0 0.00% 0.00% - - S2 F 3/20/2012 100 0 0% 29.16 1.94 0 0 0.00% 0.00% - - S2 (2) Averages 99.5 0 0% 28.88 1.93 0 0 0.00% 0.00% 0.00% 0.00% S2 G551D/F508del (+) ivacaftor S2 F 5/8/2012 82 4 5% 35.55 2.37 11 0.37 0.02% 0.06% - - S2 F 5/15/2012 90 1 1% 29.16 1.94 2 0.07 0.00% 0.01% - - S2 (+) Averages 86 2.5 3% 32.36 2.16 7 0.22 0.01% 0.04% 0.13% 2.75% S3 G551D/F508del (+) ivacaftor S3 F 7/24/2012 90 21 23% 45.03 3.00 95 3.18 0.11% 0.41% 0.58% 2.27% S5 G551D/F508del (+) ivacaftor data S5 M 11/2/2012 58 29 50% 78.94 5.26 703 23.42 0.45% 1.74% 3.07% 4.11% S6 G551D/F508del (2) ivacaftor data S6 F 2/2/2012 143 0 0% 14.37 0.96 0 0 0.00% 0.00% 0.00% 0.00% S7 G551D/F508del (2) ivacaftor data S7 F 2/13/2012 123 0 0% 32.16 2.14 0 0 0.00% 0.00% 0.00% 0.00% Summary (2) ivacaftor: 4 G551D subjects Sums or Averages 852 0 0% 36.50 2.43 0 0 0.00% 0.00% 0.00% 0.00% Summary (+) ivacaftor 4 G551D subjects Sums or Averages 906 317 29% 42.93 2.84 267 6.80 0.29% 1.15% 1.84% 4.07% Summary of results for CF subjects with G551D mutations. Login to comment 91 ABCC7 p.Gly551Asp Six adult G551D CF subjects were tested. Login to comment 100 ABCC7 p.Gly551Asp Mand C-sweat secretion (&#b1;) ivacaftor for CF subject 1: male, F508del/G551D. Login to comment 123 ABCC7 p.Gly551Asp Mand C-sweat secretion (&#b1;) ivacaftor for CF subject 2: female, F508del/G551D. Login to comment 142 ABCC7 p.Gly551Asp Results For six CF subjects each carrying one G551D mutation, CFTR-independent M-sweating was imaged for 15 minutes, allowing us to identify 58-143 sweat glands per subject and measure the M-sweat rates for each gland. Login to comment 145 ABCC7 p.Gly551Asp Images of M-sweat and C-sweat bubbles are shown for each G551D CF subject in Fig. 1. Login to comment 146 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp G551D subjects: within-subject comparisons (2) and (+) ivacaftor Subject 1 (S1, male, F508del/G551D) was tested 3 times in each condition. Login to comment 151 ABCC7 p.Gly551Asp C-sweat secretion (+) ivacaftor in CF subject 3: female, F508del/G551D. Login to comment 159 ABCC7 p.Gly551Asp C-sweat secretion (+) ivacaftor in CF subject 5: male, F508del/G551D. Login to comment 185 ABCC7 p.Gly551Asp On the log scale, M-sweat levels vary from 0 to 5.527, and the corresponding probabilities of a C-sweat response are.00006 and.23 Subject S2 (S2, female, F508del/G551D) was tested twice in each condition. Login to comment 193 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Two additional G551D subjects tested only (+) ivacaftor Subject 3 (S3, female, F508del/G551D) was tested only once. Login to comment 199 ABCC7 p.Gly551Asp Absence of C-sweat secretion (2) ivacaftor in two G551D CF subjects. Login to comment 200 ABCC7 p.Gly551Asp (A) S6: female, F508del/G551D. Login to comment 203 ABCC7 p.Gly551Asp (B) S7: female, F508del/G551D. Login to comment 207 ABCC7 p.Gly551Asp Subject 5 (S5, male, F508del/G551D), was tested only once (+) ivacaftor. Login to comment 213 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Two additional G551D subjects tested only (2) ivacaftor Two female G551D CF subjects, S6 and S7, were each tested once only (2) ivacaftor. Login to comment 218 ABCC7 p.Arg117His Summary of results for R117H-5T CF subjects and the healthy control. Login to comment 220 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117His (nl/gland) MCh Rate Estimate nl/gl/min Cktl Avg Final Vol (pl/gl) Cktl Rate (pl/gl/min) C mean/ M mean % WT % WT (Highest 12 M-sweat) % WT (Hi-12, Loss-Corrected) S4 G551D/R117H-5T (+) ivacaftor data S4 F 8/22/ 2012 37 8 22% 16.05 1.07 100 3.33 0.31% 1.22% - - S4 F 11/6/ 2012 32 6 19% 10.66 0.69 30 1.00 0.14% 0.55% - - S4 F 4/30/ 2013 37 11 30% 13.57 0.57 150 5.00 0.55% 2.16% - - S4 (+) Averages 35.33 8.33 23% 13.43 0.78 93 3.11 0.35% 1.31% 2.13% 7.83% S8 R117H/I508del (2) ivacaftor S8 M 1/31/ 2013 52 1 2% 46.76 3.12 6 0.19 0.01% 0.02% - - S8 M 2/8/2013 54 1 2% 49.88 3.33 12 0.39 0.01% 0.04% - - S8 M 2/15/ 2013 50 1 2% 59.51 3.97 4 0.14 0.00% 0.01% - - S8 (2) Averages 52 1 0% 52 3 7 0.24 0.01% 0.03% 0.06% 1.50% S8 R117H/I508del (+) ivacaftor S8 M 3/15/ 2013 53 28 53% 41.40 2.76 531 17.70 0.64% 2.42% - - S8 M 3/22/ 2013 53 45 84% 54.35 3.62 766 25.55 0.71% 2.66% - - S8 M 3/29/ 2013 49 42 86% 53.88 3.59 1146 38.20 1.06% 4.01% - - S8 M 6/17/ 2013 53 43 81% 57.84 3.86 645 21.49 0.56% 2.10% - - S8 M 6/24/ 2013 53 40 75% 53.85 3.59 474 15.80 0.44% 1.66% - - S8 (+) Averages 52.2 39.6 76% 52.26 3.48 712 23.75 0.68% 2.57% 4.51% 5.89% Healthy Control WT F 3/14/ 2013 51 50 98% 35.94 2.40 9687 323 13% 51% - - WT F 3/25/ 2013 51 51 100% 36.98 2.47 9371 312 13% 48% - - WT F 3/28/ 2013 51 51 100% 28.81 1.92 11519 384 20% 75% - - WT F 5/2/2013 51 50 98% 36.48 2.43 10540 351 14% 54% - - WT Averages 51 50.5 99% 34.55 2.30 10279 343 15% 57% 60.65% 62.97% Table is arranged using same format as Table 1 and shows data for S8, S4 and the healthy control. Login to comment 221 ABCC7 p.Arg117His ABCC7 p.Arg117His doi:10.1371/journal.pone.0088564.t002 Within-subject study of an R117H-5T subject (2) and (+) ivacaftor Subject 8 has genotype I507del/R117H-5T. Login to comment 222 ABCC7 p.Arg117His The I507del mutation does not traffic [18,19] and so is predicted to be unresponsive to ivacaftor, allowing us to assess the in vivo effect of ivacaftor on the R117H-5T mutation in isolation. Login to comment 239 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117His Study of a CF subject with two responsive mutations: G551D/R117H-5T Subject 4 (S4, female, G551D/R117H-5T) has two mutations that are each expected to respond to ivacaftor. Login to comment 259 ABCC7 p.Arg117His Images of C-sweat responses for CF subject 8: male, I507del/R117H-5T (&#b1;) ivacaftor (N of 1 trial). Login to comment 272 ABCC7 p.Arg117His Summary data for S8, R117H-5T, (&#b1;) ivacaftor. Login to comment 285 ABCC7 p.Gly551Asp doi:10.1371/journal.pone.0088564.g008 Individual differences across subjects and sensitivity of the C/M ratio to sweat gland secretory capacity Table 1 shows summary results for all 6 G551D CF subjects; Table 2 has the summary results for the other subjects. Login to comment 291 ABCC7 p.Gly551Asp The G551D subjects differed significantly from one another in the proportion of glands that produced measureable C-sweat (ANOVA, F = 54.8, p = 0.001). Login to comment 294 ABCC7 p.Gly551Asp The corresponding C/M ratios for these matched samples of glands (Fig. 11C, left axis, blue columns,) show that G551D subjects still differ widely: e.g. S5`s ratio is ,7-fold .S1`s. Login to comment 295 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Even so, the differences within G551D subjects are overshadowed by the much higher C/M ratio seen in R117H-5T subject 8, and in S4, who, with two responsive mutations, had the highest C/M ratio after partially correcting for M-sweat rate. Login to comment 304 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Discussion Our first goal in this study was to determine if this bioassay could detect improved CFTR function in CF subjects with G551D or R117H-5T mutations who were taking ivacaftor. Login to comment 305 ABCC7 p.Gly551Asp We knew before starting that ivacaftor increases G551D CFTR function sufficiently to benefit health [4,5]. Login to comment 307 ABCC7 p.Arg117His But no clinical data were available on ivacaftor treated R117H-5T subjects, so our finding of a strong ivacaftor effect for subjects with this compound allele provides in vivo support for the prediction that ivacaftor will be therapeutic in such patients [2]. Login to comment 309 ABCC7 p.Gly551Asp In fact, we observed large (,6-fold), inter-subject differences in C-sweat responses among the four G551D subjects taking ivacaftor, after correcting for M-sweat rates (Fig. 11C). Login to comment 310 ABCC7 p.Gly551Asp Individual differences could arise from different G551D-CFTR expression levels, differences in transport processes affecting CFTR-dependent sweat secretion, drug pharmacokinetics/tissue distribution, different responses of the in trans F508del allele, and compliance. Login to comment 313 ABCC7 p.Gly551Asp A third goal was to determine if the assay could quantify the extent to which ivacaftor restored function to G551D and Figure 9. Login to comment 314 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Summary data for S4, a female CF subject having two potentially responsive mutations: G551D & R117H-5T who was tested 3 times (+) ivacaftor. Login to comment 322 ABCC7 p.Arg117His doi:10.1371/journal.pone.0088564.g009 R117H-5T CFTR. Login to comment 341 ABCC7 p.Arg117His With this background, we first consider results for the R117H-5T subject. Login to comment 351 ABCC7 p.Arg117His ABCC7 p.Arg117His doi:10.1371/journal.pone.0088564.g010 Quantifying R117H-5T CFTR function in vivo and in vitro R117H-5T CFTR retains partial function and confers pancreatic sufficiency when trans to a severe allele. Login to comment 352 ABCC7 p.Arg117His Studies of heterologously expressed CFTR channels by Sheppard and colleagues [12] found that the whole-cell Cl2 conductance of R117H was reduced to 15% of WT, while single channel analysis found that both PO and c were reduced predicting ,24% WT CFTR channel function. Login to comment 353 ABCC7 p.Arg117His ABCC7 p.Arg117His In a male and a female each having R117H/R117H on a 7T/7T background, all clinical indices were normal except for CBAVD in the male, electrophysiological measurements of nasal and intestinal epithelia were also normal, and sweat chloride values were 34 mM for the male and 42 mM for the female [23]. Login to comment 354 ABCC7 p.Arg117His ABCC7 p.Arg117His However, when R117H-CFTR is in cis with a 5T allele, n = 0.1/0.7 = 14% WT, reducing R117H-5T function to 2.1-3.7% of WT function. Login to comment 357 ABCC7 p.Arg117His To estimate the amount of primary sweat in S8 that was lost in the (2) ivacaftor condition, we assigned a CFTR functional level = 1.5% WT to S8, based on the above analysis of R117H-5T function [12,13]. Login to comment 360 ABCC7 p.Arg117His ABCC7 p.Arg117His If ivacaftor restored normal PO to R117H and left n and c unchanged, it would increase R117H-5T CFTR function equivalent to 5T function on 1 allele, or 7% of WT. Login to comment 363 ABCC7 p.Arg117His Thus, in this R117H-5T subject, functional estimates based on patch-clamp [12] and mRNA transcript analysis [13] converged with those of based on C/M-sweat bubble analysis. Login to comment 364 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Quantifying G551D CFTR function in vivo and in vitro The G551D mutation severely decreases PO to near zero (,0.004) without a known effect on n or c [15]. Login to comment 365 ABCC7 p.Gly551Asp The calculations used above predict that this level of function is invisible to the sweat bubble test, consistent with the lack of any observed sweating in the 4 G551D subjects tested (2) ivacaftor. Login to comment 366 ABCC7 p.Gly551Asp The PO of G551D-CFTR (+)-ivacaftor has been measured as 0.04 [14] and 0.12 [2] or 10-30% of the WT value of 0.4. Login to comment 367 ABCC7 p.Gly551Asp Subjects S1-S3 and S5 each have one G551D and one (nonfunctional) F508del allele, so their ivacaftor-potentiated CFTR function should have been half of those values or 5-15% of WT. Login to comment 370 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Based on all the data and on comparisons with the ivacaftor-treated R117H subject, we estimate ivacaftor-potentiated CFTR function in G551D/ F508del subjects to be ,5% WT. Login to comment 371 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Subject S4 has mutations R117H-5T and G551D. Login to comment 372 ABCC7 p.Gly551Asp ABCC7 p.Arg117His In the (+) ivacaftor condition, the data from patch clamp and transcript analysis predicts CFTR function of 2.6-5% function for R117H-5T, and up to 5% for G551D, or up to 10% function for the combination. Login to comment 382 ABCC7 p.Gly551Asp ABCC7 p.Arg117His S1-S3 and S5 each has one G551D mutation, S8 has R117H-5T, and S4 has both. Login to comment 412 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Conclusions Multiple measures of C-sweating and M-sweating from individual glands revealed that oral dosing with ivacaftor improved b-adrenergic cocktail-stimulated, CFTR-dependent sweating (C-sweating) in CF subjects with G551D or R117H-5T mutations. Login to comment 431 ABCC7 p.Gly551Asp ABCC7 p.Arg117His doi:10.1371/journal.pone.0088564.g014 function was 2.6661.06% of WT for the 4 G551D subjects, 6.1% for the R117H-5T subject, and 7.7% for the subject with both mutations. Login to comment 432 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117His The finding that CFTR-dependent sweat secretion was greater in R117H-5T than in G551D subjects supports prior work suggesting that ivacaftor will be clinically useful for subjects with the R117H-5T mutation [6]. Login to comment 453 ABCC7 p.Gly551Asp Accurso FJ, Rowe SM, Clancy JP, Boyle MP, Dunitz JM, et al. (2010) Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation. Login to comment 456 ABCC7 p.Gly551Asp Ramsey BW, Davies J, McElvaney NG, Tullis E, Bell SC, et al. (2011) A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. Login to comment 487 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Bompadre SG, Sohma Y, Li M, Hwang TC (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment 511 ABCC7 p.Arg117His 23. de Nooijer RA, Nobel JM, Arets HG, Bot AG, van Berkhout FT, et al. (2011) Assessment of CFTR function in homozygous R117H-7T subjects. Login to comment