ABCMdb

PMID: 26255232

Zomer-van Ommen DD, Vijftigschild LA, Kruisselbrink E, Vonk AM, Dekkers JF, Janssens HM, de Winter-de Groot KM, van der Ent CK, Beekman JM
Limited premature termination codon suppression by read-through agents in cystic fibrosis intestinal organoids.
J Cyst Fibros. 2015 Aug 5. pii: S1569-1993(15)00169-1. doi: 10.1016/j.jcf.2015.07.007., [PubMed]

Sentences

No. Mutations Sentence Comment

31 ABCC7 p.Trp1282* ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Glu60* ABCC7 p.Glu60* Human organoid culture and functional CFTR measurements Crypts were isolated from rectal biopsies of six subjects with cystic fibrosis (E60X/4015delATTT, E60X/F508del, G542X/ F508del, R1162X/F508del, W1282X/F508del and F508del/ F508del) as previously described (20). Login to comment 39 ABCC7 p.Glu60* Results To investigate CFTR repair by PTC124 and G418, we measured swelling of human intestinal organoids compound heterozygous for a CFTR PTC allele and a frame shift allele (E60X/4015delATTT), lacking CFTR function as indicated by absence of forskolin-induced swelling (FIS) (20). Login to comment 46 ABCC7 p.Trp1282* ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Glu60* We next assessed read-through by G418 and PTC124 in organoids compound heterozygous for F508del and a nonsense mutation (E60X, G542X, R1162X and W1282X) (Fig. 2A). Login to comment 47 ABCC7 p.Arg1162* Subject genotype R1162X/F508del is a low responder, indicated by standard swelling assays performed in our laboratory (data not shown). Login to comment 58 ABCC7 p.Glu60* The G418-induced CFTR function was further stimulated by CFTR-modifying drugs VX-770 and VX-809 in organoids lacking residual CFTR function (E60X/4015delATTT), confirming G418 induced full-length CFTR protein and confirming previous data (22) (Fig. 1A, C-D). Login to comment 61 ABCC7 p.Glu60* These data indicated that E60X/4015delATTT intestinal organoids express sufficient CFTR nonsense mRNA levels for detection of read-through by pharmacological agents such as G418. Login to comment 66 ABCC7 p.Glu60* Forskolin-induced swelling (FIS) of intestinal organoids (E60X/4015delATTT and F508del/F508del), incubated with either G418, PTC124 or DMSO (vehicle), w/o VX-809 for 24 h and stimulated with forskolin, w/o VX-770. Login to comment 69 ABCC7 p.Glu60* A) Normalized E60X/4015delATTT organoid area increase in 60 min of FIS (%, &#b1;SD); B) representative images to determine total area and luminal area per well; C) luminal size (% of total organoid area) after 60 min of FIS (%, &#b1;SD), *p b 0.05, **p b 0.01; D) representative confocal images, conditions as indicated. Login to comment 70 ABCC7 p.Trp1282* ABCC7 p.Gly542* 3 of PTC124, and included CFTR mutations that were previously associated with PTC124 read-through, such as G542X (c.1624G- N T) and W1282X (c.3846G- N A) (cftr2.org). Login to comment