Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line

Abstract

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. While 70% of CF chromosomes carry a deletion of the phenylalanine residue 508 (deltaF508) of CFTR, roughly 5% of all CF chromosomes carry a premature stop mutation. We reported that the aminoglycoside antibiotics G-418 and gentamicin can suppress two premature stop mutations [a stop codon in place of glycine residue 542 (G542X) and arginine residue 553 (R553X)] when expressed from a CFTR cDNA in HeLa cells. Suppression resulted in the synthesis of full-length CFTR protein and the appearance of a cAMP-activated anion conductance characteristic of CFTR function. However, it was unclear whether this approach could restore CFTR function in cells expressing mutant forms of CFTR from the nuclear genome. We now report that G-418 and gentamicin are also capable of restoring CFTR expression in a CF bronchial epithelial cell line carrying the CFTR W1282X premature stop mutation (a stop codon in place of tryptophan residue 1282). This conclusion is based on the reappearance of cAMP-activated chloride currents, the restoration of CFTR protein at the apical plasma membrane, and an increase in the abundance of CFTR mRNA levels from the W1282X allele.

Effect of Readthrough Treatment in Fibroblasts of Patients Affected by Lysosomal Diseases Caused by Premature Termination Codons

“…Therefore, we analyzed the nucleotide context of the responsive patients and found that fibroblasts that were responsive to treatment carried PTC mutations in a slightly different nucleotide background than previously reported (Table 3). Despite the small sample size of our study, the results presented herein suggest a hierarchy of stop codon readthrough treatment efficacy similar to that reported [20]: UAG≥UGA>UAA. Also, in agreement with Floquet et al [24] we found the same response pattern in nucleotides -1 and +4.…”

“…In the last few years, other potential strategies such as pharmacological chaperones [8], or the suppression of pathogenic nonsense mutations through the induction of translational readthrough have emerged [2,[9][10][11][12][13][14][15][16][17][18]. To this effect, aminoglycoside antibiotics such as gentamicin [16,17], and small molecules such as PTC124 [18] and NB84 [19], have been described to induce PTC readthrough, eluding the NMD mechanism and allowing the formation of stable mRNAs encoding for full-length mutant, but probably still quite active, proteins [20,21]. These products reduce proofreading of codon-anticodon recognition in the ribosome, allowing the suppression of PTCs and, as a general rule, the insertion of glutamine or tryptophan at premature UAG/UAA or UGA codons, respectively, occurs [17].…”

Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment

“…Bedwell and co-workers 4,5 have also shown that G-418 and gentamicin can restore the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in a bronchial cell line with a nonsense mutation in the CFTR gene. Similarly, a Hurler syndrome fibroblast cell line heterozygous for a stop mutation showed a significant increase in alpha-L-iduronidase when cultured in the presence of gentamicin.…”

“…Similarly, a Hurler syndrome fibroblast cell line heterozygous for a stop mutation showed a significant increase in alpha-L-iduronidase when cultured in the presence of gentamicin. 4,5 The strength of this approach was first demonstrated in vivo by the work of Barton-Davis and co-workers, who demonstrated that gentamicin could partially restore expression of fulllength dystrophin in mdx mice with the X-linked muscular dystrophy mutation (Mdm mdx ), a C-T transition at position 3185, resulting in a termination codon replacing a glutamine codon in the dystrophin gene. 6 Negamycin, a dipeptide antibiotic with similar stop codon-misreading activity, has also been shown to restore dystrophin synthesis in the mdx mouse skeletal muscle.…”

Novel Animal Models of Pediatric Epilepsy

“…In vitro and in vivo studies have suggested that this strategy may be relevant for human diseases. Suppression of a nonsense mutation in the cystic fibrosis transmembrane conductance regulator gene by growth in gentamicin increased cystic fibrosis transmembrane conductance regulator expression by 10 to 20 % compared to control cells and restored cAMP-activated chloride transport [149,150]. In a mouse model of Duchenne muscular dystrophy, gentamicin was used to disrupt translational fidelity to phenotypically correct a nonsense mutation [151].…”