In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study

Abstract

Background: Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which acts as a chloride channel activated by cyclic AMP (cAMP). The most frequent mutation found in 70% of CF patients is F508del, while premature stop mutations are found in about 10% of patients. In vitro aminoglycoside antibiotics (e.g. gentamicin) suppress nonsense mutations located in CFTR permitting translation to continue to the natural termination codon. Pharmacologic suppression of stop mutations within the CFTR may be of benefit to a significant number of patients. Our pilot study was conducted to determine whether intravenous gentamicin suppresses stop codons in CF patients and whether it has clinical benefits.

Methods: A dual gene reporter system was used to determine the gentamicin-induced readthrough level of the most frequent stop mutations within the CFTR in the French population. We investigated readthrough efficiency in response to 10 mg/kg once-daily intravenous gentamicin perfusions in patients with and without stop mutations. Respiratory function, sweat chloride concentration, nasal potential difference (NPD) and CFTR expression in nasal epithelial cells were measured at baseline and after 15 days of treatment.

Results: After in vitro gentamicin incubation, the readthrough efficiency for the Y122X mutation was at least five times higher than that for G542X, R1162X, and W1282X. In six of the nine patients with the Y122X mutation, CFTR immunodetection showed protein at the membrane of the nasal epithelial cells and the CFTR-dependent Cl- secretion in NPD measurements increased significantly. Respiratory status also improved in these patients, irrespective of the gentamicin sensitivity of the bacteria present in the sputum. Mean sweat chloride concentration decreased significantly and normalised in two patients. Clinical status, NPD and sweat Cl- values did not change in the Y122X patients with no protein expression, in patients with the other stop mutations investigated in vitro and those without stop mutations.

Conclusion: Suppression of stop mutations in the CFTR gene with parenteral gentamicin can be predicted in vitro and is associated with clinical benefit and significant modification of the CFTR-mediated Cl- transport in nasal and sweat gland epithelium.

Figure 1

Sweat chloride concentration and ΔCl^- free-isoproterenol before and after gentamicin in Y122X patients (●). Previously published representative data of healthy controls (○) are shown [27].

Figure 2

Example of nasal potential difference tracing (NPD) (a, b) and CFTR immunostaining with MATG 1061 monoclonal antibody of nasal ciliated cells (c,d) before (a,c) and after (b,d) parenteral gentamicin treatment in a Y122X homozygous CF patient. In (d), insert of nasal epithelial cells from a healthy control subject. Scale bars are 40, 20 and 60 microns for (c), (d) and insert, respectively. (a) NPD before treatment showed a basal NPD of -50 mV, strong depolarisation in response to 100 μM amiloride (25 mV), and no significant response to CFTR activation by 10 μM isoproterenol in chloride-free solution. (b) At the end of treatment, basal PD was close to -45 mV, amiloride-induced depolarisation had not changed, and addition of isoproterenol in chloride-free solution induced hyperpolarisation of ~ -5 mV, indicating a CFTR-dependent chloride transport. (c) No CFTR labeling was observed before treatment. (d) After gentamicin treatment, CFTR labeling was detected in the patient's nasal cells.