(610) Evaluating the mechanism of amino acid insertion upon readthrough of CFTR nonsense mutations

Background: 10% of cystic fibrosis (CF) patients carry a nonsense mutation on at least one cystic fibrosis transmembrane conductance regulator (CFTR) allele. A nonsense mutation is a point mutation in the DNA that generates a premature termination codon (PTC) in the transcribed messenger RNA (mRNA). Suppressing termination at PTCs, also called readthrough (RT), is a therapeutic approach being explored for these patients. RT occurs when a near-cognate amino acid is incorporated into the polypeptide at the site of the PTC, allowing translation to continue and a full-length protein to be generated. However, little is known about the identities of near-cognate amino acids incorporated upon RT, how amino acid insertion is affected by the native CFTR mRNA sequence, or if amino acid insertion differs with different chemical classes of RT compounds

Methods: To address this issue, we have created a reporter system where we can express CFTR PTCs in their native mRNA context in human (HEK293) cells. We then induced RT and purified the corresponding full-length protein produced. Finally, we identified the amino acid(s) inserted at the site of the PTC via tandem mass spectroscopy.

Results: We identified the amino acid(s) inserted upon G418-mediated RT in human cells for a total of nine different CFTR PTCs. These nonsense mutations include each of the three stop codon identities (UGA, UAG, and UAA) and represent some of the most common CF-causing nonsense mutations. For the UGA PTCs, R553X mirrored the previously reported G542X with the addition of cysteine (C), tryptophan (W), and arginine (R). R1162X, however, had very little R present at the site of the PTC, inserting C or W 99% of the time. At the G550X UGA, only W was detected at the PTC. With the UAG CFTR PTCs, the differences were even more pronounced. At Q493X, glutamine (Q) and tyrosine (Y) were identified at the PTC, whereas at E60X, Q and Y were present, but the novel serine (S) was the predominant amino acid. Similarities were found at the two UAA CFTR PTCs, Y122X and Y1092X, where Q, Y, and lysine (K) were seen.

Conclusions: These data illustrate that the native local CFTR mRNA context greatly impacts the identity and frequency of amino acids incorporated at PTCs upon RT. In most cases, the CFTR produced upon RT is a variant protein like what would be created because of a missense mutation. The identity of the amino acids inserted at the site of the CFTR PTC would then impact the full-length protein’s overall processing, activity, and responsiveness to modulation. We are currently testing whether other classes of RT compounds, with mechanisms disparate from G418, can promote differences in amino acid insertion at CFTR PTCs. By understanding this mechanism and how we can influence it, we may be able to determine how we can maximize CFTR function for CF patients with nonsense mutations.

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