Deletion of CFTR Translation Start Site Reveals Functional Isoforms of the Protein in CF Patients

Abstract

Background/Aims: Mutations in the CFTR gene cause Cystic Fibrosis (CF) the most common life-threatening autosomal recessive disease affecting Caucasians. We identified a CFTR mutation (c.120del23) abolishing the normal translation initiation codon, which occurs in two Portuguese CF patients. This study aims at functionally characterizing the effect of this novel mutation. Methods: RNA and protein techniques were applied to both native tissues from CF patients and recombinant cells expressing CFTR constructs to determine whether c.120del23 allows CFTR protein production through usage of alternative internal codons, and to characterize the putative truncated CFTR form(s). Results: Our data show that two shorter forms of CFTR protein are produced when the initiation translation codon is deleted indicating usage of internal initiation codons. The N-truncated CFTR generated by this mutation has decreased stability, very low processing efficiency, and drastically reduced function. Analysis of mutants of four methionine codons downstream to M1 (M82, M150, M152, M156) revealed that each of the codons M150/M152/M156 (exon 4) can mediate CFTR alternative translation. Conclusions: The CFTR N-terminus has an important role in avoiding CFTR turnover and in rendering effective its plasma membrane traffic. These data correlate well with the severe clinical phenotype of CF patients bearing the c.120del23 mutation.

The effect of premature termination codon mutations on CFTR mRNA abundance in human nasal epithelium and intestinal organoids: a basis for read‐through therapies in cystic fibrosis

“…However, among the tissues available for testing, none had mutations occurring in the penultimate exon. The only mutations giving any indication that they might escape NMD to some degree were S4X, dele2,3_21 kb, and possibly E60X, each of which could be associated with the use of an alternative initiation codon, such as met150 (Ramalho et al., ).…”

Does extensive genotyping and nasal potential difference testing clarify the diagnosis of cystic fibrosis among patients with single-organ manifestations of cystic fibrosis?

“…The role of in silico tools to predict the functional consequences of rare CFTR mutations has also been evaluated 30 31. While in silico tools may provide insight into the potential pathogenicity of rare mutations, they were shown to predict the clinical severity of missense mutations with known clinical consequences poorly, raising considerable doubt over their diagnostic role in mutations with variable or unknown clinical consequences 32…”

Multiple Mechanisms Influence Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Gene Promoter

“…In contrast, both pSP73CFTR2-24 and pSP7321a/2-24 produced two major species that migrated at a faster mobility (Figure 2A.). We demonstrated by mutagenesis that the proteins produced from pSP73CFTR2-24 arise from the use of ATGs in exon 4 (at aas M150, M152 or M156) ( Figure E1) (32). Hence, based on their migration on SDS/ PAGE, inclusion of exon 21a spliced to exon 2 apparently generates proteins that also initiate translation at these methionines in exon 4.…”

“…The pSP73CFTR2-24 construct produced two major products resulting from initiation at in-frame downstream AUGs in exon 4 (M150, M152, and M156 [ Figure E1] and Ref. 32), and the inclusion of the upstream alternative 21a/1a exons abolished the production of these proteins in vitro. However, this might have been due to failure of translation alone or of transcription, causing lack of production of a stable mRNA.…”