The CFTR frameshift mutation 3905insT and its effect at transcript and protein level

Abstract

Cystic fibrosis (CF) is one of the most common genetic diseases in the Caucasian population and is characterized by chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, and elevation of sodium and chloride concentrations in the sweat and infertility in men. The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a protein that functions as chloride channel at the apical membrane of different epithelia. Owing to the high genotypic and phenotypic disease heterogeneity, effects and consequences of the majority of the CFTR mutations have not yet been studied. Recently, the frameshift mutation 3905insT was identified as the second most frequent mutation in the Swiss population and found to be associated with a severe phenotype. The frameshift mutation produces a premature termination codon (PTC) in exon 20, and transcripts bearing this PTC are potential targets for degradation through nonsense-mediated mRNA decay (NMD) and/or for exon skipping through nonsense-associated alternative splicing (NAS). Using RT-PCR analysis in lymphocytes and different tissue types from patients carrying the mutation, we showed that the PTC introduced by the mutation does neither elicit a degradation of the mRNA through NMD nor an alternative splicing through NAS. Moreover, immunocytochemical analysis in nasal epithelial cells revealed a significantly reduced amount of CFTR at the apical membrane providing a possible molecular explanation for the more severe phenotype observed in F508del/3905insT compound heterozygotes compared with F508del homozygotes. However, further experiments are needed to elucidate the fate of the 3905insT CFTR in the cell after its biosynthesis.

Long-term gas exchange characteristics as markers of deterioration in patients with cystic fibrosis

“…The different progression between F508del/frameshift and non- F508del/frameshift is especially striking. Based on recent findings [ 48 ] showing complete lack of CFTR protein at the apical membrane in F508del/3905insT compound heterozygous patients, we hypothesize that there may be an interaction between the plasma membrane resulting in a more severe phenotype. However, further experiments are needed to elucidate the fate of the 3905insT protein in the cell after its biosynthesis.…”

Phenotypic Characterization and Comparison of Cystic Fibrosis Rat Models Generated Using CRISPR/Cas9 Gene Editing

“…The cytoplasmic presence of CFTR in KO airways suggests that some transcripts escape nonsense-mediated decay, a phenomenon that is present in human CFTR mutations, such as 3905insT. 46 As in Phe508del rats, KO CFTR was also colocalized with the lysosome, indicating degradation of the truncated protein. The lack of critical functional domains of the CFTR protein, in conjunction with the nasal potential difference profile indicating loss of CFTR function, suggests that although the CFTR protein is present, the function is lost, leading to generation of a more severe CF phenotype.…”

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

“…A significant proportion of the ∼2000 potentially disease causing CFTR variants identified to date are Class I mutations, i.e., those introducing an in‐frame premature termination codon (PTC) into the CFTR messenger RNA (CFTR1 mutation database: http://www.genet.sickkids.on.ca). However, besides these in‐frame nonsense mutations (8% of the total in CF) PTCs can also be introduced by frameshift or splicing mutations (Oren, Pranke, Kerem, & Sermet‐Gaudelus, ; Sanz et al., ), representing approximately 2% and 12% of CFTR mutations, respectively. Translation of an mRNA transcript bearing a PTC will produce a truncated and most likely nonfunctional protein, but this is largely avoided by an mRNA surveillance mechanism termed nonsense mediated decay (NMD, Frischmeyer & Dietz, ).…”