CFTR was detected using either an antibody directed against the C-terminal or the R region … Table 1 Proportion of cells with apically localized CFTR Discussion The frameshift mutation 3905insT (c. 3773_3774insT) is one of the most common CFTR mutations in the Swiss population8 and has been associated with a severe phenotype.9, 10, 11, 12 The mutation is characterized by the introduction Pacritinib clinical trial of a PTC in exon 20 of the CFTR gene. PTCs can be recognized by the so-called NMD, which degrades transcripts bearing such PTCs thereby preventing the formation of a truncated protein.14 It has also been proposed that PTCs can be targeted by another mechanism termed NAS, in which the exon containing the PTC is excluded from the mRNA through alternative splicing reducing the levels of full-length CFTR transcripts.

22 Performing a combination of real-time RT�CPCR and fragment analysis, we could show that the 3905insT mRNA is insensitive to complete degradation by NMD (Figure 1c). Three out of four patients showed transcript levels of around 50%, whereas P4 presented with levels of 26% raising awareness of additional complex mechanisms such as individual and tissue-specific expression and/or stability of mRNAs.23 The lack of degradation by NMD was further confirmed by RT�CPCR amplification, in which we were able to detect 3905insT cDNA from skin and colon samples of a 3905insT homozygous patient (Figure 2c). Moreover, we present evidence that 3905insT transcripts are not subject to alternative splicing by NAS in different tissues of 3905insT carriers (Figure 2).

To our knowledge, this is the first CFTR frameshift mutation that has extensively been analyzed for the influence of NMD, and additionally of NAS. Furthermore, it is also the first report of a CFTR frameshift mutation that constitutes an exception to the ��50�C55 boundary rule’, which states that NMD is triggered whenever a PTC is located >50�C55 nucleotides upstream of the last EEJ.15, 16 There is a growing number of mutations that have been reported to be insensitive to NMD24, 25, 26, 27, 28, 29 supporting a novel model in which the physical distance between the PTC and the poly(A) tail is at least as crucial as the distance between the PTC and the last EEJ.30 This model might explain the resistance of 3905insT mRNA to NMD, particularly when considering the relative proximity of the emerging PTC to the poly(A) tail.

Aberrant proteins produced in the endoplasmatic reticulum (ER) are recognized and then destroyed by a process termed as ER-associated protein degradation (ERAD).31 It is well established that F508del CFTR is degraded because of a misfolding of the protein that results in an almost complete lack of protein at the plasma membrane. In spite of the degradation, some F508del protein Dacomitinib can reach the apical membrane and function as a chloride channel.