Transcript analysis of CFTR nonsense mutations in lymphocytes and nasal epithelial cells from cystic fibrosis patients

Abstract

The mutational effects at the mRNA level were investigated by RT-PCR analysis of nine different nonsense mutations (Q39X, E60X, R75X, G542X, L719X, Y1092X, R1162X, S1196X, W1282X) and one frameshift mutation (1078delT) within the CFTR gene. With the exception of mutation R1162X, reduced mRNA levels ranging from 30% to less than 5% of the wild type have been observed. In case of the R75X and E60X mutations, the mRNA reduction was accompanied by the appearance of atypical CFTR isoforms. Single exon 3 skipping, as well as joint exon 2 and 3 skipping, was observed in lymphocyte and nasal epithelial mRNA derived from R75X alleles. The analysis of mRNA transcribed from E60X alleles revealed skipping of exon 3 (lymphocytes and nasal epithelial cells) or skipping of exons 3 and 4 (nasal epithelial cells). With the exception of the E60X mutation, no obvious tissue-specific differences in the splicing pattern and ratios of mutation to wild-type transcripts were detected between lymphocytes and nasal epithelial cells. In addition to aberrant splicing, the reduction of transcripts is the most common effect of nonsense and frameshift mutations within the CFTR gene.

Assessing the Disease-Liability of Mutations in CFTR

“…PTC mutations generally cause the loss of messenger RNA because of nonsense-mediated RNA decay (NMRD) (Frischmeyer and Dietz 1999). Mutations in CFTR including G542X, R553X ( p.Arg553X), and W1282X have been shown to lead to NMRD in primary airway cells (Hamosh et al 1991(Hamosh et al , 1992bWill et al 1995). There are a few exceptions to the concept that PTC mutations cause NMRD.…”

“…There are a few exceptions to the concept that PTC mutations cause NMRD. A notable example is the R1162X ( p.Arg1162X) mutation in which the transcript containing a PTC is stable (Rolfini and Cabrini 1993;Will et al 1995). Patients homozygous for R1162X showed severe pancreatic exocrine deficiency and variable lung disease severity that overlapped with measures observed in F508del homozygotes (Gasparini et al 1992).…”

Applicability of Different Antibodies for Immunohistochemical Localization of CFTR in Sweat Glands from Healthy Controls and from Patients with Cystic Fibrosis

“…Although overreading of stop codons has been reported to occur in mammalian cells (McCaughan et al 1995), ample evidence exists that the nonsense mutations R553X and G542X of the CFTR gene result in reduced to undetectable levels of mRNA transcripts (Hamosh et al 1992;Will et al 1995) and absence of full-length protein (Howard et al 1996). With respect to the W1282X mutation, conflicting data are reported in the literature.…”

“…With respect to the W1282X mutation, conflicting data are reported in the literature. mRNA levels were found to be severely decreased by Will et al (1995) and Hamosh et al (1992) but within normal range by Shoshani et al (1994). However, expression of full-length protein has never been demonstrated under normal conditions in this stop mutation (Bedwell et al 1997).…”

Detection of a large heterozygous deletion and a splicing defect in the CFTR transcripts from nasal swab of a Japanese case of cystic fibrosis

“…This method has been used to detect aberrantly spliced transcripts and reduced mRNA levels that were associated with various types of nonsense, frameshift, stop and splicing mutations. [16][17][18][19] In fact, we detected the CFTR transcripts with or without exon 9 in samples from both the patient and healthy control (Figure 1). The probability of alternative splicing of exon 9 is related to the genotype of poly T and TG repeats in intron 8.…”