Quantitative methods for the analysis of CFTR transcripts/splicing variants

AbstractIn cystic fibrosis (CF), transcript analysis and quantification are important for diagnosis, prognosis and also as surrogate markers for some therapies including gene therapy. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods are evolving towards reliable quantification. Various protocols for the quantitative analysis of CFTR transcripts (including those resulting from splicing variants) are described and discussed here

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Atypical 5′ Splice Sites Cause CFTR Exon 9 To Be Vulnerable to Skipping

The molecular basis of the skipping of constitutive exons in many messenger RNAs is not fully understood. A well-studied example is exon 9 of the human cystic fibrosis transmembrane conductance regulator gene (CFTR), in which an abbreviated polypyrimidine tract between the branch point A and the 3′ splice site is associated with increased exon skipping and disease. However, many exons, both in CFTR and in other genes and have short polypyrimidine tracts in their 3′ splice sites, yet they are not skipped. Inspection of the 5′ splice sites immediately up- and downstream of exon 9 revealed deviations from consensus sequence, so we hypothesized that this exon may be inherently vulnerable to skipping. To test this idea, we constructed a CFTR minigene and replicated exon 9 skipping associated with the length of the polypyrimidine tract upstream of exon 9. We then mutated the flanking 5′ splice sites and determined the effect on exon skipping. Conversion of the upstream 5′ splice site to consensus by replacing a pyrimidine at position +3 with a purine resulted in increased exon skipping. In contrast, conversion of the downstream 5′ splice site to consensus by insertion of an adenine at position +4 resulted in a substantial reduction in exon 9 skipping, regardless of whether the upstream 5′ splice site was consensus or not. These results suggested that the native downstream 5′ splice site plays an important role in CFTR exon 9 skipping, a hypothesis that was supported by data from sheep and mouse genomes. Although CFTR exon 9 in sheep is preceded by a long polypyrimidine tract (Y(14)), it skips exon 9 in vivo and has a nonconsensus downstream 5′ splice site identical to that in humans. On the other hand, CFTR exon 9 in mice is preceded by a short polypyrimidine tract (Y(5)) but is not skipped in vivo. Its downstream 5′ splice site differs from that in humans by a 2-nt insertion, which, when introduced into the human CFTR minigene, abolished exon 9 skipping. Taken together, these observations place renewed emphasis on deviations at 5′ splice sites in nucleotides other than the invariant GT, particularly when such changes are found in conjunction with other altered splicing sequences, such as a shortened polypyrimidine tract. Thus, careful inspection of entire 5′ splice sites may identify constitutive exons that are vulnerable to skipping

Acquired pseudoxanthoma elasticum presenting after liver transplantation

Background: Pseudoxanthoma elasticum (PXE) is thought to be a metabolic disorder resulting from mutations in the gene encoding the cellular transporter, ABCC6, which is primarily expressed in liver and kidney. We encountered 3 patients who developed clinical and histopathological evidence of PXE after liver transplantation, suggesting that PXE could have been acquired from the transplanted organ. Objective: We sought to delineate the clinical features and screen each patient and samples of donor liver for mutations in the ABCC6 gene. Methods: Each patient underwent full clinical examination, skin biopsy, and ophthalmologic examination, and whole genome sequencing using standard techniques. Fixed samples of donor liver tissue were available for mutation analysis in two patients and of donor kidney tissue in one. Results: All 3 patients had unequivocal clinical and histopathologic evidence of PXE. No patient (or family member available for screening) had evidence of mutations in ABCC6. Neither liver specimen nor the single available kidney specimen showed evidence of mutations in ABCC6. Limitations: Liver tissue was not available from one patient and DNA was of poor quality in another, resulting in limited screening. Genetic testing does not detect ABCC6 mutations in 10% of patients with confirmed PXE. Conclusion: Although we were unable to demonstrate A.BCC6 mutations in limited screening of fixed donor livers, the absence of any PXE mutations in the affected patients, the timing of onset of PXE, and the known acquisition of other metabolic disorders and coagulopathies from donor livers suggest that PXE was likely acquired via liver transplantation