Two CFTR mutations within codon 970 differently impact on the chloride channel functionality

Abstract

Pharmacological rescue of mutant cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis (CF) depends on the specific defect caused by different mutation classes. We asked whether a patient with the rare p.Gly970Asp (c.2909G>A) mutation could benefit from CFTR pharmacotherapy since a similar missense mutant p.Gly970Arg (c.2908G>C) was previously found to be sensitive to potentiators in vitro but not in vivo. By complementary DNA transfection, we found that both mutations are associated with defective CFTR function amenable to pharmacological treatment. However, analysis of messenger RNA (mRNA) from patient's cells revealed that c.2908G>C impairs RNA splicing whereas c.2909G>A does not perturb splicing and leads to the expected p.Gly970Asp mutation. In agreement with these results, nasal epithelial cells from the p.Gly970Asp patient showed significant improvement of CFTR function upon pharmacological treatment. Our results underline the importance of controlling the effect of CF mutation at the mRNA level to determine if the pharmacotherapy of CFTR basic defect is appropriate.

Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies

“…While CRISPR/Cas and other methods of gene editing can generate cell lines expressing CFTR variants in the native genomic context [ 64 , 65 ], EMGs provide a more rapid approach for assessing a large number of variants to identify those with unexpected consequences that warrant follow-up studies. Additionally, we show here and in prior work by ourselves and others that our results in EMGs are corroborated by studies in primary cells [ 20 , 35 – 38 , 44 , 46 , 50 , 66 ].…”

“…Additionally, c.3873G>A missplices through use of a noncanonical splice donor site that has a GC dinucleotide rather than a GT, which makes this outcome more difficult to predict. We have yet to identify an explanation for the false positive called only by CryptSplice, however our in vitro results recapitulate what others have reported in primary cells [ 46 ], giving additional evidence that the in silico prediction made by CryptSplice is incorrect. The variant which was incorrectly assessed by both algorithms (c.274G>A), resulted in only very low levels of missplicing which is likely why it was a false negative.…”

“…This was of particular concern for CFTR bearing the variant c.2908G>C (predicted effect G970R), which was shown to respond well to ivacaftor in vitro , whereas individuals with CF carrying this variant that were entered into a clinical trial did not respond [ 28 , 45 ]. Indeed, c.2908G>C was recently reported to alter CFTR mRNA splicing in primary cells, thus explaining the lack of response [ 46 ]. Therefore, study of these variants also provided an opportunity to test the ability of EMGs to replicate what has been observed in vivo , and extend splicing analysis to an additional variant in this codon.…”

The novel mutation c.1210-3C>G in cis with a poly-T tract of 5T affects CFTR mRNA splicing in a Chinese patient with cystic fibrosis

“…By Sanger sequencing, we identified a low-frequency variant p.Gly970Asp (c.2909G>A, p.G970D), and a novel variant c.1210-3C>G in this patient. p.Gly970Asp is located within the third cytoplasmic loop of the CFTR protein, and its impairment of chloride conductance may lead to a potentiator-sensitive gating defect as well as a partial trafficking defect without RNA splicing alteration [7,14]. In Amato's report, p.Gly970Asp mutant plasmidtransfected HEK293 cells showed very low channel activity, demonstrating the mutation's pathogenicity [14].…”

“…p.Gly970Asp is located within the third cytoplasmic loop of the CFTR protein, and its impairment of chloride conductance may lead to a potentiator-sensitive gating defect as well as a partial trafficking defect without RNA splicing alteration [7,14]. In Amato's report, p.Gly970Asp mutant plasmidtransfected HEK293 cells showed very low channel activity, demonstrating the mutation's pathogenicity [14]. In addition, with an allele frequency of 9.8%, p.Gly970Asp is the most commonly seen hotspot mutation in CF patients of Chinese origin [9].…”