Highlights
- •Exonic variants may have various molecular mechanisms of pathogenicity.
- •The impact on splicing of exonic sequence variations should be systemically assessed.
- •Splicing default of exonic variants may hamper the efficiency of targeted pharmacotherapy.
- •Functional in vitro experiments are key tools to classify CFTR rare variants in order to offer personalized therapies.
Abstract
Background
The majority of variants of unknown clinical significance (VUCS) in the CFTR gene
are missense variants. While change on the CFTR protein structure or function is often
suspected, impact on splicing may be neglected. Such undetected splicing default of
variants may complicate the interpretation of genetic analyses and the use of an appropriate
pharmacotherapy.
Methods
We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions
on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and
maturation (western blot), cellular localization (immunofluorescence) and channel
function (patch clamp) of the CFTR protein. We also studied maturation and function
of the truncated protein, consecutive to in-frame aberrant splicing, on additional
plasmid constructs.
Results
Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing
a splicing defect and the impairment of CFTR folding and/or function related to the
amino acid substitution; deleterious missense variants that impair CFTR folding and/or
function; and variants with no consequence on the different processes tested.
Conclusion
The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of
the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the
results of laboratory experiments.
Graphical abstract
Graphical Abstract
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Article info
Publication history
Published online: December 23, 2022
Accepted:
December 5,
2022
Received in revised form:
September 30,
2022
Received:
April 20,
2022
Identification
Copyright
© 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
