- •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.
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.
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.
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.
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.
To read this article in full you will need to make a payment
Purchase one-time access:Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
One-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:Subscribe to Journal of Cystic Fibrosis
Already a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
- Performance evaluation of pathogenicity-computation methods for missense variants.Nucleic Acids Res. 2018; 46: 7793-7804https://doi.org/10.1093/nar/gky678
- Splicing in action: assessing disease causing sequence changes.J Med Genet. 2005; 42: 737-748https://doi.org/10.1136/jmg.2004.029538
- Exon identity influences splicing induced by exonic variants and in silico prediction efficacy.J Cyst Fibros. 2021; 20: 464-472https://doi.org/10.1016/j.jcf.2020.12.003
- Recommendations for the classification of diseases as CFTR-related disorders.J Cyst Fibros. 2011; 10: S86-102https://doi.org/10.1016/S1569-1993(11)60014-3
- Diagnosis of cystic fibrosis: consensus guidelines from the cystic fibrosis foundation.J Pediatr. 2017; 181 (e1): S4-S15https://doi.org/10.1016/j.jpeds.2016.09.064
- CFTR2: how will it help care?.Paediatr Respir Rev. 2013; 14: 2-5https://doi.org/10.1016/j.prrv.2013.01.006
- CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants.Hum Mutat. 2017; 38: 1297-1315https://doi.org/10.1002/humu.23276
- A new era in the treatment of cystic fibrosis: correction of the underlying CFTR defect.Lancet Respir Med. 2013; 1: 158-163https://doi.org/10.1016/S2213-2600(12)70057-7
- Transcriptomic and proteostasis networks of CFTR and the development of small molecule modulators for the treatment of cystic fibrosis lung disease.Genes (Basel). 2020; 11: E546https://doi.org/10.3390/genes11050546
- Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.J Cyst Fibros. 2008; 7: 179-196https://doi.org/10.1016/j.jcf.2008.03.009
- Human Splicing Finder: an online bioinformatics tool to predict splicing signals.Nucleic Acids Res. 2009; 37: e67https://doi.org/10.1093/nar/gkp215
- Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals.J Comput Biol. 2004; 11: 377-394https://doi.org/10.1089/1066527041410418
- A generalized hidden Markov model for the recognition of human genes in DNA.Proc Int Conf Intell Syst Mol Biol. 1996; 4: 134-142
- A classification model relative to splicing for variants of unknown clinical significance: application to the CFTR gene.Hum Mutat. 2013; 34: 774-784https://doi.org/10.1002/humu.22291
- Extensive functional analyses of RHD splice site variants: insights into the potential role of splicing in the physiology of Rh.Transfusion. 2015; 55: 1432-1443https://doi.org/10.1111/trf.13083
- Misprocessing of the CFTR protein leads to mild cystic fibrosis phenotype: misprocessing of cftr and mild phenotype.Hum Mutat. 2005; 25: 360-371https://doi.org/10.1002/humu.20156
- Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method.Methods. 2001; 25: 402-408https://doi.org/10.1006/meth.2001.1262
- Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis.Eur Respir J. 2015; 45: 116-128https://doi.org/10.1183/09031936.00113214
- Phosphorylation of the Chaperone-Like HspB5 Rescues Trafficking and Function of F508del-CFTR.IJMS. 2020; 21: 4844https://doi.org/10.3390/ijms21144844
- Development of automated patch clamp technique to investigate CFTR chloride channel function.Front Pharmacol. 2017; 8https://doi.org/10.3389/fphar.2017.00195
- The CYSMA web server: an example of integrative tool for in silico analysis of missense variants identified in Mendelian disorders.Hum Mutat. 2020; 41: 375-386https://doi.org/10.1002/humu.23941
- Ivacaftor potentiation of multiple CFTR channels with gating mutations.Journal of Cystic Fibrosis. 2012; 11: 237-245https://doi.org/10.1016/j.jcf.2011.12.005
- Two CFTR mutations within codon 970 differently impact on the chloride channel functionality.Hum Mutat. 2019; 40: 742-748https://doi.org/10.1002/humu.23741
- A novel exon in the cystic fibrosis transmembrane conductance regulator gene activated by the nonsense mutation E92X in airway epithelial cells of patients with cystic fibrosis.J Clin Invest. 1994; 93: 1852-1859https://doi.org/10.1172/JCI117172
- Spectrum of CFTR mutations in Chechen cystic fibrosis patients: high frequency of c.1545_1546delTA (p.Tyr515X; 1677delTA) and c.274G>A (p.Glu92Lys, E92K) mutations in North Caucasus.BMC Med Genet. 2019; 20: 44https://doi.org/10.1186/s12881-019-0785-z
- Phenotyping of rare CFTR mutations reveals distinct trafficking and functional defects.Cells. 2020; 9: 754https://doi.org/10.3390/cells9030754
- Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.PLoS Genet. 2014; 10e1004376https://doi.org/10.1371/journal.pgen.1004376
- Regulation of CFTR bicarbonate channel activity by WNK1: implications for pancreatitis and CFTR-Related disorders.Cell Mol Gastroenterol Hepatol. 2020; 9: 79-103https://doi.org/10.1016/j.jcmgh.2019.09.003
- p.Ser1235Arg should no longer be considered as a cystic fibrosis mutation: results from a large collaborative study.Eur J Hum Genet. 2011; 19: 36-42https://doi.org/10.1038/ejhg.2010.137
- Localization studies of rare missense mutations in cystic fibrosis transmembrane conductance regulator (CFTR) facilitate interpretation of genotype-phenotype relationships.Hum Mutat. 2008; 29: 1364-1372https://doi.org/10.1002/humu.20866
- Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies.PLoS Genet. 2020; 16e1009100https://doi.org/10.1371/journal.pgen.1009100
- G970R-CFTR mutation (c.2908G>C) results predominantly in a splicing defect.Clin Transl Sci. 2021; 14: 656-663https://doi.org/10.1111/cts.12927
- Caution advised in the use of CFTR modulator treatment for individuals harboring specific CFTR variants.J Cyst Fibros. 2022; (S1569-1993(22)00108-4)https://doi.org/10.1016/j.jcf.2022.04.019
Published online: December 23, 2022
Accepted: December 5, 2022
Received in revised form: September 30, 2022
Received: April 20, 2022
© 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.