Comparative ex vivo, in vitro and in silico analyses of a CFTR splicing mutation: Importance of functional studies to establish disease liability of mutations.
The Cystic Fibrosis p.Ile1234Val missense mutation actually creates a new dual splicing site possibly used either as a new acceptor or donor. Here, we aimed to test the accuracy of in silico predictions by comparing them with in vitro and ex vivo functional analyses of this mutation for an accurate CF diagnosis/prognosis. To this end, we applied a new in vitro strategy using a CFTR mini-gene which includes the complete CFTR coding sequence plus intron 22 (short version) which allows the assessment of alternatively spliced mRNA levels as well as the properties of the resulting abnormal CFTR protein regarding processing, intracellular localization and function. Our data demonstrate that p.Ile1234Val leads to usage of the alternative splicing donor (but not acceptor) resulting in alternative CFTR transcripts lacking 18 nts of exon 22 which produce a truncated CFTR protein with residual Cl- channel function. These results recapitulate data from native tissues of a CF patient. In conclusion, the existing in silico prediction models have limited application and ex vivo functional assessment of mutation effects should be made. Alternatively the in vitro strategy adopted here can be applied to assess the disease liability of mutations for an accurate CF diagnosis/prognosis.
University of Lisboa, Faculty of Sciences, BioISI - Biosystems and Integrative Sciences Institute, Lisboa, Portugal.
Anabela S
AS
Ramalho
University of Lisboa, Faculty of Sciences, BioISI - Biosystems and Integrative Sciences Institute, Lisboa, Portugal.
Luka A
LA
Clarke
University of Lisboa, Faculty of Sciences, BioISI - Biosystems and Integrative Sciences Institute, Lisboa, Portugal.
Marisa
M
Sousa
University of Lisboa, Faculty of Sciences, BioISI - Biosystems and Integrative Sciences Institute, Lisboa, Portugal.
Verónica
V
Felicio
Hospital de Santa Maria, Lisboa, Portugal.
Celeste
C
Barreto
Hospital de Santa Maria, Lisboa, Portugal.
Carlos
C
Lopes
University of Lisboa, Faculty of Sciences, BioISI - Biosystems and Integrative Sciences Institute, Lisboa, Portugal. Electronic address: mdamaral@fc.ul.pt.
Margarida D
MD
Amaral
D000328:Adult; D017398:Alternative Splicing; D003198:Computer Simulation; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D009154:Mutation; D012326:RNA Splicing; D015203:Reproducibility of Results
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
Alternative splicing; CFTR; Cystic fibrosis; In silico predictions; Splicing mutation