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నైరూప్య

Multi-Functional Consequences of a Single Mutation of CFTR of the Importance to Analyze Mutations by Functional Tests

Ladeveze V, Farhat R, El Seedy A and Kitzis A

Cystic Fibrosis is the most frequent rare disease in the Caucasian population. This severe hereditary recessive disease is caused by mutations in the Cystic Fibrosis transmembrane conductance regulator (CFTR) gene. This gene encodes for a protein expressed at the apical membrane of epithelial cells. Mutations are classified in six classes based on their effects and phenotype severity. The F508delmutation is the most frequent mutation of the CFTR gene; it induces a misfolding of the protein thus blocking its maturation, membrane localization and in the end, its functionality.

This study combines a clinical approach and multiple level cellular analyses to determine the physiopathological consequences of the c.1392G>T (p.Lys464Asn) CFTR exon 10 mutation, detected in a CF patient with a frame shift deletion in trans and a TG(11)T(5) in cis. First, the splicing was studied by minigene experiments, with different TG(m)T(n) alleles, and by nasal cell mRNA extracts to determine the consequence of c.1392G>T. Then, the processing of p.Lys464Asn protein was evaluated, in cellulo, by western blotting analyses.

The c.1392G>T mutation affects exon 10 splicing by inducing its complete deletion and encoding a frame shift transcript. The polymorphism TG (11)T(5) increases the effects of this mutation on aberrant splicing, suggesting the importance of complex allele. Analysis of mRNA obtained from maternal airway epithelial cells confirmed these in cellulo results. At the protein level the p.Lys464Asn protein showed no fully glycosylated form.

Thus, the c.1392G>T mutation alone or in association with the poly T tract revealed obvious impacts on splicing and CFTR protein processing. The c.[T(5); 1392G>T] complex allele contributes to the CF phenotype by affecting splicing and inducing a severe misprocessing defect. These results demonstrate that the classical CFTR mutations classification is not sufficient: in vivo and in cellulo studies of a possible complex allele in a patient are required to provide correct CFTR mutation classification, adequate medical counseling, and adapted therapeutic strategies.