Identificazione e caratterizzazione di mutazioni in regioni regolatorie del gene malattia della fibrosi cistica

Abstract

Mutation epidemiology is crucial for cystic fibrosis (CF) diagnosis and counselling. ~6%-7% of alleles from CF patients do not bear mutations in the coding regions of the Cystic Fibrosis Transmembrane Regulator (CFTR) disease gene. In these patients, mutations may be present in non-coding, regulatory regions of the gene as i) intronic regions (particularly in high conserved sequences), ii) the promoter region or iii) the area at the 3’ of the gene, which is the target of microRNA regulation. We studied these regions by gene sequencing in a group of CF patients with one or both unidentified mutations after the analysis of CFTR coding regions, and in a group of CF patients with a different clinical expression of disease to evaluate if mutations in such regions may have a role in modulating CF clinical expression. Our analysis revealed: i) a dozen of mutations (most novel) in the large promoter area of 6000 bp at the 5’ of the gene; expression studies in four cell lines demonstrated that a half of such mutations may have a pathogenic effect; ii) a series of mutations in 52 conserved sequence tags (CSTs), i.e. intronic regions with a high homology between humans and mouse; expression studies revealed the pathogenic effect of one of these mutations; iii) finally, three mutations in the 1500 bp region at the 3’ of the gene; one of this has a pathogenic role, enhancing the interaction of CFTR with two inhibitory microRNAs. To the best of our knowledge, this is the first example of such pathogenic mechanism in a monogenic disorder. On the contrary, no mutations were identified in patients with different clinical expression in any of the three non-coding regions. To conclude, the sequencing of non coding regions may improve the detection rate of molecular analysis in CF, but functional studies are required to define the pathogenic effect of novel mutations.