Studio in vitro dell'espressione di varianti del gene CFTR con ruolo patofisiologico non ancora definito

The mutational screening of the CFTR gene, performed with three progressive steps (screening of the most common mutations with a commercial diagnostic kit, analysis of deletions/duplications with MLPA assay and sequencing of the coding CFTR regions) leads to a detection rate of about 94%. The analysis of the CFTR transcripts, by RNA extraction from nasal epithelial cells, allows to identify new mutations and/or to define the pathogenic role of variants initially considered as polymorphisms. In this PhD study, three different mutations, identified in patients followed at the CF Center of Regione Lombardia, were characterized: i) the c.2679 G>T mutation, initially described as a polymorphism, causes the creation of a new donor splice site, leading to the skipping of 230 bp of exon 17 in CFTR transcript; ii) the duplication spanning from exon 7 to exon 18, identified by MLPA assay was confirmed and characterized on CFTR transcript. This rearrangement causes a 2244-bp-out-of-frame insertion, leading to the creation of a premature stop codon located 8 amino acids after the beginning of the duplicated exon 7; iii) the c.1584+18672 A>G deep-intronic mutation, identified in five different patients, was characterized by a combination of in-vitro (hybrid minigene constructs) and in-vivo (RTPCRs performed on nasal brushing RNA) approaches. These studies showed that this mutation creates a new donor splice site and activates two cryptic acceptor splice sites, causing the production of two CFTR transcript variants. The two alternative splicing products include a 104-bp pseudoexon and a 65-bp pseudoexon located between exon 11 and exon 12. Allele-specific measurement of wild-type and aberrant splicings from the nasal brushing RNA of the probands showed that the c.1584+18672 A>G mutation did not completely abolish the wild-type splicing in vivo. In particular, about 3,5% of the mRNA molecules produced by the c.1584+18672 A>G allele were completely wild-type; these data well correlate with the mild clinical phenotype of patients. In the second part of the PhD project, two pilot studies, aiming at the improvement of the molecular analysis of CFTR gene, have been developed: i) analysis of the CFTR transcript by RNA extraction from lymphocytes and lymphoblastoid cell lines, to evaluate the possibility to use a target tissue alternative to the nasal epithelium, not available in pediatric patients (age < 9 years) and in patients refusing the nasal brushing. The data obtained, however, suggest that the very low level of CFTR expression in lymphocytes does not allow a correct analysis of transcript profiles. ii) Development of a custom new generation sequencing (NGS) kit, using the platform HiSeq 2000 (Illumina), in order to scan the entire CFTR gene. After a careful evaluation of the possible alternatives, we chose a target enrichment strategy and designed a set of probes covering the full-length CFTR to be used for DNA capture (custom capture kit, Nimblegen SeqCap EZ Library, Roche). The application of NGS will lead to the improvement of time and cost of molecular diagnosis, that is nowadays increasingly relevant thanks to the recent advances obtained in mutation-specific therapie