Intragenic modulators of CFTR gene expression involved in cystic fibrosis clinical variability

Alterations in Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene cause Cystic Fibrosis (CF), a disease whose complications lead to death due to respiratory failure. CFTR produces a primary transcript of 6132 bases, with a complex transcriptional regulation that depends on the entire locus of the gene. The correlation between genotype and phenotype in this pathology is still difficult to understand. We evaluated the possible mechanisms involved in the intragenic modulation of CFTR expression in CF and CF-like populations, already extensively characterized at the mutational level. Our study is based on three experimental approaches, applied to the CFTR gene of these populations: analysis of intragenic haplotypes; analysis of variant tracts (TG)mTn and effects on mRNA splicing; analysis of 3'-UTR for potential miRNAs recognition sites. We performed the structural analysis on DNA (by primer extension and Sanger sequencing) and the functional analysis on RNA (by reverse transcriptase PCR, densitometric analysis and Real-time PCR). Among the haplotypes characterized, one showed a significantly higher frequency in the populations of interest (CF and CF-like) compared to control populations (general population and normospermic men), with a trend that varies according to the severity of the phenotypic manifestations. It has been highlighted that no anomalous splicing is produced by the presence of the individual sequence variations of the haplotype. Future studies will evaluate whether the simultaneous presence in cis of all the variations that compose the haplotype may influence the levels of CFTR mRNA. Some variant tracts (TG)mTn resulted more frequent in CF-like populations than in classical CF or in controls. Functional analysis at RNA level evidenced a higher percentage of anomalous splicing produced by some specific tracts. This correlates with the clinical phenotype of these patients. We evidenced some variants within the CFTR 3’-UTR. We did not highlight 3’-UTR sequence variations that could influence the binding of miRNAs currently known in the literature. Future studies will evaluate what effect the variations we found in this area can have.