Gene mutation in microRNA target sites of CFTR gene: a novel pathogenetic mechanism in cystic fibrosis?

Abstract

Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on alterations of a chloride channel expressed by most epithelial cells and encoded by CFTR gene. Also using scanning techniques to analyze the whole coding regions of CFTR gene, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTR-related disorders (CFTR-RD). Other gene regions may be the site of causing-disease mutations. We searched for genetic variants in the 1500 bp of CFTR 3' untranslated region, typical target of microRNA (miRNA) posttranscriptional gene regulation, in either CF patients with the F508del homozygous genotype and different clinical expression (n = 20), CF (n = 32) and CFTR-RD (n = 43) patients with one or none mutation after CFTR scanning and in controls (n = 50). We identified three SNPs, one of which, the c.*1043A>C, was located in a region predicted to bind miR-433 and miR-509-3p. Such mutation was peculiar of a CFTR-RD patient that had Congenital Bilateral Absence of Vas Deferens (CBAVD), diffuse bronchiectasis, a borderline sweat chloride test and the heterozygous severe F508del mutation on the other allele. The expression analysis demonstrated that the c.*1043A>C increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Both miRNAs cause in vitro a reduced expression of CFTR protein. Thus, the c.*1043A>C may act as a mild CFTR mutation enhancing the affinity for inhibitory miRNAs as a novel pathogenetic mechanism in CF.

Figure 1. Bioinformatics prediction of c*1043A>C mutation position and effect on miRNA affinity.

A–B) schematic diagram of the two in silico predicted miRNAs aligned with either wild-type (in green) or c.*1043A>C (in red) mutation of CFTR 3′UTR. C–D) predicted wild-type or CFTR 3′UTR-c.*1043A>C mutation Hybrid structure with miR-433. E–F) predicted wild-type or CFTR 3′UTR-c.*1043A>C mutaion Hybrid structure with miR-509-3p, for each hybrid structure is shown the mean free energy (mfe) calculated using the RNAHybrid server.

Figure 2. MiRNAs expression analysis.

Relative expression of miR-433 A) and miR-509-3p B) in different cell line and brushed nasal epithelial cell (N.E.) compared to Hela cell line.

Figure 3. Effect of miR-433 and miR-509-3p on CFTR-3′UTR and CFTR protein expression.A

: luciferase expression from pLuc-CFTR-3′UTR co-transfected with relative miRNA mimics and miRNA mimics plus anti-miRNA both in HEK293 and Panc1 cell lines, ***p<0.001 B: expression of CFTR protein is decreased to about 25% in Panc1 cells transfected with miR-433 mimic (** p<0.01) and about 45% in miR-509-3p (*** p<0.001) transfected cells.

Figure 4. Experimental evaluation of c*1043A>C mutation effect on miRNAs affinity.A

: luciferase reporter constructs derived from pGL3-Control vector. Underlined and in bold is shown the location of the variant c.*1043A>C or altered nucleotide bases to destroy the seed overlapping region of miR-433 and miR-509-3p in the CFTR 3′UTR. B: luciferase expression from pLuc-CFTR-3′UTR or mutated form constructs transient transfected in different Panc-1 cells stably espressing the miR-433, miR-509-3p or the sh-3′UTR. Data reported here are the means ± St.Dev., **P<0.01