Background: Cystic Fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians with one child in every 3000 newborns affected by disease. 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, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTR-Related Disorders (CFTR-RD). This suggests that other gene regions, i.e. 3'Untranslated region (3'UTR) of CFTR gene, may be the site of causing- disease mutations. Methods: We set up a multistep analysis: 1) Analysis of 1500 bp of CFTR 3'UTR region to search for genetic variants 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). 2) Identification of miRNA binding sites within the CFTR 3'UTR using software, i.e. TargetScan, miRBase, PITA. 3) Cloning of the 3'UTR of CFTR into pGL3-Control vector for luciferase assay to validate the activity of putative miRNA, using both miRNA mimic or Lentiviral particle pre-miRNA expressing. 4) Western Blot analysis to verify the CFTR protein level in cell transient transfected with miRNA mimics. Results: We identified three SNPs, one of which located in a region predicted to bind miR-433 and miR-509-3p. Western blot analysis revealed that the miRNAs are able to downregulate the CFTR protein expression and the Luciferase assay that this down regulation is due to the interaction of miRNA with the 3'UTR of CFTR gene. Further, this variant was peculiar of a CFTR-RD patient and the expression analysis demonstrated that such SNP increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Conclusion: These data show that at least two new miRNAs are able to regulate the expression of CFTR protein. But, more importantly, demonstrate that the presence of a SNP in the 3'UTR region can affect the binding of miRNAs likely leading to a pathological effect.

Gene Mutation In MicroRNA Target Sites Of CFTR Gene: A Novel Pathogenetic Mechanism In Cystic Fibrosis? / Amato, Felice; M., Seia; S., Giordano; A., Elce; F., Zarrilli; G., Castaldo; Tomaiuolo, Rossella. - (2013). (Intervento presentato al convegno 20 th IFCC-EFLM European Congress of Clinical Chemistry and Laboratory Medicine (EuroMedLab) tenutosi a Milano nel 19-23 Maggio 2013).

Gene Mutation In MicroRNA Target Sites Of CFTR Gene: A Novel Pathogenetic Mechanism In Cystic Fibrosis?

TOMAIUOLO, ROSSELLA
2013

Abstract

Background: Cystic Fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians with one child in every 3000 newborns affected by disease. 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, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTR-Related Disorders (CFTR-RD). This suggests that other gene regions, i.e. 3'Untranslated region (3'UTR) of CFTR gene, may be the site of causing- disease mutations. Methods: We set up a multistep analysis: 1) Analysis of 1500 bp of CFTR 3'UTR region to search for genetic variants 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). 2) Identification of miRNA binding sites within the CFTR 3'UTR using software, i.e. TargetScan, miRBase, PITA. 3) Cloning of the 3'UTR of CFTR into pGL3-Control vector for luciferase assay to validate the activity of putative miRNA, using both miRNA mimic or Lentiviral particle pre-miRNA expressing. 4) Western Blot analysis to verify the CFTR protein level in cell transient transfected with miRNA mimics. Results: We identified three SNPs, one of which located in a region predicted to bind miR-433 and miR-509-3p. Western blot analysis revealed that the miRNAs are able to downregulate the CFTR protein expression and the Luciferase assay that this down regulation is due to the interaction of miRNA with the 3'UTR of CFTR gene. Further, this variant was peculiar of a CFTR-RD patient and the expression analysis demonstrated that such SNP increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Conclusion: These data show that at least two new miRNAs are able to regulate the expression of CFTR protein. But, more importantly, demonstrate that the presence of a SNP in the 3'UTR region can affect the binding of miRNAs likely leading to a pathological effect.
2013
Gene Mutation In MicroRNA Target Sites Of CFTR Gene: A Novel Pathogenetic Mechanism In Cystic Fibrosis? / Amato, Felice; M., Seia; S., Giordano; A., Elce; F., Zarrilli; G., Castaldo; Tomaiuolo, Rossella. - (2013). (Intervento presentato al convegno 20 th IFCC-EFLM European Congress of Clinical Chemistry and Laboratory Medicine (EuroMedLab) tenutosi a Milano nel 19-23 Maggio 2013).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/595726
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