Studio in vitro dell'espressione di varianti del gene CFTR con ruolo patofisiologico non ancora definito
type: CreativeWork
description: 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 correl…
abstract: 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 correl…
author: RUSCONI, DAMIANA
language: ita
keywords: Fibrosi cistica, analisi in vitro, brushing nasale, cystic fibrosis, in vitro analysis, mutations, mutazioni, nasal brushing, Settore MED/03 - Genetica Medica
date published: 2013-05-29
date modified: 2026-07-24T01:30:01Z
provider: Università di Cagliari
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Università degli Studi di Cagliari

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property ID: Handle
value: http://hdl.handle.net/11584/266120