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Giorgi, Gianpietro (2013) Utilizzo di minigeni ibridi per la validazione di mutazioni di splicing. [Tesi di dottorato]

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Abstract (inglese)

The interpretation of the numerous sequence variants of unknown biological and clinical significance (UV for “unclassified variant”) found in genetic screenings or patients represents a major challenge in the molecular diagnosis of genetic disease, including cancer susceptibility. A fraction of UVs may be deleterious because they affect mRNA splicing. The most direct approach to determine whether disease-causing mutations are associated with splicing is to perform a reverse transcription PCR (RT-PCR) analysis on RNA from the relevant tissues of affected individuals. However, tissue samples are often not available because the expression of these genes are tissue specific (for example only in brain or heart) or patients are too far from laboratory. An alternative approach is to test the effects of the splicing mutations using minigenes. Here, we describe a functional splicing assay based on a minigene construct that assesses the impact of sequence variants on splicing. A genomic segment encompassing the variant sequence of interest along with flanking intronic sequences is PCR-amplified from patients genomic DNA and is cloned into a minigene vector. After transient transfection into cultured cells, the splicing patterns of the transcripts generated from the wild-type and from the mutated constructs are compared by reverse transcription-PCR analysis and sequencing. With classical minigenes is possible have difficulties to test mutations because is difficult clone the fragment that contains this mutation in the minigene. Many times the DNA fragment containing restriction sites incompatible with restriction sites of minigene, or the ligation reaction between the fragment and minigene is hardly for the size of the fragment. For bypass this problem we cloned a Gateway cassette that allow fast and easy cloning. Whit this assay we tested 16 various splicing mutation in different genes (NF1, CFTR, AIP, COQ6, STK11) with conclusive results

Abstract (italiano)

L’ interpretazione di numerose varianti di sequenza di significato biologico e clinico sconosciuto (UV) trovate in screening di popolazione o pazienti rappresenta una sfida nella diagnosi molecolare delle malattie genetiche, inclusa la predisposizione al cancro. Una parte di UV potrebbe avere effetto deleterio perchè possono interessare lo splicing dell’mRNA. L’approccio diretto per determinare se la malattia è associata alle mutazioni di splicing è attuare un’analisi dell’mRNA dei tessuti dei pazienti affetti dalla patologia tramite retrotrascrizione (RT-PCR). Tuttavia, i campioni di tessuto non sono sempre disponibili perchè l’espressione di questi geni possono essere tessuto-specifica (per esempio in cervello o cuore) o i pazienti sono troppo lontani dal laboratorio di analisi. Un approccio alternativo è testare la mutazione di splicing con l’uso dei minigeni. Qui, descriviamo un metodo funzionale di studio di splicing basato sull’utilizzo di minigeni che valutano l’impatto sullo splicing di varianti di sequenza. Un segmento genomico che comprende la variante di splicing di interesse comprese le zone introniche fiancheggianti, amplificata dal paziente mediante PCR è clonata nel minigene. Dopo una trasfezione transiente in culture cellulari umane, il pattern dei trascritti generati dal wild-type e dal mutato sono confrontati mediante PCR da retrotrascrizione e successivo sequenziamento. Con l’utilizzo di minigeni classici è possibile incontrare delle difficoltà in quanto si possono trovare ostacoli nel clonaggio del frammento che contiene la mutazione nel minigene. Molte volte il frammento di DNA contiene dei siti di restrizione incompatibili con quelli del minigene, o la reazione di ligazione può essere complicata data la grandezza dell’inserto. Per superare questi problemi abbiamo clonato una cassetta Gateway per permettere un veloce e facile clonaggio. Con questo metodo abbiamo testato 16 mutazioni in differenti geni (NF1, CFTR, AIP, COQ6, STK11) con risultati conclusivi

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Tipo di EPrint:Tesi di dottorato
Relatore:Salviati, Leonardo
Dottorato (corsi e scuole):Ciclo 25 > Scuole 25 > MEDICINA DELLO SVILUPPO E SCIENZE DELLA PROGRAMMAZIONE > EMATOONCOLOGIA, IMMUNOLOGIA E GENETICA
Data di deposito della tesi:20 Gennaio 2013
Anno di Pubblicazione:18 Gennaio 2013
Parole chiave (italiano / inglese):splicing/splicing Mutations/mutazioni minigene/minigene
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/03 Genetica medica
Struttura di riferimento:Dipartimenti > Dipartimento di Salute della Donna e del Bambino
Codice ID:5374
Depositato il:16 Ott 2013 08:40
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