In questo lavoro di tesi, il lievito Saccharomyces cerevisiae è stato utilizzato per allestire i saggi funzionali per caratterizzare le varianti missenso di significato incerto dei geni
MSH6 e BRCA1 associati a tumore, poiché i metodi computazionali si sono rivelati
insufficienti e non completamente affidabili. La valutazione sperimentale utilizzando saggi funzionali può essere di aiuto nella classificazione delle varianti ed è uno strumento di conferma delle predizioni computazionali. Data la conservazione tra lievito e uomo dei pathway di riparazione del DNA in cui sono coinvolti i geni presi in considerazione, il lievito può essere utile per determinare le conseguenze funzionali delle varianti genetiche trovate nel cancro.
Il sistema costruito per caratterizzare le mutazioni nel gene MSH6 ha previsto la creazione del ceppo RSY6msh6::pCORE, in cui il gene è interrotto dall’inserimento di una cassetta CORE risultando non più funzionale. Il ceppo presenta un fenotipo mutatore, caratterizzato dall’aumento statisticamente significativo della frequenza di reversione rispetto al ceppo d’origine. La sostituzione del gene non funzionale con una variante genica e la successiva analisi della frequenza di reversione permettono di classificare la variante come neutra quando viene restaurato il fenotipo wild type e come patogenetica quando non viene osservato il fenotipo mutatore. Nel nostro caso la variante K177S che è stata introdotta nel ceppo RSY6msh6::pCORE ha mostrato un fenotipo simile alla forma wild type che ha confermato la predizione in silico come variante neutra. In futuro, ci impegneremo ad introdurre nel ceppo RSY6msh6::pCORE altre varianti sia patogenetiche che neutre in modo da avere dati statisticamente sufficienti per poter validare il saggio funzionale sviluppato in questo sistema modello.
In questo lavoro ho preso parte ad un altro progetto che si pone l’obiettivo di validare il saggio funzionale allestito in lievito per la caratterizzazione delle varianti missenso di BRCA1. Ho contribuito ad ampliare il numero di varianti analizzate per la validazione del saggio di ricombinazione omologa. I risultati ottenuti considerando tutte le varianti fino ad ora analizzate sono promettenti, in particolare per il saggio di ricombinazione intracromosomica dove abbiamo ottenuto una sensibilità del 100% e un’accuratezza dell’81%, e ci consentono di affermare che in futuro questo sistema modello potrà essere impiegato per classificare le VUS di BRCA1 associate ai tumori.
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