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Digital archive of theses discussed at the University of Pisa


Thesis etd-03222013-111547

Thesis type
Tesi di dottorato di ricerca
Thesis title
BRCA-1 partner genes in cell proliferation and DNA repair
Academic discipline
Course of study
relatore Dott.ssa Caligo, Maria Adelaide
tutor Prof. Bevilacqua, Generoso
  • BRCA-1 partner genes
  • DNA repair
  • genetic predisposition to breast cancer
  • homologous recombination mechanism
Graduation session start date
M1775R and A1789T are two missense variants located within the BRCT domain of BRCA1 gene, both isolated from familial breast cancers patients. The M1775R variant has widely been described as deleterious by both in silico analysis and functional assays, while the A1789T variant has been identified and classified as probably deleterious for the first time by our group. To investigate the molecular mechanism that may underlie a pathogenetic role for these two mutations, we compared the expression profile, obtained by microarray, of HeLa G1 cells transfected with the two variants. Compared to BRCA1 wt, the M1775R and the A1789T variants showed 201 and 313 differentially expressed genes respectively.
Pathway analysis showed that many pathways are modulated by both mutations and that many of them are involved in cancer onset and progression. This evidence indicates that the M1775R and A1789T variants may predispose to cancer through similar mechanisms and this is consistent with the co-localization of the two mutations in the BRCT domain of BRCA1.
The expression levels of nine genes (CDKN1A, EDN1, GPR56, NFkB1, PML, SOD2, MRE11A, EEFE1E, OBFC2B), selected because of their involvement in neoplastic transformation, were validated by RT-qPCR. Moreover the expression level of five proteins was confirmed by Western Blotting (WB).
Our hypothesis is that some genes, coming out from microarray experiment ,are probably implicated in BRCA1 pathway and could be considered as candidate genes of susceptibility involved in familial breast cancer.
We chose three out of nine validated genes (MRE11A, EEF1E1, OBFC2B) for a further validation and test their involvement in DNA double strand breaks repair and in particular in the “Homologous Recombination” (HR) process. The knockdown of OBFC2B, MRE11A and EEF1E1 reduces the HR rate respectively of 28,6%, 41,8 % and 42,3% compared to the controls. Our results together with the knowledge obtained from literature suggest that those three genes might be involved in BRCA1-pathway.So we decided to do a mutational analysis on two of these genes in individuals belonging to Breast/Ovarian cancer families tested negative for BRCA1 and BRCA2 germline mutation. For the gene EEF1E1 we found three variants known in literature and one never described previously, but none of them are reported as pathogenic.