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Digital archive of theses discussed at the University of Pisa


Thesis etd-03292017-225443

Thesis type
Tesi di dottorato di ricerca
Thesis title
Evaluation of BRCA1 related DNA repair genes in breast and ovarian carcinogenesis
Academic discipline
Course of study
tutor Dott.ssa Caligo, Maria Adelaide
  • breast cancer
  • crispr/cas9
  • dna repair
  • functional assay
  • next generation sequencing
Graduation session start date
Breast cancer in one of the most common cancer worldwide. Among women this tumor accounts for over 25% of cancer diagnosis and 15% of cancer related deaths.
5-10% of breast cancer cases are hereditary, with a germline mutation in a known cancer susceptibility gene. Inherited susceptibility to developing breast and ovarian cancer most commonly results from mutation in BRCA1 and BRCA2 genes which are inherited in an autosomal dominant way.
BRCA1 is a tumor suppressor gene which encodes for a nuclear protein involved in a wide array of cellular functions, including cell cycle regulation, DNA damage response, maintenance of the genomic stability, transcription regulation, replication, recombination and higher chromatin hierarchical control.
BRCA1 is a component of a protein complex termed BASC (BRCA1-Associated Genome Surveillance Complex) and, therefore it has been associated with a variety of proteins implicated in DNA damage response and repair mechanism, such as MLH1, MSH2, MSH6, BLM and the MRN complex constituted by RAD50, MRE11A and NBS1. Many proteins of this complex can bind damaged DNA structures and act as sensors of DNA damage.
The aim of this study is to determine if some of the DNA repair partners of BRCA1 may contribute to breast and ovarian cancer development and progression.
Preliminary results obtained by our research group through functional assays in S. cerevisiae suggested that MSH2, MSH6, MRE11A, RAD50 and RAD51 genes might have a role in genomic instability induced by BRCA1 in yeast. Moreover a mutational screening revealed a surprisingly high frequency of MSH2 somatic mutations in breast and ovarian tumors from carriers of BRCA1 VUS (variants of unknown significance).
On the basis of these preliminary results the first task of this project is to perform a mutational analysis by next generation sequencing of MSH6, MRE11A, RAD50 and RAD51 genes in selected breast and/or ovarian tumors from BRCA1 VUS carriers, BRCA1 mutation carriers and BRCA1 wt individuals. This mutational screening identified 8 rare somatic variants pathogenic or predicted pathogenic distributed on 7 patients. 4/8 variants were located on MSH6 gene, 2/8 on RAD51, 1/8 on RAD50 and 1/8 on MRE11A.
In the second part of this study it was performed a GFP dependent homology directed repair assay whereby a recombination substrate is integrated into the genome. This functional assay was carried out in HeLa and MCF7 cell lines transfected with BRCA1 wild type and four BRCA1 VUS (p.Y179C, p.N550H, p.M1775R and p.A1789T). The results of this assay showed an increase in homologous recombination frequency associated to the pathogenic variants p.M1775R and p.A1789T in both cell lines.
Finally, the CRISPR-Cas9 system was used in order to obtain HeLa and MCF7 cell lines knockout for MSH2, but so far it was possible to isolate only HeLa MSH2 knockout clones. The MSH2 knockout clones will be tested with the homology directed repair assay to investigate the combined effect of MSH2 deficiency and BRCA1 VUS on homologous recombination.
On the basis of these results it is possible to conclude that the mismatch repair system seems to be frequently impaired in breast and ovarian tumors. Moreover the homologous recombination assay could be another powerful tool to help in the classification of BRCA1 VUS.