logo SBA


Digital archive of theses discussed at the University of Pisa


Thesis etd-10172016-181819

Thesis type
Tesi di dottorato di ricerca
Thesis title
Identification and analysis of pathogenic regulatory variants in promoters, introns and in 5’ and 3’ untranslated regions of BRCA1 and BRCA2 genes
Academic discipline
Course of study
tutor Dott.ssa Caligo, Maria Adelaide
  • BRCA1
  • BRCA2
  • breast cancer
  • next generation sequencing
  • untraslated regions
Graduation session start date
Release date
Background - Germline mutations in breast cancer susceptibility genes BRCA1/2 confer a substantially increased lifetime risk of developing breast and/or ovarian cancer. The BRCA1/2 routinely clinical mutation screening is usually limited to the coding regions and intron–exon boundaries, which precludes the identification of mutations within the noncoding regulatory regions. These observations led us to assume that unknown genomic variations in the 5’, 3’ untranslated regions (UTRs) and introns of BRCA genes could be implicated in the inherited breast or ovarian cancer through their effect on BRCA gene expression. The activity of 5’ UTR and genomic flanking regions of BRCA1/2 is highly controlled by a self-regulating mechanism and by several transcription factors that act as activators or repressors. Aberrations in these regulatory regions may be associated with a deregulation in the transcriptional activity and in the mRNA levels of BRCA genes. Moreover the functional activity of BRCA genes is also regulated at the post transcriptional level: in particular the 3’UTR contains RNA-binding proteins and miRNA binding sites, and some germline variants in 3’UTR have been associated with breast cancer risk. In order to test this hypothesis, specific regulatory introns and 5’/3’ UTRs of BRCA genes were analyzed in patients highly selected for breast/ovarian cancer family history and tested negative for exonic mutations in these genes.
Materials and Methods - Genotyping analysis was performed by Next Generation Sequencing on genomic DNA of 80 index cases selected from breast and/or ovarian cancer patients with strong family history and wild type for BRCA1 and BRCA2. All variants emerged from the mutational screening of 5’/3’ UTRs and introns of BRCA1/2 were prioritized by bioinformatic tools. Following the bioinformatic prioritization, the effect of the interesting variants on gene regulation was evaluated by functional assays. A multifactorial analysis was performed to translate information from multiple sources to estimate the associated cancer risk of each regulatory variant.
Results - The mutation analysis of 80 patients showed 34 different variants: 19 in BRCA1 and 15 in BRCA2. 5 variants in BRCA1 and 8 variants in BRCA2 were novel and not present in dbSNP. 9 out of the 13 “no dbSNP” variants were resulted with significant change in bioinformatic prioritization.
3 variants in BRCA1 and 2 in BRCA2 were found in the promoter regions and were predicted to alter TF (transcription factors) binding site affinity. To examine the effect of these variants on promoter activity, luciferase reporter assays were performed.
For c.-232-176A>T and c.-232-55G>A BRCA1 variants, electrophoretic mobility shift assays (EMSA) also revealed different DNA-protein complex when the nuclear extracts were incubated with the biotinylated variant probe. The variants produced a significant change in the mobility of DNA-protein complexes compared to wild type.
Conclusion - The major challenge is to determine the significance and the contribution of non-coding variants in breast and ovarian cancer susceptibility. This could be relevant to identify inherited mutations in such patients with a strong family history without alterations in routinely analyzed BRCA1/2 regions, to propose an adaptive follow-up for them and their families and to design targeted therapeutic strategies.
Research is urgently needed to reduce the incidence and mortality of breast cancer. Genetic testing represents a powerful tool to increase the efficiency of prevention, through an effective risk prediction; moreover it may help in diagnosis and in the development of new and more specific therapeutic approaches. The final goal of this project is to develop clinically useful tools for the classification of sequence variants that map in regulatory regions of BRCA1/2 genes. Gaining deeper insights in breast cancer susceptibility will improve the ability to identify those families at increased risk and the efficiency of cancer prevention for BRCA carrier.