492PD - Functional analysis of the BRCA1 H1686Q sequence variant: From biology to clinic

Date 27 September 2014
Event ESMO 2014
Session Genetic predisposition to cancer
Topics Pathology/Molecular Biology
Personalised Medicine
Presenter Arcangela De Nicolo
Citation Annals of Oncology (2014) 25 (suppl_4): iv165-iv166. 10.1093/annonc/mdu332
Authors A. De Nicolo1, A. Giobbie-Hurder2, S. Pathania3, E. Parisini4, R.S. Gelman5, J.A.A. Ladias6, V. Joukov7
  • 1Medicine/cancer Biology, Harvard Medical School/Dana-Farber Cancer Institute, 02115 - Boston/US
  • 2Biostatistics And Computational Biology, Dana-Farber Cancer Institute, Boston/US
  • 3Medicine/cancer Biology, Harvard Medical School/Dana-Farber Cancer Institute, Boston/US
  • 4Center For Nano Science And Technology, Italian Institute of Technology, Milan/IT
  • 5Medicine/biostatistics And Computational Biology, Harvard Medical School/Dana-Farber Cancer Institute, Boston/US
  • 6Molecular Medicine Laboratory And Macromolecular Crystallography Unit/medicine, Harvard Medical School, Boston/US
  • 7Biological Chemistry And Molecular Pharmacology, Harvard Medical School, Boston/US

Abstract

Aim

Germ line mutations in the BRCA1 gene confer a significantly increased lifetime risk of breast and ovarian cancer. Discrimination between pathogenic and benign sequence alterations is paramount for cancer risk estimation, prevention, and therapy. Variants of uncertain significance (VUS), nevertheless, abound. Along with high-throughput screens developed to help classify rare VUS, in-depth analyses of single variants can be valuable to delve into BRCA1 pathobiology. The BRCA1 protein maintains genome stability by serving as a hub in DNA repair and cell cycle checkpoint pathways. This role critically depends on the integrity of its carboxyl (C)-terminal BRCT domain, which binds known cognate phosphoproteins. We previously demonstrated the functionally deleterious and neutral effect, respectively, of two BRCT-resident VUS, the Italian founder sequence alteration BRCA1 V1688del and the neighboring BRCA1 V1687I identified in a patient with triple negative breast cancer.

Methods

Here we report a multimodal functional analysis of the adjacent BRCA1 H1686Q, described in Italian and Dutch/Belgian families.

Results

We found that BRCA1 H1686Q, likely by triggering allosteric conformational changes, impacts BRCT/ligand recognition with different severity depending on the strength of the interaction. Specifically, in pull down assays, the BRCA1 H1686Q protein displayed a dramatically reduced binding to CtIP yet maintaining substantial affinity towards BRIP1/FANCJ and Abraxas. Moreover, the BRCA1 H1686Q C-terminal fragment exhibited peculiar temperature sensitivity in vitro because its binding properties were impaired at 37°C but not at 4°C. Notably, the BRCA1 H1686Q protein localized to the nucleus but failed to support homology-directed DNA double strand break repair. Thus, BRCA1 H1686Q causes a selective perturbation of BRCT-mediated interactions that is associated with a loss-of-function DNA repair phenotype.

Conclusions

Our work offers new insights into the mechanisms of BRCA1 dysfunction and advocates interaction-specific investigative approaches to unravel the complexity of BRCA1-mediated tumor suppression. Such studies could, in turn, help devise functional tests and highlight new routes to targeted therapy.

Disclosure

All authors have declared no conflicts of interest.