PgmNr Z6008: Studying the functionality of the homologous repair pathway in zebrafish embryos: heading for an in vivo functional test to evaluate the pathogenicity of BRCA2 variants identified in breast/ovarian cancer patients.

Authors:
Jeroen Vierstraete 1,2 ; Andy Willaert 1 ; Kris Vleminckx 1 ; Petra Vermassen 1 ; Paul Coucke 1 ; Leen Pieters 2 ; Anne Vral 2 ; Kathleen Claes 1


Institutes
1) Center for Medical genetics Ghent, Ghent University Hospital, Belgium; 2) Department of Basic Medical Sciences, Ghent University, Belgium.


Abstract:

Aims:

Since the introduction of next generation sequencing, the challenge for genetic testing moved from developping mutation detection methodologies towards adequate variant interpretation. We propose a novel in vivo approach to study the functionality of BRCA2 missense variants in zebrafish. We aim to develop an in vivo functional assay to measure in zebrafish embryos the capacity of homologous recombination (HR) for human BRCA2 mRNA containing variants of unknown clinical significance (VUS).

Methods:

To evaluate the efficiency of HR repair we induce DNA double strand breaks (DSB) in zebrafish embryos by irradiation. We use yH2AX and RAD51 foci assays as markers for DSB and HR repair respectively. We generated zebrafish brca2 knockdown models by morpholino injection and Crispr-Cas9 mutagenesis, and obtained a mutant line from the European Zebrafish Resource Center (EZRC). Rescue experiments will be performed with wild type human BRCA2 mRNA and mRNA containing the VUS of interest.

Results:

We developed a protocol for visualising and quantifying RAD51 foci in zebrafish embryonic tissue. Brca2 knockdown by morpholino results in an almost complete absence of RAD51 foci. Similar results have been generated in the EZRC line, and are currently being generated in the Crispr-Cas9 knockout model. In a next step we will rescue the phenotype by microinjection of wild type human BRCA2 mRNA and mRNA containing VUS to study the effect of these VUS on HR capacity.

Conclusions:

The zebrafish genome contains nearly all genes involved in different DNA repair pathways in eukaryotes, including HR, in which BRCA2 plays a major role. Therefore, zebrafish provides an ideal in vivo model for studying variants in genes involved in DNA damage and repair.



ZFIN Genetics Index
1. brca2