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

Objectives: Since the introduction of next-generation sequencing, the challenge for genetic testing moved from development of mutation detection methodologies towards adequate interpretation of (rare) variants. Here, 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 γ-H2AX 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. After synthesis of human BRCA2 mrna, rescue experiments will be performed with wild-type mrna and mrna containing the vus of interest. Results: We developed a protocol for visualizing and quantifying RAD51 foci in tissues of wild-type zebrafish embryos. Knockdown of brca2 by a morpholino results in an almost complete absence of RAD51 foci in irradiated embryos. Similar results are currently being generated in the Crispr-Cas9 brca2 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 the hr capacity. Conclusions: The zebrafish genome contains nearly all the genes involved in various dna repair pathways in eukaryotes, including homologous recombination, 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 repai

http://ift.tt/2qhvOWA