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FIELD OF EXPERTISE
- Breast Cancer
- Zebrafish
- Prime editing
- Classification of VUS
- Hypomorphic variants
PROJECTS
Background
BRCA2 is a critical cancer predisposition gene with pathogenic variants significantly increasing breast and ovarian cancer risk. However, many BRCA2 variants remain classified as variants of unknown significance (VUS), posing major challenges for clinical decision-making. Moreover, not all pathogenic variants in BRCA2 carry the same level of disease risk. For these hypomorphic or reduced-penetrance variants, there is an urgent need for cost-effective in vivo models. Existing approaches, such as clinical data analysis, in vitro functional assays, and mouse models, are often insufficient or too resource-intensive to accurately assess the cancer risk associated with these variants. However, precise classification is essential for implementing risk-reducing interventions, guiding targeted therapies and providing presymptomatic testing for at-risk family members.
Aim
The general aim of this project is to validate an in vivo assay that classifies BRCA2 VUS by knocking in the corresponding missense variants in the DNA-binding domain of zebrafish brca2 using CRISPR-mediated prime editing. Brca2 functionality will be tested using different functional tests based on the concept of synthetic lethality between PARP inhibitors and BRCA2 deficiency. Additionally, this project aims to evaluate whether hypomorphic BRCA2 variants can be modeled in vivo in zebrafish to determine if their phenotypes are distinguishable from highly penetrant loss-of-function and neutral variants, and to assess whether these variants are associated with delayed tumor formation. Lastly, we want to develop a faster workflow with a readout in the F0 generation. This will enable more reliable classification of BRCA2 VUS and improve genetic counselling as well as clinical and therapeutic care for affected individuals and their families.
Strategy
We will knock-in different known pathogenic and benign variants and functionally test them using the acridine orange (AO) assay, the eye size test and the RAD51 foci assay. This way, we can see if our in vivo assay confirms the pathogenicity/benignity of the human variants. Secondly, we will knock-in hypomorphic BRCA2 variants that previously have been knocked-in into mice and compare the results of the functional assays for these variants with those from highly penetrant loss-of-function and neutral variants. Additionally, we will see if a link can be found with delayed tumor formation for these variants. For a new readout in the F0 generation, brca2 variants will be knocked-in in heterozygous brca2+/- zebrafish using prime editing. Deep sequencing will determine relative allele frequencies in zebrafish treated with PARPi versus untreated (DMSO). Lower frequencies are expected for pathogenic variants treated with PARPi.
BRCA2 is a critical cancer predisposition gene with pathogenic variants significantly increasing breast and ovarian cancer risk. However, many BRCA2 variants remain classified as variants of unknown significance (VUS), posing major challenges for clinical decision-making. Moreover, not all pathogenic variants in BRCA2 carry the same level of disease risk. For these hypomorphic or reduced-penetrance variants, there is an urgent need for cost-effective in vivo models. Existing approaches, such as clinical data analysis, in vitro functional assays, and mouse models, are often insufficient or too resource-intensive to accurately assess the cancer risk associated with these variants. However, precise classification is essential for implementing risk-reducing interventions, guiding targeted therapies and providing presymptomatic testing for at-risk family members.
Aim
The general aim of this project is to validate an in vivo assay that classifies BRCA2 VUS by knocking in the corresponding missense variants in the DNA-binding domain of zebrafish brca2 using CRISPR-mediated prime editing. Brca2 functionality will be tested using different functional tests based on the concept of synthetic lethality between PARP inhibitors and BRCA2 deficiency. Additionally, this project aims to evaluate whether hypomorphic BRCA2 variants can be modeled in vivo in zebrafish to determine if their phenotypes are distinguishable from highly penetrant loss-of-function and neutral variants, and to assess whether these variants are associated with delayed tumor formation. Lastly, we want to develop a faster workflow with a readout in the F0 generation. This will enable more reliable classification of BRCA2 VUS and improve genetic counselling as well as clinical and therapeutic care for affected individuals and their families.
Strategy
We will knock-in different known pathogenic and benign variants and functionally test them using the acridine orange (AO) assay, the eye size test and the RAD51 foci assay. This way, we can see if our in vivo assay confirms the pathogenicity/benignity of the human variants. Secondly, we will knock-in hypomorphic BRCA2 variants that previously have been knocked-in into mice and compare the results of the functional assays for these variants with those from highly penetrant loss-of-function and neutral variants. Additionally, we will see if a link can be found with delayed tumor formation for these variants. For a new readout in the F0 generation, brca2 variants will be knocked-in in heterozygous brca2+/- zebrafish using prime editing. Deep sequencing will determine relative allele frequencies in zebrafish treated with PARPi versus untreated (DMSO). Lower frequencies are expected for pathogenic variants treated with PARPi.
DEGREES
- Master of Science in Biomedical Sciences (Ghent University, 2025)
- Bachelor of Science in Biomedical Sciences (Ghent University, 2023)