Download THE G2 MICRONUCLEUS ASSAY FOR THE ANALYSIS OF IN

THE G2 MICRONUCLEUS ASSAY FOR THE ANALYSIS OF IN
VITRO CHROMOSOMAL RADIOSENSITIVITY IN
INDIVIDUALS CARRYING A BRCA1 OR BRCA2 MUTATION.
Annelot Baert1, Julie Depuydt1, Bruce Poppe2, Tom Van Maerken2,
Fransiska Malfait2, Tim Van Damme2, Sylvia De Nobele2, Kim De
Leeneer2, Kathleen Claes2, Anne Vral1.
1
Department of Basic medical Sciences, Ghent University, Ghent,
Belgium, 2Center for Medical Genetics, Ghent University Hospital,
Ghent, Belgium.
[email protected]
BRCA1 and BRCA2 play an important role in the DNA damage response pathway.
Mutations in these genes may be associated with increased radiosensitivity, an
important issue as several guidelines advise regular mammography screening from
young age in BRCA1 and BRCA2 mutation carriers.
To determine the degree of radiosensitivity in BRCA1 and BRCA2 mutation carriers,
different assays have been developed, which however failed to provide consistent
results. For instance, the classic G0 micronucleus assay, based on the irradiation of
quiescent lymphocytes, is not suitable to unequivocally determine radiosensitivity in
BRCA1 and BRCA2 mutation carriers. In G0, only non-homologous end joining is
available for the repair of radiation induced double strand breaks (DSB). BRCA1&2,
however, are mainly involved in the homologous recombination (HR) pathway for
repair of DSB and/or in G2/M checkpoint control. These two pathways are only
active within the late S and G2 phase of the cell cycle.
For this study we recently optimized a novel G2 micronucleus assay in lymphocytes
allowing detection of defects in HR and G2/M cell cycle control (ref).
Results obtained with this assay in a pilot study showed significantly increased
radiation-induced micronucleus yields in 10 BRCA1 mutation carriers compared to
healthy controls, suggesting a decreased functionality of the HR. Furthermore, the
preliminary results of the G2/M checkpoint parameter revealed an impaired G2/M
checkpoint in these 10 BRCA1 mutation carriers.
We are currently extending this study to a larger group of BRCA1 mutation carriers
and we’re also evaluating BRCA2 mutation carriers. Our results may aid to establish
whether benefits from early mammographic screening in asymptomatic mutation
carriers outweigh the risks linked to increased cumulative doses of radiation.
Furthermore, our test may have the potential to be used as a universally applicable
functional test for analysis of variants of unknown clinical significance, at least in the
BRCA1 gene, without the need of cloning and transfection of these variants in cell
lines.
Reference: Claes et al., Variant ataxia telangiectasia: clinical and molecular findings
and evaluation of radiosensitive phenotypes in a patient and relatives. Neuromolecular
Medicine 2013; 15(3): 447-457