Analyzing Variants of Uncertain Significance in BRCA1 and BARD1
Over the past 50 years, the ease and accessibility of full genome sequencing has greatly increased. With this, there is an abundance of sequencing data; however, a problem that arises during sequencing analysis is that variants are uncovered, but their clinical significance is unknown. This research is designed to identify whether certain variants of uncertain significance (VUS) in the protein BARD1 and BRCA1 are functional in homology directed repair (HDR). The protein BARD1 forms a dimer with BRCA1, and together they function as a mediator of HDR at double stranded breaks (DBS) in the DNA. A green-fluorescent protein (GFP) repair assay was utilized to measure HDR function. In this assay, HeLa DR cells are integrated with two non- functional copies of GFP coding sequence. One copy of the GFP sequence contains a recognition site for the endonuclease I-SceI. When I-SceI is transfected into the cell, it induces a DSB, and if the cell repairs via HDR by copying the second GFP sequence, the repaired gene becomes functional. Cells are subsequently analyzed by flow cytometry to determine the percentage of cells producing GFP, or the amount of cells that successfully underwent HDR. The results of the GFP assay allowed the identification of variants that are deficient in HDR. Protein variants that are unable to repair DNA correctly can lead to mutations and cell damage that could potentially contribute to cancer. Overall, these data allow for a better clinical classification of BARD1 and BRCA1 variants in patients’ genome sequences and can help guide clinicians when advising and treating patients.
I am so proud of you!