Abstract
Genetic testing for BRCA1 and BRCA2 genes has led to the identification of many unique variants of uncertain significance (VUS). Multifactorial likelihood models that predict the odds ratio for VUS in favor or against cancer causality, have been developed, but their use is conditioned by the amount of necessary data, which are difficult to obtain if a variant is rare. As an alternative, variants mapping to the coding regions can be examined using in vitro functional assays. BRCA1 and BRCA2 proteins promote genome protection by interacting with different proteins. In this study, we assessed the functional effect of two sets of variants in BRCA genes by exploiting the green fluorescent protein (GFP)-reassembly in vitro assay, which was set-up to test the BRCA1/BARD1, BRCA1/UbcH5a, and BRCA2/DSS1 interactions. Based on the findings observed for the validation panels of previously classified variants, BRCA1/UbcH5a and BRCA2/DSS1 binding assays showed 100% sensitivity and specificity in identifying pathogenic and non-pathogenic variants. While the actual efficiency of these assays in assessing the clinical significance of BRCA VUS has to be verified using larger validation panels, our results suggest that the GFP-reassembly assay is a robust method to identify variants affecting normal protein functioning and contributes to the classification of VUS.
Highlights
Genetic testing for the breast cancer susceptibility genes, BRCA1 (MIM# 113705) and BRCA2(MIM# 600185), has become part of routine clinical care for patients with a personal or family history indicative of Hereditary Breast/Ovarian Cancer Syndrome (HBOC)
We evaluated the performance of these approaches in correctly discriminating between pathogenic and non-pathogenic analysis of a panel of variants classified according to the IARC 5-class model (Leiden Open Variation Database, URL: http://hci-exlovd.hci.utah.edu/variants.php)
We selected a total of 58 BRCA1 (n = 36) and BRCA2 (n = 22) variants mapping to the gene regions coding for the conserved motifs encompassing the RING-finger domain of BRCA1 and the DSS1/ssDNA Binding Domain (DBD) of BRCA2 [13] (Figure 1)
Summary
Genetic testing for the breast cancer susceptibility genes, BRCA1 (MIM# 113705) and BRCA2(MIM# 600185), has become part of routine clinical care for patients with a personal or family history indicative of Hereditary Breast/Ovarian Cancer Syndrome (HBOC). Genetic testing for the breast cancer susceptibility genes, BRCA1 (MIM# 113705) and BRCA2. A recent prospective study estimates the average cumulative risks by age 70 years of BRCA1 carriers to be 60% and 59% for breast and ovarian cancer, respectively. Splice site variants with a similar effect on the protein have been identified [2] In addition to these clearly loss-of-function alterations, a growing number of variants of uncertain significance (VUS) are currently being identified. These include missense substitutions, small in-frame deletions or insertions, alterations of both exonic and intronic sites potentially affecting splicing, and variants in regulatory sequences. The inability to classify the VUS either as disease causing or as rare non-pathogenic (benign) variation represents a challenge for genetic counselling, risk evaluation, and adoption of preventive and risk-reduction measures
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