However, at present it is not known whether this is due to GDC-0879 structure protein instability, inability to interact with the E2 enzyme, impaired shuttling into the nucleus, or low levels of BRCA1:BARD1 complex. It is even possible that the substitution of serine by tyrosine generates a target for another kinase that alters phosphorylation events. The known pathogenic p.Ile26Ala variant, is located within the binding interface with E2 enzyme and this substitution abolishes this interaction E2, whereas p.Cys61Gly disrupts the interaction with BARD1. We believe that the substitution of serine 36 by tyrosine disrupts the b-helix of the RING domain of BRCA1 causing an overall conformational change that affects both interactions with BARD1 as well as with E2 enzyme. The functional data in this work suggest that the abrogated protein function exhibited by the p.Ser36Tyr BRCA1 variant is due to a combination of effects on the interaction with BARD1 and E2. The E3 ligase LDN-193189 activity of the BRCA1:BARD1 heterodimer is believed to be implicated in tumor suppression, as missense mutations in the RING domain of BRCA1 that disrupt the interaction with E2 have been reported in patients. However, it was demonstrated in a mouse model that mutations in the BRCT domain and not E3 ligase inactivating mutations cause a high rate of tumor formation. This observation prompted the investigators to suggest that mutations in the RING domain that disrupt the E3 ligase activity of BRCA1 are not linked to breast cancer predisposition, whereas those that disrupt the interaction with BARD1 are linked to tumor development. Therefore it is still unclear which of the known functions of BRCA1 are directly involved in tumor suppression and this further complicates the accurate assessment of cancer risk for BRCA1 VUS. In consideration of the findings of the present study in combination with the classification of other variants that exhibit similar behavior to that of the p.Ser36Tyr, our experimental evidence supports that the p.Ser36Tyr substitution abrogates the function of BRCA1 protein. The superiority of functional assays over bioinformatics tools for classifying a VUS, is highlighted in this work, as discrepancies were exhibited using two of the most widely applied software analysis tools PolyPhen and Align-GVGD, to classify the p.Ser36Tyr variant. PolyPhen predicted that both p.Ser36Tyr and p.Cys61Gly are possibly damaging, whereas AlignGVGD predicted that the probability of the p.Ser36Tyr mutation interfering with the function of the protein is very low, unlike p.Cys61Gly which impairs the interaction of BRCA1 with BARD1. In contrast to the bioinformatics tools, the functional work we have described demonstrates that the p.Ser36Tyr mutation interferes with the function of the BRCA1 protein. We have used our expression system of full length BRCA1 protein in HEK293T cells and S-phase cell cycle synchronization of BRCA1BARD1 co-transfected cells to classify a novel BRCA1 VUS identified in Cypriot families, following the guidelines for functional assays recently suggested by Millot and colleagues. Our results indicate that the p.Ser36Tyr BRCA1 variant has abrogated function in functional assays because it is produced at lower levels than wild-type protein and hence it exhibits reduced ability to interact with BARD1.