Considering all data available for the p.Ser36Tyr BRCA1 variant, it should be classified as a moderate cancer risk mutation. We believe that the described system is reliable for assessing the functional significance of any specific VUS and it can be easily adapted for the classification of VUS identified in the different domains of the BRCA1 gene. Its advantage is that high protein levels of full-length BRCA1 can be achieved in a transient expression system, thus avoiding long periods of selecting positive clones as described by Chang and colleagues in their mouse embryonic stem cell system. The use of a bicistronic expression vector further facilitates analysis, as through the incorporation of a gene that encodes a fluorescent protein in one of the multiple cloning sites, transfection efficiency can be calculated and immunofluorescence analysis can be restricted to transfected cells only. This system is versatile as it allows the simultaneous evaluation of many of the known BRCA1 key cellular functions. These include screening for protein expression levels, interaction with BARD1, sub-cellular localization and ability to induce the formation of conjugated ubiquitin chains at sites of DNA damage. This system is potentially very powerful as a wide range of information can be generated from just two independent experiments; immunoprecipitation and immunofluorescence staining of transfected cells. For example, using a repertoire of appropriate antibodies against other BRCA1 interacting proteins, the ability of BRCA1 to form the BRCC complex or interact with Abraxas, BACH1 and CtIP at specific stages of the cell cycle can also be investigated. From an immunofluorescence experiment of transiently transfected cells, information such as sub-cellular localization and co-localization with other proteins involved in the DNA repair machinery can be extracted. In addition, this system can be easily adapted for other assays and for different variants. The VUS can be created by site directed mutagenesis of the wild type encoding construct and for the DNA repair of DSBs by homologous recombination or transcriptional activation assays, certain features of the constructs including the promoter can be incorporated into the appropriate vectors thus examining full-length BRCA1 variant. Furthermore drug sensitivity assays can be performed after modifying the constructs. For example the fluorescent protein marker can be removed from the vector and replaced with an antibiotic resistance marker. This would facilitate the selection of positive clones and the generation of SAR131675 citations stable transfectants required for drug sensitivity assays. Examining known pathogenic variants in regions other than the BRCA1 RING domain using the above described system will establish this system as a robust method for the clinical evaluation of any BRCA1 VUS. It is our aim to further pursue this and examine a number of different VUS detected in the different functional domains of the BRCA1 protein. Accurate classification of BRCA1 VUS is important for appropriate genetic counseling and further management of mutation carriers. We have demonstrated via several functional assays that the BRCA1 p.Ser36Tyr GW-572016 231277-92-2 variant abrogates BRCA1 protein function. However, the in silico, clinical, genetic and epidemiological data which accompany this variant are inconsistent with features of a high-risk pathogenic mutation.