Functional Investigation of the Novel BRCA1variant (Glu1661Gly) byComputationalTools andYeastTranscription Activation Assay

Introduction: Mutations in the BRCA1 gene are major risk factors for breast and ovarian cancers. However, the relationship between some BRCA1 mutations and cancer risk remains largely unknown. Cancer risk predictions could be improved by evaluation of the impairment degree in the BRCA1 functions due to a specific mutation. This study aimed to assess the functional effect of a novel variant (Glu1661Gly) by a combination of in silico tools, structural analysis, and also experimental functional assay based on yeast transcription activation. Methods: Computational tools including PROVEAN, PolyPhen2, AlignGVGD, Mutation Taster, and also structural analysis were used for prediction of the impact of Glu1661Gly on protein function. To perform the yeast functional assay, the BRCA1 Cterminal (BRCT domain) was cloned into pLexA plasmid inframe with the DNAbinding domain of LexA to generate a functional transcription activator. The resulted construct was transformed into EGY48/ pRB1840 yeast and positive colonies were assayed for #946;galactosidase activity. Wildtype BRCA1 and Ser1613Gly were used as positive controls and Met1775Arg as negative control. Results: #160;The #160;Glu1661Gly #160;variant #160;was #160;predicted #160;to #160;be #160;neutral #160;by #160;PROVEAN, #160;disease causing by Mutation Taster, probably damaging by Polyphen2, and intermediate effect by AlignGVGD. The yeast functional assay revealed that Glu1661Gly activity was comparable to wildtype BRCA1 Conclusions: Observed discrepancies between in silico tools make it difficult to interpret the results. Based on structural analysis, the Glu1661Gly on #945;1 helix of the Cterminal domain does not seem to impair function due to #945;1 helix is far from the BRCTBRCT interface and phosphopeptidebinding site. This variant was also classified as neutral; using yeast functional assay.