Development of a Functional Assay for Determination of BRCA1 Carrier Status
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Given the high risk of cancer development associated with BRCA1 mutation carriers, it is important that they be identified early and accurately. Identification is impeded however, by the large number of variants of unknown significance (VUS) and the pleiotropic nature of BRCA1 function. Our lab has developed a novel functional assay that identifies BRCA1 mutation carriers based on gene signatures in Epstein-Barr virus (EBV)-transformed lymphoblastoid cells (LCLs). Given that this assay was developed using blood-derived cells, we hypothesized that the same abnormally regulated genes would be detected in fresh blood samples and that this could be used to develop a novel blood-based functional assay for the determination of BRCA1 status in a patient population. The first part of the study used selected reaction monitoring-mass spectrometry (SRM-MS) as well as flow cytometry (FC) to determine protein expression in LCLs and identify potential targets for the development of a protein-based functional assay. Interestingly, many of the strong predictors of BRCA1 carriers were proteins involved in two signalling pathways: Interferon (IFN)-regulated signalling and B cell development. If validated in fresh blood samples, these observations represent potentially novel findings and suggest yet unexplored functions for BRCA1. Next, this study established the methodology to identify the desired cell population within a heterogeneous whole-blood sample by determining that LCLs express the pan B cell surface marker CD20. To determine which targets were detectable in CD20+ B cells isolated from fresh blood samples, mRNA and protein expression of the selected targets was examined using a sample cohort of BRCA1 mutation carrier and non-carrier control patients. The results showed that four mRNA targets, CXCR3, GLDC, IFIT1 and TBX21 were detectable in these fresh blood samples identifying them as appropriate targets for further development of a qRT-PCR-based assay. However, given poor RNA quality and changes in transcript expression levels as the age of the blood samples increased, the development of a protein-based assay is the best approach moving forward. Moreover, four proteins, IGHM, IGHD, CD24 and CXCR3, had high expression on CD20+ B cells, identifying them as the best targets for further development of a FC-based functional assay.