Validation of a Gene-Expression Based Assay for BRCA1 Function

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Uy, Paolo Miguel
Breast cancer , Clinical assay , qPCR , BRCA1 , Gene expression , Variants of Unknown Significance
Breast cancer is a disease that afflicts a significant proportion of women globally. 5-10% of breast cancer cases are linked to inherited polymorphisms in critical genes such as BRCA1, a tumour suppressor essential for genomic stability. A dysfunctional BRCA1 gene can increase breast cancer risk by 60-80%. Previous microarray analysis established that differential gene expression in unperturbed Epstein-Barr virus transformed lymphocyte cell lines (EBV-LCL) was able to distinguish BRCA1 mutation carriers from controls with a high degree of accuracy. A 43-gene radiation-independent classifier for BRCA1 status was constructed. We hypothesize that this differential gene expression can be observed in a subset of these genes using quantitative PCR (qPCR) in both EBV-LCL and B-lymphocytes isolated from patients with known BRCA1 mutation carrier status. The 43-gene classifier was analyzed using gene ontology analysis and 4 target genes selected based on predictive value, expression intensity and gene ontology similarity. Genes selected were CXCR3, TBX21, MX2, and IFIT1, with GusB as an endogenous reference gene. EBV-LCL established from known BRCA1 mutation carriers and from BRCA1 wildtype individuals were obtained and RT-qPCR (reverse transcriptase qPCR) performed on isolated RNA. Our results showed significant downregulation of CXCR3 and TBX21 in BRCA1 mutation carriers (p=0.018 and p=0.003, respectively), as expected from previous microarray results. IFIT1, while showing a non-significant upregulation (p=0.183), agreed with the expected trend. MX2 did not show significant differential expression. These results indicate that differential gene expression has the potential to accurately distinguish pathogenic variants, even if it may require EBV immortalization of B-lymphocytes. To determine whether the assay could be extended to fresh blood samples, B-lymphocytes were isolated from patients with known BRCA1 mutation carrier status from North York General Hospital in Toronto, ON. An optimized protocol to enrich the B-lymphocyte population using magnetic separation was developed for this purpose. RT-qPCR using RNA isolated from these lymphocytes showed no significant differential gene expression in CXCR3 and TBX21. However, a low sample size, use of non-sequenced controls and a need for further qPCR optimization may call these results into question. In addition, problems with blood sample transportation from off-site sources resulted in an unacceptable drop in RNA integrity. While this gene expression assay may be limited to screening a small number of blood samples, results indicate that may still have clinical relevance that can be explored. This would necessitate further optimization of the qPCR methodology and resolution of the issues surrounding RNA integrity and sample transport.
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