Abstract
Women carrying pathogenic (disease causing) DNA variants in the BRCA1 gene have an approximate 72% increase in lifetime risk of developing breast cancer. However, the increase in breast cancer risk for a given pathogenic variant can be altered based on variant type and its location within the gene. These differences in risk can result in women, who are predicted to be at high risk of developing breast cancer (and cancers), presenting with different outcomes. In a large genetic association study, our laboratory recently identified that deletions overlapping BRCA1 were associated with greater risk (OR=1.21, 95% CI 1.09-1.35) of breast cancer compared to single nucleotide variants, including missense, nonsense, splicing and indel variants. The mechanism underpinning this difference in risk outcome is currently unknown. A recently discovered process, termed transcriptional adaptation, has been described as a potential mechanism of genetic compensation, which minimises the deleterious effect of a variant. Transcriptional adaptation is the process of compensatory genes being upregulated in response to the degradation of mRNA containing a premature termination codon variant. This mechanism has not yet been described in humans. The aim of this project was to develop a BRCA1 in vitro transcription system that would test the hypothesis that transcriptional adaptation could be occurring in some BRCA1 variants, explaining the lower risk. Bioinformatic assessment of CNVs overlapping BRCA1 using data from the genetic association study showed that all exons are represented in the cohort so there is no bias. Long-range PCR was optimised to generate the full-length BRCA1 coding sequence and attach a T7 promoter adapter to the amplicons. Using the products from long- range PCR, T7 in vitro transcription was used to generate sense and antisense BRCA1 mRNA transcripts. These methods were the first step in creating an in vivo assay to investigate the potential transcriptional adaptation phenomena. Future research will enable BRCA1 transcripts to be transfected into cells to induce degradation and measure the transcriptional adaptation response by using RNAseq to detect differential expression. Such analyses will provide greater understanding into the mechanisms underlying the risk of developing breast cancer associated with BRCA1 pathogenic variants.