Assessing the Impact of Non-coding Mutations on Hereditary Breast Cancer Gene Function and Risk

Background:
The genetic predisposition of BRCAx patients (women diagnosed with Hereditary Breast or Ovarian Cancer (HBOC) of unknown genetic cause) are poorly understood. We have identified mutations in gene regulatory regions of known HB predisposition genes (many of which function within the DNA damage response (DDR) pathway), in a significant proportion of Northern Irish (NI) BRCAx patients. This study aims to examine the impact of these non-coding mutations on risk gene expression and/or DDR deficiency.

Methods:
Initially, anonymised clinico-pathological data was gathered for the NI BRCAx cohort and linked to sequencing results to assess linked mutation/tumour phenotypes. Targeted panel RNA-Seq analysis will be carried out in tumour samples from patients with gene regulatory region mutations and a group of wild-type matched controls. Effects of mutations on mRNA splicing will also be assessed. For optimisation, a pilot experiment was performed on 8 patient samples. Additionally, publically available RNA-expression gene signatures, such as the DDRD signature, will be used to assess for DNA repair deficiencies in these tumours. Identified promoter mutations are being modelled in vitro using transcription reporter assays.

Results:
502 tumours were available for analysis. The median age at BC diagnosis was10-years lower than the NI population. Targeted RNA-Seq analysis of FFPE tumour samples has been optimised and is ongoing. Methods for calculating DDRD scores from microarray data were adapted and, utilising RNA-Seq data of 1,211 breast tumours from TCGA, a robust method was designed for DDRD scoring RNA-Seq data. These scores correlated strongly with those generated by independent groups. This template was applied to RNA-Seq data generated from our NI BRCAx pilot experiment.

Conclusion:
Our NI BRCAx database is a unique and comprehensive resource to further study genotype/phenotype relationships. Methods outlined will develop our understanding of the contribution of non-coding mutations on HBC gene expression, splicing and risk.