A recent analysis using family history weighting and co-observation classification modeling indicated that BRCA1 c.594-2A > C (IVS9-2A > C), previously described to cause exon 10 skipping (a truncating alteration), displays characteristics inconsistent with those of a high risk pathogenic BRCA1 variant. We used large-scale genetic and clinical resources from the ENIGMA, CIMBA and BCAC consortia to assess pathogenicity of c.594-2A > C. The combined odds for causality considering case-control, segregation and breast tumor pathology information was 3.23 × 10-8 Our data indicate that c.594-2A > C is always in cis with c.641A > G. The spliceogenic effect of c.[594-2A > C;641A > G] was characterized using RNA analysis of human samples and splicing minigenes. As expected, c.[594-2A > C; 641A > G] caused exon 10 skipping, albeit not due to c.594-2A > C impairing the acceptor site but rather by c.641A > G modifying exon 10 splicing regulatory element(s). Multiple blood-based RNA assays indicated that the variant allele did not produce detectable levels of full-length transcripts, with a per allele BRCA1 expression profile composed of ≈70-80% truncating transcripts, and ≈20-30% of in-frame Δ9,10 transcripts predicted to encode a BRCA1 protein with tumor suppression function.We confirm that BRCA1c.[594-2A > C;641A > G] should not be considered a high-risk pathogenic variant. Importantly, results from our detailed mRNA analysis suggest that BRCA-associated cancer risk is likely not markedly increased for individuals who carry a truncating variant in BRCA1 exons 9 or 10, or any other BRCA1 allele that permits 20-30% of tumor suppressor function. More generally, our findings highlight the importance of assessing naturally occurring alternative splicing for clinical evaluation of variants in disease-causing genes.

Many germline DNA sequence variants in gene regulatory elements are associated with an increased risk of cancer. These include large genomic deletions of the promoter of the BRCA1 gene, through to single nucleotide variants in the promoter, UTRs and long-range enhancers of multiple other cancer related genes. Next generation sequence analysis of early onset and familial breast cancer cases is currently identifying an escalating number of variants in non-coding regions of the genome, however for most of these the significance in unknown. This project aims to determine the functional and clinical significance of breast cancer associated variants in regulatory regions of BRCA1 and BRCA2, as part of an international collaborative project arising from the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. To date over two hundred variants mapping to promoter, UTR and deep intronic regions have been identified in early onset or familial breast cancer patients that have no identifiable coding or splicing changes in BRCA1 or BRCA2. Through a pipeline of bioinformatics analyses using ENCODE datasets, these variants have been prioritized for experimental evaluation. This has lead to the identification of several variants that significantly alter the regulation of BRCA1 or BRCA2 expression, through mechanisms including altered binding of transcription factors and microRNAs. These studies will lay the groundwork for comprehensive statistical analyses, in which bioinformatics and experimental data is combined with clinical and genetic data to establish multifactorial risk prediction models that can ultimately be used in a clinical setting. Citation Format: Jan Sevcik, Leslie Burke, Gaetana Gambino, Brooke L. Brewster, Emma Tudini, Philip J. Whiley, Siranoush Manoukian, ENIGMA Consortium, Thomas van Overeem Hansen, Marta Santamarina Pena, Ana Vega, Maria A. Caligo, Paolo Radice, Paolo Peterlongo, Etienne Rouleau, Amanda B. Spurdle, Melissa A. Brown. Bioinformatic and experimental evaluation of regulatory variants in breast cancer susceptibility genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1859.