Combined genetic and splicing analysis of BRCA1 c.[594-2A>C; 641A>G] highlights the relevance of naturally occurring in-frame transcripts for developing disease gene variant classification algorithms

Abstract

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 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.

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

“…This suggests that promoter region variants, irrespective of bioinformatic prediction or functional assay results, are unlikely to be associated with a high risk of cancer. This is consistent with current evidence from ENIGMA studies (de la Hoya et al., ), which suggest that an allele resulting in only ∼20–30% expression of BRCA1 transcript/s encoding functional transcripts is not associated with high risk of BC. The low impact of these variants on risk is likely to reflect the complex interplay of TFs and DNA elements, and possible redundancy in the system.…”

Screening of BRCA1/2 deep intronic regions by targeted gene sequencing identifies the first germline BRCA1 variant causing pseudoexon activation in a patient with breast/ovarian cancer

“…However, whether this is a true contribution from variant allele or a PCR artefact caused by unspecific primer hybridisation cannot be concluded from our results. In any case, the production of functional BRCA1 protein from the variant allele would be lower than the rescue threshold (≈30%) proposed in de la Hoya work,37 supporting a likely pathogenic role for our variant. Taking this into account and from an analytical point of view, the variant should be classified as likely pathogenic (Class-4).…”

Power of pedigree likelihood analysis in extended pedigrees to classify rare variants of uncertain significance in cancer risk genes

“…If the quantitative results from co-segregation analysis are in conflict with in vitro data, this may indicate that the in vitro assay does not capture the underlying cancer biology and perhaps further, or different, in vitro analysis is warranted. [34, 35]. …”