Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using <i>BRCA2</i> Exon 3 as a Model System

Abstract

<i>BRCA2</i> is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all <i>BRCA2</i> VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored <i>BRCA2</i> exon 3 (<i>BRCA2</i>e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for <i>BRCA2</i> haploinsufficiency. <i>In silico</i> predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL <i>BRCA2</i> transcripts. Of 100 <i>BRCA2</i>e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL <i>BRCA2</i> transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL <i>BRCA2</i> with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL <i>BRCA2</i> exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of <i>BRCA2</i> variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that <i>BRCA2</i> tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of <i>BRCA2</i> leaky splicing variants, some of which may not increase cancer risk.