Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using Exon 3 as a Model System.

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 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 exon 3 (e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for haploinsufficiency. 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 transcripts. Of 100 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 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 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 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of leaky splicing variants, some of which may not increase cancer risk.