The c.516G>A variant in BRCA2 is a synonymous variant (p.(Lys172=)). This variant is absent from gnomAD v4.1 (read depth ≥25x in >90% samples, PM2_Supporting met). This BRCA2 synonymous (silent) variant has a SpliceAI score of 0.84, predicting an impact on splicing (score threshold ≥0.2) (PP3 not applied because a PVS1 code is met). This variant is reported to result in aberrant mRNA splicing. RT-PCR with patient-derived mRNA demonstrated that the variant impacts splicing by skipping of exon 6 and skipping of exon 5 and 6 (both out of frame). Confirmation with a minigene assay showed no full-length transcript to be produced by the variant allele, using a semi-quantitative assessment with gel electrophoresis, cloning and sequence analysis (PMID: 19267246). RT-PCRSeq confirmed the aberrant transcripts and reported that no full-length transcript was produced by the variant allele (Ambry internal data). Appropriate code strength determined by comparison of results to PVS1 decision tree (PVS1 (RNA)). Multifactorial likelihood ratio analysis using clinically calibrated data produced a combined LR for this variant of 0.92 (based on Co-occurrence LR=1.05; Family History LR=0.88), which is above the ENIGMA BRCA1/2 VCEP threshold for BP5 (>0.48) and below PP4 (<2.08) (BP5 and PP4 not met; PMID: 31853058). In summary, this variant meets the criteria to be classified as a Likely pathogenic variant for BRCA2-related cancer predisposition based on the ACMG/AMP criteria applied as specified by the ENIGMA BRCA1/2 VCEP (v1.2) (PM2_Supporting, PVS1 (RNA)).