From a variant of unknown significance to pathogenic: Reclassification of a large novel duplication in BRCA2 by high‐throughput sequencing

BACKGROUND: Germline mutations in BRCA1/2 significantly contribute to hereditary breast and/or ovarian cancer. Here, we report a novel BRCA2 duplication of exons 22–24 in a female patient with bilateral breast cancer at age 35 and 44. The duplicated region was initially detected by gene panel sequencing and multiplex ligation‐dependent probe amplification. However, the location and orientation of the duplicated region was unknown. Therefore, it was initially classified as a variant of unknown significance. METHODS: The spatial directional characterization of the BRCA2 duplication was achieved by targeted enrichment of the whole‐genomic BRCA2 locus including exons and introns, and subsequent high‐throughput sequencing. Subsequently, bioinformatics tools and a breakpoint‐spanning PCR were used for identification of location and orientation of the duplication. RESULTS: The duplicated region was arranged in tandem and direct orientation (Chr13(GRCh37):g.32951579_32960394dup; NM_000059.3 c.8754 + 651_9256+6112dup p.(Ala3088Phefs*3)). It is predicted to result in a frameshift and a premature stop codon likely triggering nonsense‐mediated mRNA decay. Consequently, it is regarded as pathogenic. CONCLUSION: This case study demonstrates that a comprehensive characterization of a structural variant by breakpoint assessment is crucial for its correct classification. Therefore, sequencing strategies including non‐coding regions might be necessary to identify cancer predispositions in affected families.