Offering genetic testing to identify pathogenic variants in individuals with clinicallypresumed hereditary breast and/or ovarian cancer is currently routine clinicalpractice. In case a pathogenic... Show moreOffering genetic testing to identify pathogenic variants in individuals with clinicallypresumed hereditary breast and/or ovarian cancer is currently routine clinicalpractice. In case a pathogenic variant is identified, carriers might benefit from riskreducing measures as intensified screening programmes or prophylactic surgery.Pathogenic variants associated with high cancer risk typically disrupt protein functionvia the introduction of a premature stop codon due to a nonsense mutation or via frame shifting insertions/deletions. However, for many of the variants identified by genetic testing it is uncertain if the function of the variant protein is impaired to such an extent that cancer risk is enhanced. Those variants are therefore classified as variants of uncertain significance (VUS) and they represent a major challengefor genetic counselling and clinical management of the families in which they are identified. Most VUS are rare and insufficient information can be mined to compute a reliable cancer risk estimation, leaving large numbers of families in uncertainty. Driven by a clear clinical need to classify variants in relevant cancer risk categories (i.e. high, moderate and population level), we optimized and validated a biological assay. This assay allows functional characterization of all types of variants (intronic and exonic), including those that may affect mRNA splicing. Show less
Mesman, R.L.S.; Calleja, F.M.G.R.; Hoya, M. de la; Devilee, P.; Asperen, C.J. van; Vrieling, H.; Vreeswijk, M.P.G. 2020
Purpose Current interpretation guidelines for germline variants in high-risk cancer susceptibility genes consider predicted loss-of-function (LoF) variants, such as nonsense variants and variants... Show morePurpose Current interpretation guidelines for germline variants in high-risk cancer susceptibility genes consider predicted loss-of-function (LoF) variants, such as nonsense variants and variants in the canonical splice site sequences of BRCA2, to be associated with high cancer risk. However, some variant alleles produce alternative transcripts that encode (partially) functional protein isoforms leading to possible incorrect risk estimations. For accurate classification of variants it is therefore essential that alternative transcripts are identified and functionally characterized. Methods We systematically evaluated a large panel of human BRCA2 variants for the production of alternative transcripts and assessed their capacity to exert BRCA2 protein functionality. Evaluated variants included all single-exon deletions, various multiple-exon deletions, intronic variants at the canonical splice donor and acceptor sequences, and variants that previously have been shown to affect messenger RNA (mRNA) splicing in carriers. Results Multiple alternative transcripts encoding (partially) functional protein isoforms were identified (e.g., increment [E4-E7], increment [E6-E7], increment E[6q39_E8], increment [E10], increment [E12], increment E[12-14]). Expression of these transcripts did attenuate the impact of predicted LoF variants such as the canonical splice site variants c.631+2T>G, c.517-2A>G, c.6842-2A>G, c.6937+1G>A, and nonsense variants c.491T>A, c.581G>A, and c.6901G>T. Conclusion These results allow refinement of variant interpretation guidelines for BRCA2 by providing insight into the functional consequences of naturally occurring and variant-related alternative splicing events. Show less
