Alternative splicing and ACMG-AMP-2015-based classification of PALB2 genetic variants: an ENIGMA report

Lopez-Perolio, I.; Leman, R.; Behar, R.; Lattimore, V.; Pearson, J.F.; Castera, L.; Martins, A.; Vaur, D.; Goardon, N.; Davy, G.; Garre, P.; Garcia-Barberan, V.; Llovet, P.; Perez-Segura, P.; Diaz-Rubio, E.; Caldes, T.; Hruska, K.S.; Hsuan, V.; Wu, S.T.; Pesaran, T.; Karam, R.; Vallon-Christersson, J.; Borg, A.; Valenzuela-Palomo, A.; Velasco, E.A.; Southey, M.; Vreeswijk, M.P.G.; Devilee, P.; Kvist, A.; Spurdle, A.B.; Walker, L.C.; Krieger, S.; Hoya, M. de la; kConFab Investigators

doi:doi:10.1136/jmedgenet-2018-105834

Background PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. Methods Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional...Show moreBackground PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. Methods Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212-1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant. Results We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies. Conclusions PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.Show less

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Alternative splicing and ACMG-AMP-2015-based classification of PALB2 genetic variants: an ENIGMA report