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

Abstract

<i>PALB2</i> monoallelic <i>loss-of-function</i> germ-line variants confer a breast cancer risk comparable to the average <i>BRCA2</i> pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify <i>loss-of-function</i> variants in <i>PALB2-</i>without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in <i>PALB2</i>, 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. Alternative splicing was characterised in RNAs extracted from blood, breast and <i>fimbriae</i>/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 <i>PALB2</i> 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. 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. PVS1 is not necessarily warranted for splice site variants targeting four <i>PALB2</i> acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed <i>pathogenic</i>/<i>likely pathogenic</i> without further evidences. Our study puts a warning in up to five <i>PALB2</i> genetic variants that are currently reported as <i>pathogenic/likely pathogenic</i> in ClinVar.