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

BACKGROUND

monoallelic germ-line variants confer a breast cancer risk comparable to the average pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify variants in without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in , 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 /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 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 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed / without further evidences. Our study puts a warning in up to five genetic variants that are currently reported as in ClinVar.