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

Irene Lopez-Perolio (1) , Raphaël Leman (2, 3) , Raquel Behar (1) , Vanessa Lattimore (4) , John F. Pearson (4) , Laurent Castéra (2, 3) , Alexandra Martins (3) , Dominique Vaur (2, 3) , Nicolas Goardon (2, 3) , Grégoire Davy (2, 3) , Pilar Garre (1) , Vanesa García-Barberán (1) , Patricia Llovet (1) , Pedro Pérez-Segura (1) , Eduardo Díaz-Rubio (1) , Trinidad Caldés (1) , Kathleen S. Hruska (5) , Vickie Hsuan (6) , Sitao Wu (6) , Tina Pesaran (6) , Rachid Karam (6) , Johan Vallon-Christersson (7) , Ake Borg (7) , Kconfab Investigators (8) , Alberto Valenzuela-Palomo (9) , Eladio A. Velasco (9) , Melissa Southey (8) , Maaike P. G. Vreeswijk (10) , Peter Devilee (10) , Anders Kvist (7) , Amanda B Spurdle (11) , Logan C. Walker (4) , Sophie Krieger (2, 3) , Miguel de La Hoya (1)

1 IdISSC - Instituto de Investigación Sanitaria del Hospital Clínico San Carlos [Madrid, Spain]
2 UNICANCER/CRLC - Centre Régional de Lutte contre le Cancer François Baclesse [Caen]
3 GPMCND - Génomique et Médecine Personnalisée du Cancer et des Maladies Neuropsychiatriques
4 University of Otago [Dunedin, Nouvelle-Zélande]
5 GeneDx [Gaithersburg, MD, USA]
6 Ambry Genetics [Aliso Viejo, CA, USA]
7 Lund University [Lund]
8 University of Melbourne
9 INGEBI - Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, “Dr. Héctor N. Torres” [Buenos Aires]
10 Leiden University Medical Center (LUMC)
11 QIMR Berghofer Medical Research Institute

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 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.

Domains: Life Sciences [q-bio]; Human health and pathology

Irene Lopez-Perolio, Raphaël Leman, Raquel Behar, Vanessa Lattimore, John F. Pearson, et al.. Alternative splicing and ACMG-AMP-2015-based classification of PALB2 genetic variants: an ENIGMA report. Journal of Medical Genetics, 2019, 56 (7), pp.453-460. ⟨10.1136/jmedgenet-2018-105834⟩. ⟨hal-02194498⟩