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J Med Genet. 2019 Jul;56(7):453-460. doi: 10.1136/jmedgenet-2018-105834. Epub 2019 Mar 19.

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

Author information

Molecular Oncology Laboratory CIBERONC, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), Madrid, Spain.
Laboratory of Clinical Biology and Oncology, Centre François Baclesse, Inserm U1245 Genomics and Personalized Medicine in Cancer and Neurological Disorders, Normandy University, Caen, France.
Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.
Inserm U1245 Genomics and Personalized Medecine in Cancer and Neurological Disorders, UNIROUEN, Normandie Université, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
GeneDx, Gaithersburg, Maryland, USA.
Ambry Genetics, Aliso Viejo, CA, USA.
Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.
Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.
The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Australia.
Splicing and genetic susceptibility to cancer, Instituto de Biología y Genética Molecular (CSIC-UVa), Valladolid, Spain.
Genetic Epidemiology Laboratory, Department of Clinical Pathology, The University of Melbourne, Melbourne, VIC, Australia.
Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Molecular Cancer Epidemiology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia.
Contributed equally



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.


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.


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 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.


acmg-amp guidelines; palb2; pvs1; splicing; variant classification

Conflict of interest statement

Competing interests: VH, SW, PT, RK were employees of Ambry Genetics when they were engaged with this project. KSH was employee of GeneDx when she was engaged with this project. EDR has consulting or advisory roles in Amgen, Bayer, Genómica, Servier and Merck. EDR has got research funding from: Roche, Merck-Serono, Amgen, AstraZeneca and Sysmex.

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