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Original article
Alternative splicing and ACMG-AMP-2015-based classification of PALB2 genetic variants: an ENIGMA report
  1. Irene Lopez-Perolio1,
  2. Raphaël Leman2,
  3. Raquel Behar1,
  4. Vanessa Lattimore3,
  5. John F Pearson3,
  6. Laurent Castéra2,
  7. Alexandra Martins4,
  8. Dominique Vaur2,
  9. Nicolas Goardon2,
  10. Grégoire Davy2,
  11. Pilar Garre1,
  12. Vanesa García-Barberán1,
  13. Patricia Llovet1,
  14. Pedro Pérez-Segura1,
  15. Eduardo Díaz-Rubio1,
  16. Trinidad Caldés1,
  17. Kathleen S Hruska5,
  18. Vickie Hsuan6,
  19. Sitao Wu6,
  20. Tina Pesaran6,
  21. Rachid Karam6,
  22. Johan Vallon-Christersson7,
  23. Ake Borg7,
  24. kConFab Investigators8,9,
  25. Alberto Valenzuela-Palomo10,
  26. Eladio A Velasco10,
  27. Melissa Southey11,
  28. Maaike P G Vreeswijk12,
  29. Peter Devilee12,
  30. Anders Kvist7,
  31. Amanda B Spurdle13,
  32. Logan C Walker3,
  33. Sophie Krieger2,
  34. Miguel de la Hoya1
  1. 1 Molecular Oncology Laboratory CIBERONC, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), Madrid, Spain
  2. 2 Laboratory of Clinical Biology and Oncology, Centre François Baclesse, Inserm U1245 Genomics and Personalized Medicine in Cancer and Neurological Disorders, Normandy University, Caen, France
  3. 3 Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand
  4. 4 Inserm U1245 Genomics and Personalized Medecine in Cancer and Neurological Disorders, UNIROUEN, Normandie Université, Normandy Centre for Genomic and Personalized Medicine, Rouen, France
  5. 5 GeneDx, Gaithersburg, Maryland, USA
  6. 6 Ambry Genetics, Aliso Viejo, CA, USA
  7. 7 Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden
  8. 8 Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
  9. 9 The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Australia
  10. 10 Splicing and genetic susceptibility to cancer, Instituto de Biología y Genética Molecular (CSIC-UVa), Valladolid, Spain
  11. 11 Genetic Epidemiology Laboratory, Department of Clinical Pathology, The University of Melbourne, Melbourne, VIC, Australia
  12. 12 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
  13. 13 Molecular Cancer Epidemiology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia
  1. Correspondence to Dr Miguel de la Hoya, Laboratorio OncologíaMolecular, Hospital Clínico San Carlos, Madrid 28040, Spain; mdhoya{at}


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.

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

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  • SK and MdlH are joint senior authors.

  • IL-P and RL contributed equally.

  • Contributors IL-P, RL, RB, VL, JFP, LC, AM, DV, NG, GD, PG, VG-B, PLl, PP-S, ED-R, TC, KSH, VH, SW, TP, RK, JV-C, AV-P and MS, contributed to data acquisition, revised the manuscript for important intellectual content and approved the final version. kConFAB provided research resources used in this study. ABS coordinated the ENIGMA consortium. AB, EAV, MPV, PD, AK, ABS, LW and SK contributed to the conception and design of the study, contributed to obtain all necessary approvals and clearances to conduct the research, contributed to data acquisition, contributed to data analysis, contributed to grant funding, revised the manuscript for important intellectual content and approved the final version. MdH contributed to obtain all necessary approvals and clearances to conduct the research, contributed to the conception and design of the study, contributed to data acquisition, contributed to data analysis, contributed to grant funding, wrote the manuscript and approved the final version.

  • Funding PD, MPGW, EAV, AB, AK and MH have received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 634935. RL is supported by a Normandy-University, Federation-Hospitalo-Universitaire (FHU) grant. VL is supported by a Mackenzie Family Cancer Postdoctoral Fellowship. RB is supported by funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 634935. AM is supported by a French Cancéropôle Nord-Ouest (CNO) grant. AB is supported by Mrs Berta Kamprad Foundation. EAV and MH are supported by Spanish Instituto de Salud Carlos III (ISCIII) funding (grants PI17/00227 to EAV and PI15/00059 to MH), an initiative of the Spanish Ministry of Economy and Innovation partially supported by European Regional Development FEDER Funds. ABS is supported by an NHMRC Senior Research Fellowship (ID1061779). LCW is supported by the Rutherford Discovery Fellowship. SK is supported by Ligue Contre le Cancer, Normandie. kConFab is supported by a grant from the National Breast Cancer Foundation, and previously by the National Health and Medical Research Council (NHMRC), the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia and the Cancer Foundation of Western Australia.

  • 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.

  • Ethics approval Ethics approval Academic Hospital San Carlos ethics committee (reference numbers 15/139 E and 16/505 E). The SCAN-B study has been approved by the Lund Regional Ethical Review Board, Sweden (approval number 2009/658). The fimbriae tissue samples were obtained and analysed with approval by the Lund Regional Ethical Review Board, Sweden (approval number 2014/717). French Biomedicine Agency. CASOHAR trial ethic committee (NTC NTC02560818). Ambry Genetics’ patient’s information has been de-identified, and this study has been approved and carried out in accordance with the recommendations of the Western Institutional Review Board (WIRB; IRB Tracking Number:20171324). Whole-transcriptome RNAseq study was approved by the New Zealand Southern Health and Disability Ethics Committee (12/STH/44).

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data sharing statement Targeted RNAseq data contributed by laboratory 1 is available from Dr Sophie Krieger on reasonable request. Whole-transcriptome RNAseq data generated by laboratory 3 is available from Dr Logan Walker on reasonable request. Targeted RNAseq data contributed by laboratory 2 is available from SCAN-B and Ingrid Hedenfalk, respectively, but restrictions apply to the availability of these data, which were used under licence for the current study, and so are not publicly available. Data are however available from the authors on reasonable request and with permission of SCAN-B or Ingrid Hedenfalk.

  • Correction notice This article has been corrected since it was published Online First. The affiliations have been corrected.

  • Patient consent for publication Not required.