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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Genet. | doi: 10.3389/fgene.2019.01139

COMPREHENSIVE ASSESSMENT OF BARD1 mRNA SPLICING WITH IMPLICATIONS FOR VARIANT CLASSIFICATION

  • 1University of Otago, Christchurch, New Zealand
  • 2Lund University, Sweden
  • 3Charles University, Czechia
  • 4Instituto de Investigación Sanitaria del Hospital Clínico San Carlos, Spain
  • 5Centre Hospitalier Universitaire (CHU) de Rouen, France
  • 6Sir Peter MacCallum Department of Oncology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Australia
  • 7Peter MacCallum Cancer Centre, Australia
  • 8QIMR Berghofer Medical Research Institute, Australia

Introduction
Case–control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 mRNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines.

Methods
Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants.

Results
We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or RT-PCR analysis performed for this study. Quantative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterised 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splice events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5.

Conclusions
No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/-1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate.

Keywords: breast cancer, mRNA splicing, nanopore sequencing, RNAseq analysis, Variant classification, ACMG

Received: 12 Aug 2019; Accepted: 21 Oct 2019.

Copyright: © 2019 Walker, Lattimore, Kvist, Kleiblova, Zemankova, de Jong, Wiggins, Hakkaart, Cree, Behar, Houdayer, Investigators, Parsons, Kennedy, Spurdle and De La Hoya. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Dr. Logan C. Walker, University of Otago, Christchurch, Christchurch, 8140, Canterbury, New Zealand, logan.walker@otago.ac.nz
Mx. KconFab Investigators, Sir Peter MacCallum Department of Oncology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria, Australia, heather.thorne@petermac.org