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

López‐Perolio, Irene; Leman, Raphaël; Behar, Raquel; Lattimore, Vanessa; Pearson, John; Castéra, Laurent; Martins, Alexandra; Vaur, Dominique; Goardon, Nicolas; Davy, Grégoire; Garré, Pilar; García-Barberán, Vanesa; Llovet, Patricia; Pérez‐Segura, Pedro; Dı́az-Rubio, Eduardo; Caldés, Trinidad; Hruska, Kathleen S.; Hsuan, Vickie; Wu, Sitao; Pesaran, Tina; Karam, Rachid; Vallon‐Christersson, Johan; Borg, Åke; Investigators, kConFab; Valenzuela-Palomo, Alberto; Velasco, Eladio A.; Southey, Melissa C.; Vreeswijk, Maaike P.G.; Devilee, Peter; Kvist, Anders; Spurdle, Amanda B.; Walker, Logan C.; Krieger, Sophie; Hoya, Miguel de la

doi:10.1136/jmedgenet-2018-105834

Cited by 25 publications

(38 citation statements)

References 26 publications

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“…Libraries were paired-end sequenced on an Illumina HiSeq 2000 (2x50 bp, two breast samples) or a NextSeq 500 (2x75 bp, remaining seven samples). Sequence reads were analyzed as described previously (Lopez-Perolio et al, 2019).…”

Section: Methodsmentioning

confidence: 99%

“…Massively parallel complementary DNA sequencing (RNA-seq) has further advanced our ability to characterize and quantify gene transcripts, and will therefore become a key technology for measuring gene expression changes in clinical diagnostics. Recent studies have begun to demonstrate the utility of RNA-seq for identifying mRNA splicing events in breast cancer susceptibility genes, including BRCA1 (Davy et al, 2017; de Jong et al, 2017; Hojny et al, 2017), BRCA2 (Davy et al, 2017), PALB2 (Lopez-Perolio et al, 2019), and BARD1 (Davy et al, 2017). These studies revealed that key advantages of using RNA-seq over RT-PCR is the ability to quantitatively assess multiple splicing events across the whole transcript in one sequencing assay.…”

Section: Introductionmentioning

confidence: 99%

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Comprehensive Assessment of BARD1 Messenger Ribonucleic Acid Splicing With Implications for Variant Classification

et al. 2019

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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 messenger RNA 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 reverse transcription PCR analysis performed for this study. Quantitative 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 characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing 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.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comprehensive Assessment of BARD1 Messenger Ribonucleic Acid Splicing With Implications for Variant Classification

et al. 2019

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“…This is of course important since some splice variants can result in the expression of a transcript that may be (partially) functional. For instance, several exons in PALB2 (exons 1, 2, 4, 6, 7, 9, 10, and 11-12 combined) can be skipped due to splice site variants and still result in an in-frame transcript (Lopez-Perolio et al, 2019). Such transcripts may still express an isoform of PALB2 with an entire exon deleted, yet retain partial protein function.…”

Section: Toward the Functional Analysis Of Palb2 Vus In Rna Splicingmentioning

confidence: 99%

Functional Characterization of PALB2 Variants of Uncertain Significance: Toward Cancer Risk and Therapy Response Prediction

Boonen

Vreeswijk

Attikum

2020

Front. Mol. Biosci.

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In recent years it has become clear that pathogenic variants in PALB2 are associated with a high risk for breast, ovarian and pancreatic cancer. However, the clinical relevance of variants of uncertain significance (VUS) in PALB2, which are increasingly identified through clinical genetic testing, is unclear. Here we review recent advances in the functional characterization of VUS in PALB2. A combination of assays has been used to assess the impact of PALB2 VUS on its function in DNA repair by homologous recombination, cell cycle regulation and the control of cellular levels of reactive oxygen species (ROS). We discuss the outcome of this comprehensive analysis of PALB2 VUS, which showed that VUS in PALB2's Coiled-Coil (CC) domain can impair the interaction with BRCA1, whereas VUS in its WD40 domain affect PALB2 protein stability. Accordingly, the CC and WD40 domains of PALB2 represent hotspots for variants that impair PALB2 protein function. We also provide a future perspective on the high-throughput analysis of VUS in PALB2, as well as the functional characterization of variants that affect PALB2 RNA splicing. Finally, we discuss how results from these functional assays can be valuable for predicting cancer risk and responsiveness to cancer therapy, such as treatment with PARP inhibitor-or platinumbased chemotherapy.

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“…Variants and transcripts were annotated according to the Human Genome Variation Society (HGVS) guidelines on basis of the BRCA2 GenBank sequence NM000059.1. In order to simplify, we identified transcripts with a shortened code that combines the following symbols (Lopez-Perolio et al, 2019): Δ (skipping of reference exonic sequences), (inclusion of reference intronic sequences), E (exon), p (acceptor shift), q (donor shift). When necessary, the exact number of skipped or retained nucleotides is indicated.…”

Section: Methodsmentioning

confidence: 99%

Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15

et al. 2019

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A relevant fraction of BRCA2 variants is associated with splicing alterations and with an increased risk of hereditary breast and ovarian cancer (HBOC). In this work, we have carried out a thorough study of variants from BRCA2 exons 14 and 15 reported at mutation databases. A total of 294 variants from exons 14 and 15 and flanking intronic sequences were analyzed with the online splicing tools NNSplice and Human Splicing Finder. Fifty-three out of these 294 variants were selected as candidate splicing variants. All variants but one, were introduced into the minigene MGBR2_ex14-20 (with exons 14–20) by site-directed mutagenesis and assayed in MCF-7 cells. Twelve of the remaining 52 variants (23.1%) impaired splicing at different degrees, yielding from 5 to 100% of aberrant transcripts. Nine variants affected the natural acceptor or donor sites of both exons and three affected putative enhancers or silencers. Fluorescent capillary electrophoresis revealed at least 10 different anomalous transcripts: (E14q5), Δ (E14p10), Δ(E14p246), Δ(E14q256), Δ(E14), Δ(E15p12), Δ(E15p13), Δ(E15p83), Δ(E15) and a 942-nt fragment of unknown structure. All transcripts, except for Δ(E14q256) and Δ(E15p12), are expected to truncate the BRCA2 protein. Nine variants induced severe splicing aberrations with more than 90% of abnormal transcripts. Thus, according to the guidelines of the American College of Medical Genetics and Genomics, eight variants should be classified as pathogenic (c.7008-2A > T, c.7008-1G > A, c.7435+1G > C, c.7436-2A > T, c.7436-2A > G, c.7617+1G > A, c.7617+1G > T, and c.7617+2T > G), one as likely pathogenic (c.7008-3C > G) and three remain as variants of uncertain clinical significance or VUS (c.7177A > G, c.7447A > G and c.7501C > T). In conclusion, functional assays by minigenes constitute a valuable strategy to primarily check the splicing impact of DNA variants and their clinical interpretation. While bioinformatics predictions of splice site variants were accurate, those of enhancer or silencer variants were poor (only 3/23 spliceogenic variants) which showed weak impacts on splicing (∼5–16% of aberrant isoforms). So, the Exonic Splicing Enhancer and Silencer (ESE and ESS, respectively) prediction algorithms require further improvement.

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

Cited by 25 publications

References 26 publications

Comprehensive Assessment of BARD1 Messenger Ribonucleic Acid Splicing With Implications for Variant Classification

Comprehensive Assessment of BARD1 Messenger Ribonucleic Acid Splicing With Implications for Variant Classification

Functional Characterization of PALB2 Variants of Uncertain Significance: Toward Cancer Risk and Therapy Response Prediction

Minigene Splicing Assays Identify 12 Spliceogenic Variants of BRCA2 Exons 14 and 15

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