Splicing predictions, minigene analyses, and ACMG-AMP clinical classification of 42 germline PALB2 splice-site variants.
PALB2
VUS
aberrant splicing
breast cancer
clinical interpretation
functional assay
minigene
splicing
susceptibility genes
Journal
The Journal of pathology
ISSN: 1096-9896
Titre abrégé: J Pathol
Pays: England
ID NLM: 0204634
Informations de publication
Date de publication:
03 2022
03 2022
Historique:
revised:
18
11
2021
received:
16
07
2021
accepted:
26
11
2021
pubmed:
1
12
2021
medline:
1
3
2022
entrez:
30
11
2021
Statut:
ppublish
Résumé
PALB2 loss-of-function variants confer high risk of developing breast cancer. Here we present a systematic functional analysis of PALB2 splice-site variants detected in approximately 113,000 women in the large-scale sequencing project Breast Cancer After Diagnostic Gene Sequencing (BRIDGES; https://bridges-research.eu/). Eighty-two PALB2 variants at the intron-exon boundaries were analyzed with MaxEntScan. Forty-two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1-12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site-directed mutagenesis and assayed in MCF-7/MDA-MB-231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full-length transcripts of each minigene. More than 30 different variant-induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in-house adaptation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) variant classification scheme. Up to 23 variants were classified as pathogenic/likely pathogenic. Remarkably, three ±1,2 variants (c.49-2A>T, c.108+2T>C, and c.211+1G>A) were classified as variants of unknown significance, as they produced significant amounts of either in-frame transcripts of unknown impact on the PALB2 protein function or the minigene full-length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG-AMP rationale. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Identifiants
pubmed: 34846068
doi: 10.1002/path.5839
pmc: PMC9306493
doi:
Substances chimiques
Biomarkers, Tumor
0
Fanconi Anemia Complementation Group N Protein
0
PALB2 protein, human
0
Protein Isoforms
0
RNA Splice Sites
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
321-334Commentaires et corrections
Type : ErratumIn
Informations de copyright
© 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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