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Splicing predictions, minigene analyses, and ACMG-AMP clinical classification of 42 germline PALB2 splice-site variants
- Source :
- Digital.CSIC. Repositorio Institucional del CSIC, instname, Journal of Pathology, 256(3), 321-334. WILEY
- Publication Year :
- 2021
- Publisher :
- WILEY, 2021.
-
Abstract
- 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 ~113,000 women of the large-scale sequencing project BRIDGES (Breast Cancer After Diagnostic Gene Sequencing; 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 ACMG/AMP (American College of Medical Genetics and Genomics and the Association for Molecular Pathology) 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 since 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.<br />PD, MPGV, DFE, MdlH, and EAV have received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 634935. The laboratory of EAV is supported by grants from the Spanish Ministry of Science and Innovation, Plan Nacional de I+D+I 2013-2016, ISCIII (PI17/00227 and PI20/00225) co-funded by FEDER from Regional Development European Funds (European Union) and from the Consejería de Educacion, Junta de Castilla y Leon, ref. CSI242P18 (actuacion cofinanciada P.O. FEDER 2014-2020 de Castilla y Leon). The laboratory of MdlH is supported by a grant from the Spanish Ministry of Science and Innovation, Plan Nacional de I+D+I 2013-2016, ISCIII (PI20/00110) co-funded by FEDER from Regional Development European Funds (European Union). Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Escalera de Excelencia, Junta de Castilla y León (ref. CLU-2019-02). AV-P is supported by a predoctoral fellowship from the Consejería de Educación, Junta de Castilla y León (2018–2022). LS-M is supported by a predoctoral fellowship from the AECC-Scientific Foundation, Sede Provincial de Valladolid (2019–2023). AE-S is supported through the Operational Program for Youth Employment and Youth Employment Initiative (YEI), called by the Community of Madrid in 2020, and co-financed by the European Social Fund.
- Subjects :
- Susceptibility genes
Breast Neoplasms
Splicing
Aberrant splicing
aberrant splicing
splicing
Breast cancer
Minigene
breast cancer
Databases, Genetic
Biomarkers, Tumor
Humans
Protein Isoforms
susceptibility genes
minigene
Functional assay
Clinical interpretation
Exons
VUS
clinical interpretation
Alternative Splicing
Case-Control Studies
PALB2
MCF-7 Cells
Female
RNA Splice Sites
Fanconi Anemia Complementation Group N Protein
functional assay
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Digital.CSIC. Repositorio Institucional del CSIC, instname, Journal of Pathology, 256(3), 321-334. WILEY
- Accession number :
- edsair.dedup.wf.001..fa212c013096bfd6577348a2230424a8