Dataset Information

Splicing predictions, minigene analyses, and ACMG-AMP clinical classification of 42 germline PALB2 splice-site variants.

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 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.

SUBMITTER: Valenzuela-Palomo A

PROVIDER: S-EPMC9306493 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The ataxia telangiectasia-mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss-of-function variants are associated with 2-fold increased breast cancer risk. We aimed at identifying and classifying spliceogenic ATM variants detected in subjects of the large-scale sequencing project BRIDGES. A total of 381 variants at the intron-exon boundaries were identified, 128 of which were predicted to be spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis. Four functional minigenes (mgATM) spanning exons 4-9, 11-17, 25-29, and 49-52 were constructed in the splicing plasmid pSAD. Selected variants were genetically engineered into the four constructs and assayed in MCF-7/HeLa cells. Forty-eight variants (85.7%) impaired splicing, 32 of which did not show any trace of the full-length (FL) transcript. A total of 43 transcripts were identified where the most prevalent event was exon/multi-exon skipping. Twenty-seven transcripts were predicted to truncate the ATM protein. A tentative ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme that integrates mgATM data allowed us to classify 29 ATM variants as pathogenic/likely pathogenic and seven variants as likely benign. Interestingly, the likely pathogenic variant c.1898+2T>G generated 13% of the minigene FL-transcript due to the use of a noncanonical GG-5'-splice-site (0.014% of human donor sites). Circumstantial evidence in three ATM variants (leakiness uncovered by our mgATM analysis together with clinical data) provides some support for a dosage-sensitive expression model in which variants producing ≥30% of FL-transcripts would be predicted benign, while variants producing ≤13% of FL-transcripts might be pathogenic. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Project description: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.

Dataset Information

Splicing predictions, minigene analyses, and ACMG-AMP clinical classification of 42 germline PALB2 splice-site variants.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets