Minigene-based splicing analysis and ACMG/AMP-based tentative classification of 56 ATM variants.
ATM
VUS
aberrant splicing
hereditary breast cancer
minigenes
splicing
splicing assay
susceptibility genes
variant classification
Journal
The Journal of pathology
ISSN: 1096-9896
Titre abrégé: J Pathol
Pays: England
ID NLM: 0204634
Informations de publication
Date de publication:
09 2022
09 2022
Historique:
revised:
11
05
2022
received:
24
03
2022
accepted:
08
06
2022
pubmed:
19
6
2022
medline:
16
8
2022
entrez:
18
6
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 35716007
doi: 10.1002/path.5979
pmc: PMC9541484
doi:
Substances chimiques
Ataxia Telangiectasia Mutated Proteins
EC 2.7.11.1
ATM protein, human
EC 2.7.11.1
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
83-101Informations de copyright
© 2022 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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