RT info:eu-repo/semantics/article
T1 Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants
A1 Sanoguera-Miralles, Lara
A1 Valenzuela-Palomo, Alberto
A1 Bueno-Martínez, Elena
A1 Esteban-Sánchez, Ada
A1 Lorca, Víctor
A1 Llinares-Burguet, Inés
A1 García-Álvarez, Alicia
A1 Pérez-Segura, Pedro
A1 Infante, Mar
A1 Easton, Douglas F
A1 Devilee, Peter
A1 Vreeswijk, Maaike P G
A1 de la Hoya, Miguel
A1 Velasco-Sampedro, Eladio A
AB BackgroundDisrupted pre-mRNA splicing is a frequent deleterious mechanism in hereditary cancer. We aimed to functionally analyze candidate spliceogenic variants of the breast cancer susceptibility gene CHEK2 by splicing reporter minigenes.MethodsA total of 128 CHEK2 splice-site variants identified in the Breast Cancer After Diagnostic Gene Sequencing (BRIDGES) project (https://cordis.europa.eu/project/id/634935) were analyzed with MaxEntScan and subsetted to 52 variants predicted to impact splicing. Three CHEK2 minigenes, which span all 15 exons, were constructed and validated. The 52 selected variants were then genetically engineered into the minigenes and assayed in MCF-7 (human breast adenocarcinoma) cells.ResultsOf 52 variants, 46 (88.5%) impaired splicing. Some of them led to complex splicing patterns with up to 11 different transcripts. Thirty-four variants induced splicing anomalies without any trace or negligible amounts of the full-length transcript. A total of 89 different transcripts were annotated, which derived from different events: single- or multi-exon skipping, alternative site-usage, mutually exclusive exon inclusion, intron retention or combinations of the abovementioned events. Fifty-nine transcripts were predicted to introduce premature termination codons, 7 kept the original open-reading frame, 5 removed the translation start codon, 6 affected the 5′UTR (Untranslated Region), and 2 included missense variations. Analysis of variant c.684-2A > G revealed the activation of a non-canonical TG-acceptor site and exon 6 sequences critical for its recognition.ConclusionsIncorporation of minigene read-outs into an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme allowed us to classify 32 CHEK2 variants (27 pathogenic/likely pathogenic and 5 likely benign). However, 20 variants (38%) remained of uncertain significance, reflecting in part the complex splicing patterns of this gene.
PB Oxford Academic
SN 0009-9147
YR 2024
FD 2024
LK https://uvadoc.uva.es/handle/10324/73288
UL https://uvadoc.uva.es/handle/10324/73288
LA spa
NO Clinical Chemistry, Volume 70, Issue 1, January 2024, Pages 319–338
DS UVaDOC
RD 05-feb-2025