Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants.
Background Disrupted 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. Methods A total of 128 CHEK2 splice-site variants identifie...
| Publicado en: | Clinical Chemistry Vol. 70; no. 1; pp. 319 - 339 |
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| Autores principales: | , , , , , , , , , , , , , |
| Formato: | Journal Article |
| Publicado: |
Oxford University Press / USA
Jan2024
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=174979920&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 174979920 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00099147 10CS jtl: Clinical Chemistry issn: 00099147 maglogo: N pubinfo: dt: Jan2024 vid: 70 iid: 1 pid: 622 pub: Oxford University Press / USA artinfo: ui: 174979920 10.1093/clinchem/hvad125 174979920 ppf: 319 ppct: 20 formats: fmt: – @attributes: type: T – @attributes: type: P tig: atl: Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants. aug: au: Sanoguera-Miralles, Lara Valenzuela-Palomo, Alberto Bueno-Martínez, Elena Esteban-Sánchez, Ada Lorca, Víctor Llinares-Burguet, Inés García-Álvarez, Alicia Pérez-Segura, Pedro Infante, Mar Easton, Douglas F Devilee, Peter Vreeswijk, Maaike P G de la Hoya, Miguel Velasco-Sampedro, Eladio A affil: Splicing and Genetic Susceptibility to Cancer, Unidad de Excelencia Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas—Universidad de Valladolid (CSIC-UVa) , Valladolid , Spain sug: ab: Background Disrupted 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. Methods A 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. Results Of 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. Conclusions Incorporation 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. pubtype: Academic Journal doctype: Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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