Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants

Clinical Chemistry, vol. 70, pp. 319–338

Abstract

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.

Authors 14

1. Lara Sanoguera‐Miralles

   Universidad de Valladolid · Consejo Superior de Investigaciones Científicas · Instituto de Biomedicina y Genética Molecular de Valladolid

   Affiliation as printed

   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

2. Alberto Valenzuela‐Palomo

   Universidad de Valladolid · Consejo Superior de Investigaciones Científicas · Instituto de Biomedicina y Genética Molecular de Valladolid

   Affiliation as printed

   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

3. Elena Bueno Martínez

   Universidad de Valladolid · Consejo Superior de Investigaciones Científicas · Instituto de Biomedicina y Genética Molecular de Valladolid

   Affiliation as printed

   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

4. Ada Esteban‐Sánchez

   Hospital Clínico San Carlos · Instituto de Investigación Sanitaria del Hospital Clínico San Carlos · Centro de Investigación Biomédica en Red de Cáncer

   Affiliation as printed

   Molecular Oncology Laboratory CIBERONC, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos) , Madrid , Spain

5. Víctor Lorca

   Hospital Clínico San Carlos · Instituto de Investigación Sanitaria del Hospital Clínico San Carlos · Centro de Investigación Biomédica en Red de Cáncer

   Affiliation as printed

   Molecular Oncology Laboratory CIBERONC, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos) , Madrid , Spain

6. Inés Llinares‐Burguet

   Universidad de Valladolid · Consejo Superior de Investigaciones Científicas · Instituto de Biomedicina y Genética Molecular de Valladolid

   Affiliation as printed

   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

7. Alicia García‐Álvarez

   Universidad de Valladolid · Consejo Superior de Investigaciones Científicas · Instituto de Biomedicina y Genética Molecular de Valladolid

   Affiliation as printed

   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

8. Pedro Pérez‐Segura

   Hospital Clínico San Carlos · Instituto de Investigación Sanitaria del Hospital Clínico San Carlos · Centro de Investigación Biomédica en Red de Cáncer

   Affiliation as printed

   Molecular Oncology Laboratory CIBERONC, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos) , Madrid , Spain

9. Mar Infante

   Consejo Superior de Investigaciones Científicas · Instituto de Biomedicina y Genética Molecular de Valladolid

   Affiliation as printed

   Cancer Genetics, Instituto de Biología y Genética Molecular (CSIC-UVa) , Valladolid , Spain

10. Douglas F. Easton

    University of Cambridge

    Affiliation as printed

    Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge , CB1 8RN, Cambridge , United Kingdom

11. Peter Devilee Aachen Department of Human Genetics

    Leiden University Medical Center

    Affiliation as printed

    Department of Human Genetics, Leiden University Medical Center , Leiden , the Netherlands

12. Maaike P.G. Vreeswijk Aachen Department of Human Genetics

    Leiden University Medical Center

    Affiliation as printed

    Department of Human Genetics, Leiden University Medical Center , Leiden , the Netherlands

13. Miguel de la Hoya corresponding

    Hospital Clínico San Carlos · Instituto de Investigación Sanitaria del Hospital Clínico San Carlos · Centro de Investigación Biomédica en Red de Cáncer

    Affiliation as printed

    Molecular Oncology Laboratory CIBERONC, Hospital Clínico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos) , Madrid , Spain

14. Eladio A. Velasco corresponding

    Universidad de Valladolid · Consejo Superior de Investigaciones Científicas · Instituto de Biomedicina y Genética Molecular de Valladolid

    Affiliation as printed

    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