Minigene‐based splicing analysis and ACMG / AMP ‐based tentative classification of 56 ATM variants

The Journal of Pathology, vol. 258, pp. 83–101

Abstract

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.

Authors 15

1. Elena Bueno Martínez

   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 (CSIC‐UVa) Valladolid Spain

2. Lara Sanoguera‐Miralles

   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 (CSIC‐UVa) Valladolid Spain

3. Alberto Valenzuela‐Palomo

   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 (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

   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 (CSIC‐UVa) Valladolid Spain

7. Jamie Allen

   University of Cambridge

   Affiliation as printed

   Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care University of Cambridge Cambridge UK

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

8. Alicia García‐Álvarez

   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 (CSIC‐UVa) Valladolid Spain

9. 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

10. M. Durán

    Instituto de Biomedicina y Genética Molecular de Valladolid

    Affiliation as printed

    Cancer Genetics, Instituto de Biología y Genética Molecular Valladolid Spain

11. Douglas F. Easton

    University of Cambridge

    Affiliation as printed

    Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care University of Cambridge Cambridge UK

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

12. Peter Devilee Aachen Department of Human Genetics Leiden University Medical Center Leiden The Netherlands Department of Human Genetics

    Leiden University Medical Center

    Affiliation as printed

    Department of Human Genetics Leiden University Medical Center Leiden The Netherlands

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

13. Maaike P.G. Vreeswijk Aachen Department of Human Genetics Leiden University Medical Center Leiden The Netherlands Department of Human Genetics

    Leiden University Medical Center

    Affiliation as printed

    Department of Human Genetics Leiden University Medical Center Leiden The Netherlands

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

14. Miguel de la Hoya

    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

    MdlH and EAV-S are joint senior authors

15. Eladio A. Velasco corresponding

    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 (CSIC‐UVa) Valladolid Spain

    MdlH and EAV-S are joint senior authors