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Functional and in silico assessment of MAX variants of unknown significance

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Abstract

The presence of germline mutations affecting the MYC-associated protein X (MAX) gene has recently been identified as one of the now 11 major genetic predisposition factors for the development of hereditary pheochromocytoma and/or paraganglioma. Little is known regarding how missense variants of unknown significance (VUS) in MAX affect its pivotal role in the regulation of the MYC/MAX/MXD axis. In the present study, we propose a consensus computational prediction based on five “state-of-the-art” algorithms. We also describe a PC12-based functional assay to assess the effects that 12 MAX VUS may have on MYC’s E-box transcriptional activation. For all but two of these 12 VUS, the functional assay and the consensus computational prediction gave consistent results; we classified seven variants as pathogenic and three as nonpathogenic. The introduction of wild-type MAX cDNA into PC12 cells significantly decreased MYC’s ability to bind to canonical E-boxes, while pathogenic MAX proteins were not able to fully repress MYC activity. Further clinical and molecular evaluation of variant carriers corroborated the results obtained with our functional assessment. In the absence of clear heritability, clinical information, and molecular data, consensus computational predictions and functional models are able to correctly classify VUS affecting MAX.

Key messages

  • A functional assay assesses the effects of MAX VUS over MYC transcriptional activity.

  • A consensus computational prediction and the functional assay show high concordance.

  • Variant carriers’ clinical and molecular data support the functional assessment.

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Acknowledgments

This work was supported in part by the Fondo de Investigaciones Sanitarias (projects PI12/00236 and PI11/01359 to A.C. and M.R., respectively), the Fundación Mutua Madrileña (project AP2775/2008 to M.R.), and a grant from the European Community’s Seventh Framework Programme (ENS@T-CANCER; HEALTH-F2-2010-259735). Aguirre A. de Cubas and Veronika Mancikova are predoctoral fellows in “la Caixa”/CNIO International PhD Programme. Lucía Inglada-Pérez and Iñaki Comino-Méndez are predoctoral fellows with the CIBERER and the Fundacion Ferrer, respectively.

Conflict of Interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain

    Iñaki Comino-Méndez, Luis J Leandro-García, Lucía Inglada-Pérez, Aguirre A. de Cubas, María Currás-Freixes, Rocío Letón, Álvaro Gómez-Graña, Veronika Mancikova, María Apellániz-Ruiz, Cristina Rodríguez-Antona, Mercedes Robledo & Alberto Cascón

  2. Macromolecular Crystallography Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

    Guillermo Montoya

  3. Structural Biology Group, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Guillermo Montoya

  4. ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Valencia, Spain

    Lucía Inglada-Pérez, Cristina Rodríguez-Antona, Mercedes Robledo & Alberto Cascón

  5. Department of Molecular Genetics, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK

    Carolyn Tysoe

  6. Department of Clinical Genetics, Guy’s and St Thomas’ Foundation Trust, Guy’s Hospital, London, UK

    Louise Izatt

  7. Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy

    Massimo Mannelli

  8. Familial Cancer Clinic and Oncoendocrinology, Veneto Institute of Oncology IRCCS, Padova, Italy

    Francesca Schiavi

  9. INSERM, UMR970, Paris-Cardiovascular Research Center, and Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France

    Judith Favier & Anne-Paule Gimenez-Roqueplo

  10. Department of Medicine, Division of Endocrinology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

    Henri J. L. M. Timmers

  11. Monoclonal Antibodies Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

    Giovanna Roncador

  12. Department of Pathology, M D Anderson Cancer Center Madrid, Madrid, Spain

    Juan F. Garcia

Authors
  1. Iñaki Comino-Méndez
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  2. Luis J Leandro-García
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  3. Guillermo Montoya
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  4. Lucía Inglada-Pérez
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  20. Cristina Rodríguez-Antona
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  22. Alberto Cascón
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Corresponding author

Correspondence to Alberto Cascón.

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Comino-Méndez, I., Leandro-García, L.J., Montoya, G. et al. Functional and in silico assessment of MAX variants of unknown significance. J Mol Med 93, 1247–1255 (2015). https://doi.org/10.1007/s00109-015-1306-y

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  • DOI: https://doi.org/10.1007/s00109-015-1306-y

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