Functional and in silico assessment of MAX variants of unknown significance

J Mol Med (Berl). 2015 Nov;93(11):1247-55. doi: 10.1007/s00109-015-1306-y.

Iñaki Comino-Méndez ¹, Luis J Leandro-García ¹, Guillermo Montoya ² ³, Lucía Inglada-Pérez ¹ ⁴, Aguirre A de Cubas ¹, María Currás-Freixes ¹, Carolyn Tysoe ⁵, Louise Izatt ⁶, Rocío Letón ¹, Álvaro Gómez-Graña ¹, Veronika Mancikova ¹, María Apellániz-Ruiz ¹, Massimo Mannelli ⁷, Francesca Schiavi ⁸, Judith Favier ⁹, Anne-Paule Gimenez-Roqueplo ⁹, Henri J L M Timmers ¹⁰, Giovanna Roncador ¹¹, Juan F Garcia ¹², Cristina Rodríguez-Antona ¹ ⁴, Mercedes Robledo ¹ ⁴, Alberto Cascón ¹³ ¹⁴

Affiliations

1 Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
2 Macromolecular Crystallography Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
3 Structural Biology Group, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
4 ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Valencia, Spain.
5 Department of Molecular Genetics, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK.
6 Department of Clinical Genetics, Guy's and St Thomas' Foundation Trust, Guy's Hospital, London, UK.
7 Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy.
8 Familial Cancer Clinic and Oncoendocrinology, Veneto Institute of Oncology IRCCS, Padova, Italy.
9 INSERM, UMR970, Paris-Cardiovascular Research Center, and Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France.
10 Department of Medicine, Division of Endocrinology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
11 Monoclonal Antibodies Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
12 Department of Pathology, M D Anderson Cancer Center Madrid, Madrid, Spain.
13 Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain. acascon@cnio.es.
14 ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Valencia, Spain. acascon@cnio.es.

PMID: 26070438 DOI: 10.1007/s00109-015-1306-y

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.

Keywords

MAX; PC12 cells; Paraganglioma; Pheochromocytoma; Variants of unknown significance.

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