title: Functional and in silico assessment of MAX variants of unknown significance.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
authors.forename: Iñaki
authors.initials: I
authors.lastname: Comino-Méndez
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
authors.forename: Luis J
authors.initials: LJ
authors.lastname: Leandro-García
authors.affiliation: Macromolecular Crystallography Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.|Structural Biology Group, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Montoya
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.|ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Valencia, Spain.
authors.forename: Lucía
authors.initials: L
authors.lastname: Inglada-Pérez
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
authors.forename: Aguirre A
authors.initials: AA
authors.lastname: de Cubas
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Currás-Freixes
authors.affiliation: Department of Molecular Genetics, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Tysoe
authors.affiliation: Department of Clinical Genetics, Guy's and St Thomas' Foundation Trust, Guy's Hospital, London, UK.
authors.forename: Louise
authors.initials: L
authors.lastname: Izatt
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
authors.forename: Rocío
authors.initials: R
authors.lastname: Letón
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
authors.forename: Álvaro
authors.initials: Á
authors.lastname: Gómez-Graña
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
authors.forename: Veronika
authors.initials: V
authors.lastname: Mancikova
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Apellániz-Ruiz
authors.affiliation: Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Mannelli
authors.affiliation: Familial Cancer Clinic and Oncoendocrinology, Veneto Institute of Oncology IRCCS, Padova, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Schiavi
authors.affiliation: INSERM, UMR970, Paris-Cardiovascular Research Center, and Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France.
authors.forename: Judith
authors.initials: J
authors.lastname: Favier
authors.affiliation: INSERM, UMR970, Paris-Cardiovascular Research Center, and Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France.
authors.forename: Anne-Paule
authors.initials: AP
authors.lastname: Gimenez-Roqueplo
authors.affiliation: Department of Medicine, Division of Endocrinology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Henri J L M
authors.initials: HJ
authors.lastname: Timmers
authors.affiliation: Monoclonal Antibodies Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Roncador
authors.affiliation: Department of Pathology, M D Anderson Cancer Center Madrid, Madrid, Spain.
authors.forename: Juan F
authors.initials: JF
authors.lastname: Garcia
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.|ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Valencia, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Rodríguez-Antona
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.|ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Valencia, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Robledo
authors.affiliation: Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain. acascon@cnio.es.|ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Valencia, Spain. acascon@cnio.es.
authors.forename: Alberto
authors.initials: A
authors.lastname: Cascón
mesh_terms: D000310:Adrenal Gland Neoplasms / Q000235:genetics*; D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003198:Computer Simulation; D024721:E-Box Elements; D018095:Germ-Line Mutation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D016716:PC12 Cells; D010235:Paraganglioma / Q000235:genetics*; D010673:Pheochromocytoma / Q000235:genetics*; D051381:Rats
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; C499330:MAX protein, human; C067850:Myc associated factor X
keywords: MAX; PC12 cells; Paraganglioma; Pheochromocytoma; Variants of unknown significance