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Functional and in silico assessment of MAX variants of unknown significance
Iñaki Comino-Méndez1, Luis J Leandro-García1, Guillermo Montoya2,3
1Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
Unlabelled:
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.
Insights
Germline mutations in the MAX gene are linked to hereditary pheochromocytoma. A new functional assay and computational predictions accurately classify MAX variants of unknown significance, aiding in risk assessment.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- Oncology
Background:
- Germline mutations in the MAX gene are a known risk factor for hereditary pheochromocytoma and paraganglioma.
- The role of MAX variants of unknown significance (VUS) in regulating the MYC/MAX/MXD axis is not well understood.
Purpose of the Study:
- To develop and validate a method for classifying MAX VUS.
- To assess the impact of MAX VUS on MYC transcriptional activity.
Main Methods:
- Utilized five computational prediction algorithms to assess MAX VUS.
- Developed a PC12 cell-based functional assay to evaluate MYC E-box transcriptional activation by MAX VUS.
- Correlated in silico predictions with functional assay results and clinical data.
Main Results:
- A consensus computational prediction and functional assay showed high concordance for 12 MAX VUS.
- Seven variants were classified as pathogenic, and three as nonpathogenic.
- Pathogenic MAX variants failed to fully repress MYC activity, unlike wild-type MAX.
Conclusions:
- A combined computational and functional approach accurately classifies MAX VUS.
- This methodology aids in understanding the pathogenicity of MAX variants in hereditary cancer syndromes.
- Clinical and molecular data from variant carriers support the functional assessment's reliability.
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