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. 2025 Aug 1;26(15):7455.
doi: 10.3390/ijms26157455.

GnomAD Missense Variants of Uncertain Significance: Implications for p53 Stability and Phosphorylation

Fernando Daniel García-Ayala  1   2 , María de la Luz Ayala-Madrigal  1   2 , Jorge Peregrina-Sandoval  2   3 , José Miguel Moreno-Ortiz  1   2 , Anahí González-Mercado  1   2 , Melva Gutiérrez-Angulo  1   2   4
Affiliations

GnomAD Missense Variants of Uncertain Significance: Implications for p53 Stability and Phosphorylation

Fernando Daniel García-Ayala et al. Int J Mol Sci. .

Abstract

The TP53 gene, frequently mutated across multiple cancer types, plays a pivotal role in regulating the cell cycle and apoptosis through its protein, p53. Missense variants of uncertain significance (VUSs) in TP53 present challenges in understanding their impact on protein function and complicate clinical interpretation. This study aims to analyze the effects of missense VUSs in p53, as reported in the gnomAD database, with a specific focus on their impact on protein stability and phosphorylation. In this study, 33 missense VUSs in TP53 reported in the gnomAD database were analyzed using in silico tools, including PhosphositePlus v6.7.4, the Kinase Library v0.0.11, and Dynamut2. Of these analyzed variants, five disrupted known phosphorylation sites, while another five created new consensus sequences for phosphorylation. Moreover, 20 variants exhibited a moderate destabilizing effect on the protein structure. At least three missense VUSs were identified as potentially affecting p53 function, which may contribute to cancer development. These findings highlight the importance of integrating in silico structural and functional analysis to assess the pathogenic potential of missense VUSs.

Keywords: TP53 gene; VUS; gnomAD; phosphorylation sites; protein stability.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The distribution of 33 variants. Those in bold are located at recognition sites for proteins interacting with p53, which are shown at the top of the figure. TAD1: transactivation domain 1. TAD2: transactivation domain 2. DBD: DNA-binding domain. TET: tetramerization domain. Created with BioRender.com.
Figure 2
Figure 2
Distribution of loss and new phosphorylation sites on p53. PhS: phosphorylation sites. TAD1: transactivation domain 1. TAD2: transactivation domain 2. DBD: DNA-binding domain. TET: tetramerization domain. * Both threonine and serine are susceptible to phosphorylation, and thus the variant may not result in the loss of a phosphorylation site. Created with BioRender.com.
Figure 3
Figure 3
Structural stability analysis and three-dimensional structure of wild-type p53 protein. ΔΔG values for variants in (a) N-terminal, (b) central, and (c) C-terminal regions. (d) A three-dimensional representation of the p53 structure with residues affected by variants highlighted in green. Modeled with ChimeraX 1.10. Created with BioRender.com.
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