Functional Analysis Of Novel Germline TP53 Variants
Badr ID SAID, The Hospital for Sick Children, Canada
KIM H. 4
, TRAN J. 1,2
, NOVOKMET A. 1
, MALKIN D. 1,2,3
1 Division of Hematology/Oncology, The Hospital for Sick Children, Toronto, Canada
2 Department of Medical Biophysics, University of Toronto, Toronto, Canada
3 Department of Pediatrics, University of Toronto, Toronto, Canada
4 Department of Molecular Genetics and Microbiology, University of Toronto. Toronto, Canada
Purpose: Li-Fraumeni Syndrome (LFS) is a rare autosomal dominant familial cancer syndrome, characterized by multiple malignancies and frequent germline alterations in TP53. In this study, we highlight four unclassified exonic p53 variants detected in patients with a suspected diagnosis of LFS. Given the unknown clinical significance of these variants, we sought to uncover their impact on p53 function and tumor development.
Methods: Using site-directed mutagenesis, we generated pCMV-plasmids carrying the aforementioned p53 mutants. TP53-null H1299 and SAOS-2 cells were subsequently assayed using the Dual-Luciferase Assay to examine p53 transactivation levels. The tumor suppressive capacity of these p53 variants was also assessed by colony formation and Western blotting.
Results: We report for the first time the discovery of two novel functional variants in codons 191(c.572C>G; p.P191R) and 360 (c.1079G>T; p.G360V), located, respectively, in the DNA binding domain and in a linker region near the tetramerization domain of TP53. Our data revealed that while the P191R variant decreased the transactivation levels of several p53 targets (RGC, p21 and BAX), it failed to segregate with disease status. The G360V variant, on the other hand, behaved in a paradoxical fashion by causing a stark upregulation in the activity of the same p53 response elements. This enhanced tumor suppressive effect was also observed at the level of colony formation and caspase-3 activation.
Conclusions: We report the discovery of two novel functional p53 polymorphisms, P191R and G360V, which may act as phenotypic modifiers in LFS. While the P191R led to a decrease in p53 transactivation, the G360V variant caused a dramatic activation of p53 response elements. In the future, the enhanced transactivation effects of G360V-p53 may prove useful in designing more efficacious p53-based gene therapies.
Funding source: Canadian Institutes of Health Research (#MOP-300105).