Clinical and Functional Significance of TP53 Exon 4-Intron 4 Splice Junction Variants

Abstract

Germline TP53 splicing variants are uncommon, and their clinical relevance is unknown. However, splice-altering variants at exon 4-intron 4 junctions are relatively enriched in pediatric adrenocortical tumors (ACT). Nevertheless, family histories of cancer compatible with classic Li-Fraumeni syndrome are rarely seen in these patients. We used conventional and in silico assays to determine protein stability, splicing, and transcriptional activity of 10 TP53 variants at exon 4-intron 4 junctions and analyzed their clinical correlates. We reviewed public databases that report the impact of TP53 variants in human cancer and examined individual reports, focusing on family history of cancer. TP53 exon 4-intron 4 junction germline variants were identified in 9 of 75 pediatric ACTs enrolled in the International Pediatric Adrenocortical Tumor Registry and Children's Oncology Group ARAR0332 study. An additional eight independent TP53 variants involving exon 4 splicing were identified in the Pediatric Cancer Genome Project (n = 5,213). These variants resulted in improper expression due to ineffective splicing, protein instability, altered subcellular localization, and loss of function. Clinical case review of carriers of TP53 exon 4-intron 4 junction variants revealed a high incidence of pediatric ACTs and atypical tumor types not consistent with classic Li-Fraumeni syndrome. Germline variants involving TP53 exon 4-intron 4 junctions are frequent in ACT and rare in other pediatric tumors. The collective impact of these germline TP53 variants on the fidelity of splicing, protein structure, and function must be considered in evaluating cancer susceptibility. IMPLICATIONS: Taken together, the data indicate that splice variants at TP53 codon 125 and surrounding bases differentially impacted p53 gene expression and function.

Trial registration: ClinicalTrials.gov NCT00700414 NCT00304070.

Conflict of interest statement

The authors declare no potential conflicts of interest.

©2021 American Association for Cancer Research.

Figures

Figure 1.

TP53 variants at codon 125 and intron 4 boundaries included in this study cohort. Consensus splice donor and acceptor sites are represented by letters in red. Each variant is identified at its location. Protein domains are schematically represented. Colored circles represent proband tumor type (n=26).

Figure 2.

Stability analysis (a) The sidechain of threonine 125 (shown in magenta) makes hydrogen bonds with the backbone carbonyl of glycine 117 and the sidechain of arginine 282, depicted as yellow dashed lines. Mutations at this residue destabilize the protein, either through steric clashes or the inability to form these favorable contacts. Carbon, Nitrogen, and Oxygen atoms are shown in grey, blue, and red, respectively. The coordinated zinc atom is shown as a grey sphere. The phosphate backbone of DNA is shown in orange with the bases depicted as sticks. PDB code: 1tup. (b) Circular dichroism (CD) melting curves of various TP53 T125 mutants show that all mutations at T125 position are destabilizing.

Figure 3.

Experimental evidence of altered splicing for the variant p.T125T (c.375G>A) in a pediatric adrenocortical tumor (#Patient 1). (a) Chromatogram of the p.T125T (c.375G>A) in tumor DNA. (b) Schematic representation of the normal splicing pattern of p53 transcript (purple arrow). RNA-seq of tumor DNA showing retention of intron 4 due to loss of donor splicing (green arrow). Sequence analysis of TP53 cDNA from the same tumor showing the use of cryptic donor site in exon 4 (orange arrow). (c) Western blot analysis of patient #1 ACT (p.T125T) and pediatric ACTs with p.G266E and p.R337H variants. (d) Loss of p53 expression (right panel) in patient #1 ACT (H&E left panel) harboring the p.T125T (c.375G>A) variant.

Figure 4.

cDNA transcripts from transfected H1299 cells. Agarose gel products (upper panel) and representative canonical and cryptic splice use (lower panel) for the TP53 variants included in this study.

Figure 5.

Transcriptional activity of TP53 variants. (a) Attenuated p53 luciferase activity in T125A and T125M but compromised in other variants in Saos-2 and H1299 transfected cells compared to that in the hotspot DBD mutant R175H. (b) Corresponding p53 protein expression was determined by western blot analysis. Data represent two independent experiments, with each experiment including three biologic replicates. Asterisks indicate statistical significance, as determined by unpaired t-test. **** = P<0.0001 and ** = P=0.0086.

Figure 6.

Subcellular localizations of TP53 variants. Western blot analysis of p53, PARP, (nuclear marker), and HSP90 (cytoplasmic marker) after separation of nuclear (Nuc) and cytoplasmic (Cyt) protein fractions. Negative control was CMV only. Wild-type TP53 variant constructs are shown at the top. Peptide sizes (in kDa) are shown to the left.