Missense but mis-spliced: germline TP53 variant c.671A > C (p.E224A) and the path from uncertainty to pathogenicity.
TL;DR
Findings supported the classification of the TP53 germline variant c.671A>C (p.E224A) as likely pathogenic, providing a definitive molecular diagnosis for family counselling and sheds light on how certain predicted TP53 missense variants can be linked to disease mechanisms through RNA splicing disruption.
Abstract
The TP53 gene encodes the well-known p53 tumor suppressor protein, which plays a crucial role in preventing cancer development. Germline TP53 variants cause Li-Fraumeni Syndrome (LFS), an autosomal dominant disorder associated with early-onset cancers, including breast cancer, brain tumors, leukemias, bone cancers, and soft tissue sarcomas. Here, we described a germline TP53 variant c.671A>C, located at the penultimate nucleotide of exon 6 and predicted to result in the missense substitution p.E224A. The variant was identified in a 2-year-old child with retroperitoneal rhabdomyosarcoma and with a strong family history suggestive of LFS. Functional assays in yeast and human cells demonstrated wild type-like activity of the protein p.E224A; however, in silico splicing analysis indicated potential splice defects (e.g., SpliceAI score = 0.77). Given this discrepancy, we further investigated this variant using a minigene approach, demonstrating that it causes the skipping of exon 6, likely resulting in a frameshift and the introduction of a premature stop codon. These findings supported the classification of the TP53 germline variant c.671A>C (p.E224A) as likely pathogenic, providing a definitive molecular diagnosis for family counselling. Additionally, the present results sheds light on how certain predicted TP53 missense variants can be linked to disease mechanisms through RNA splicing disruption.