Missense but mis-spliced: germline TP53 variant c.671A > C (p.E224A) and the path from uncertainty to pathogenicity.
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.