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

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. Functional studies in yeast and human cells demonstrated that TP53 variants can have various effects, such as partial or complete loss of function and even gain of pro-oncogenic activities. Here, we identified a germline TP53 variant c.671A>C, resulting in the missense mutant protein p.E224A in the context of early-onset retroperitoneal rhabdomyosarcoma occurring in a child with a notable family history of cancer, suggestive of LFS. The variant was initially classified as a variant of uncertain significance (VUS). Functional assays in yeast and human cells demonstrated wild type-like activity of the protein p.E224A; however, in silico analysis predicted at RNA level a splicing defect, which we further investigated using a minigene approach. This analysis showed that the variant c.671A>C causes the skipping of exon 6, potentially introducing a frameshift in cDNA and a premature stop codon, which likely triggers nonsense-mediated mRNA decay; the loss of heterozygosity at the c.671 position in the parent’s TP53 transcript further confirmed the splicing impairment. In summary, these findings supported reclassifying the TP53 germline variant c.671A>C (p.E224A) from VUS to likely pathogenic, providing a definitive molecular diagnosis for family counseling. Additionally, this study sheds light on how certain TP53 variants that are defined as missense, can be linked to disease mechanisms through RNA splicing disruption, highlighting the need for their deep functional assessment. Competing Interest Statement The authors have declared no competing interest. Funding Statement This work was partially funded by the Italian Ministry of Health (5×1000 funds 2020 to P.Menichini and P.Monti; Current Research 2022-24 to P. Menichini); AIRC IG Grant ID 27852 to P. Malatesta; E.T.S. Engineering Ship Technology Pte Ltd Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was conducted in accordance with the Declaration of Helsinki, and Ethics approval was obtained from the medical ethics committee established by Gaslini Children Hospital (Comitato Etico Territoriale della Regione Liguria approval code: CER Liguria 399/2021). Written informed consent was obtained from parents for clinical testing and publication of genetic and clinical data, as well as clinical photographs. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes