Abstract
Background Li-Fraumeni syndrome (LFS) predisposes individuals to a wide range of cancers from childhood onwards, underscoring the crucial need for accurate interpretation of germline TP53 variants for optimal clinical management of patients and families. Several unclassified variants, particularly those potentially affecting splicing, require specialised testing. One such example is the NM_000546.6:c.1101-2A>C (rs587781664) variant, located at the splice acceptor site of the last intron of TP53 , identified in a female patient with breast cancer diagnosed in her 20s. Methods To interpret this variant, which has been classified as a variant of uncertain significance (VUS), we developed specific assays including a p53 functional assay, RT-QMPSF, Splice and Expression Analyses by exon Ligation and High-Throughput Sequencing and long RT-droplet digital PCR. Results We demonstrated a loss of p53 transcriptional activity, and a half reduction in TP53 mRNA expression. Additionally, we detected the use of a novel alternative last exon downstream of exon 11, which we have named exon 12. This transcript, typically detectable at low levels in most individuals, was found to be more highly expressed in the c.1101-2A>C carrier, predominantly transcribed from the mutant allele due to the disruption of the splice acceptor site in intron 10. Conclusion By combining these approaches, we successfully reclassified this intronic VUS as ‘pathogenic’, enabling appropriate genetic counselling for the patient and her family. Additionally, we identified a novel TP53 alternative transcript that is expressed in both physiological and pathological contexts, with heightened expression in the patient with LFS. This discovery provides a basis for further investigation into the role of TP53 isoforms in LFS oncogenesis.