Blood functional assay for rapid clinical interpretation of germline variants.

Background: The interpretation of germline variants is critical to ensure appropriate medical management of patients with cancer and follow-up of variant carriers. This interpretation remains complex and is becoming a growing challenge considering the exponential increase in tests. We developed a functional assay directly performed on patients' blood.

Methods: Peripheral blood mononuclear cells were cultured, activated, exposed to doxorubicin and the p53-mediated transcriptional response was quantified using reverse transcription-multiplex ligation probe amplification and RT-QMPSF assays, including 10 p53 targets selected from transcriptome analysis, and two amplicons to measure p53 mRNA levels. We applied this blood functional assay to 77 patients addressed for analysis.

Results: In 51 wild-type individuals, the mean p53 functionality score was 12.7 (range 7.5-22.8). Among eight individuals harbouring likely pathogenic or pathogenic variants, the scores were reduced (mean 4.8, range 3.1-7.1), and p53 mRNA levels were reduced in patients harbouring truncating variants. We tested 14 rare unclassified variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro191Arg), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del), c.-117G>T) and 12 yielded functionally abnormal scores. Remarkably, the assay revealed that the c.*1175A>C polymorphic variant within poly-adenylation site can impact p53 function with the same magnitude as a null variant, when present on both alleles, and may act as a modifying factor in pathogenic variant carriers.

Conclusion: This blood p53 assay should therefore be a useful tool for the rapid clinical classification of germline variants and detection of non-coding functional variants.