Browsing by Author "Braithwaite AW"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Accessing a New Dimension in TP53 Biology: Multiplex Long Amplicon Digital PCR to Specifically Detect and Quantitate Individual TP53 Transcripts
    (MDPI (Basel, Switzerland), 2020-03-24) Lasham A; Tsai P; Fitzgerald SJ; Mehta SY; Knowlton NS; Braithwaite AW; Print CG
    TP53, the most commonly-mutated gene in cancer, undergoes complex alternative splicing. Different TP53 transcripts play different biological roles, both in normal function and in the progression of diseases such as cancer. The study of TP53's alternative RNA splice forms and their use as clinical biomarkers has been hampered by limited specificity and quantitative accuracy of current methods. TP53 RNA splice variants differ at both 5' and 3' ends, but because they have a common central region of 618 bp, the individual TP53 transcripts are impossible to specifically detect and precisely quantitate using standard PCR-based methods or short-read RNA sequencing. Therefore, we devised multiplex probe-based long amplicon droplet digital PCR (ddPCR) assays, which for the first time allow precise end-to-end quantitation of the seven major TP53 transcripts, with amplicons ranging from 0.85 to 1.85 kb. Multiple modifications to standard ddPCR assay procedures were required to enable specific co-amplification of these long transcripts and to overcome issues with secondary structure. Using these assays, we show that several TP53 transcripts are co-expressed in breast cancers, and illustrate the potential for this method to identify novel TP53 transcripts in tumour cells. This capability will facilitate a new level of biological and clinical understanding of the alternatively-spliced TP53 isoforms.
  • Thumbnail Image
    Item
    Breast Cancer Patient Prognosis Is Determined by the Interplay between TP53 Mutation and Alternative Transcript Expression: Insights from TP53 Long Amplicon Digital PCR Assays
    (MDPI (Basel, Switzerland), 2021-03-26) Lasham A; Knowlton N; Mehta SY; Braithwaite AW; Print CG
    The TP53 gene locus is capable of producing multiple RNA transcripts encoding the different p53 protein isoforms. We recently described multiplex long amplicon droplet digital PCR (ddPCR) assays to quantify seven of eight TP53 reference transcripts in human tumors. Here, we describe a new long amplicon ddPCR assay to quantify expression of the eighth TP53 reference transcript encoding ∆40p53α. We then applied these assays, alongside DNA sequencing of the TP53 gene locus, to tumors from a cohort of New Zealand (NZ) breast cancer patients. We found a high prevalence of mutations at TP53 splice sites in the NZ breast cancer cohort. Mutations at TP53 intron 4 splice sites were associated with overexpression of ∆133TP53 transcripts. Cox proportional hazards survival analysis showed that interplay between TP53 mutation status and expression of TP53 transcript variants was significantly associated with patient outcome, over and above standard clinical and pathological information. In particular, patients with no TP53 mutation and a low ratio of TP53 transcripts t2 to t1, which derive from alternative intron 1 acceptor splice sites, had a remarkably good outcome. We suggest that this type of analysis, integrating mutation and transcript expression, provides a step-change in our understanding of TP53 in cancer.