Background: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3'ss) and are essential for splicing of premature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3'ss. Results: We used a large set of constitutive and alternative human 3'ss collected from Ensembl (n = 264,787 3'ss) and from in-house RNAseq experiments (n = 51,986 3'ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3'ss (99.48 and 65.84% accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17%. Conclusions: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3'ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area.

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Li-Fraumeni Syndrome (LFS) results from heterozygous germline mutations of TP53, encoding a key transcriptional factor activated in response to DNA damage. We have recently shown, from a large LFS series, that dominant-negative missense mutations are the most clinically severe and, thanks to a new p53 functional assay in lymphocytes, that they alter the p53 transcriptional response to DNA damage more drastically than null mutations. In this study, we first confirmed this observation by performing the p53 functional assay in lymphocytes from 56 TP53 mutation carriers harbouring 35 distinct alterations. Then, to compare the impact of the different types of germline TP53 mutations on DNA binding, we performed chromatin immunoprecipitation-sequencing (ChIP-Seq) in lymphocytes exposed to doxorubicin. ChIP-Seq performed in wild-type TP53 control lymphocytes accurately mapped 1287 p53-binding sites. New p53-binding sites were validated using a functional assay in yeast. ChIP-Seq analysis of LFS lymphocytes carrying TP53 null mutations (p.P152Rfs*18 or complete deletion) or the low penetrant ‘Brazilian’ p.R337H mutation revealed a moderate decrease of p53-binding sites (949, 580 and 620, respectively) and of ChIP-Seq peak depths. In contrast, analysis of LFS lymphocytes with TP53 dominant-negative missense mutations p.R273H or p.R248W revealed only 310 and 143 p53-binding sites, respectively, and the depths of the corresponding peaks were drastically reduced. Altogether, our results show that TP53 mutation carriers exhibit a constitutive defect of the transcriptional response to DNA damage and that the clinical severity of TP53 dominant-negative missense mutations is explained by a massive and global alteration of p53 DNA binding.

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BackgroundThe interpretation of germline TP53 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 TP53 tests. We developed a functional assay directly performed on patients’ blood.MethodsPeripheral 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 TP53 analysis.ResultsIn 51 wild-type TP53 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 TP53 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.ConclusionThis blood p53 assay should therefore be a useful tool for the rapid clinical classification of germline TP53 variants and detection of non-coding functional variants.

Background: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3’ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3’ss. Results: We used a large set of constitutive and alternative human 3’ss collected from Ensembl (n = 264,787 3’ss) and from in-house RNAseq experiments (n = 51,986 3’ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3’ss (99.48 % and 65.84 % accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17 %. Conclusions: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3’ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area. Keywords: Branch Point, Prediction, RNA, Benchmark, HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR, RNABPS, Variants

No abstract

Branch points (BPs) map within short motifs upstream of acceptor splice sites (3’ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3’ss. We used a large set of constitutive and alternative human 3’ss collected from Ensembl (n = 264,787 3’ss) and from in-house RNAseq experiments (n = 51,986 3’ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3’ss (99.48 % and 65.84 % accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17 %. Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3’ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area.