Germline TP53 mutations result into a constitutive defect of p53 DNA binding and transcriptional response to DNA damage

Abstract

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.

Methylation Tolerance-Based Functional Assay to Assess Variants of Unknown Significance in the MLH1 and MSH2 Genes and Identify Patients With Lynch Syndrome

“…When applied to a prospective set of 48 MLH1 or MSH2 VUS, 40 (83%) could be classified unambiguously as damaging or neutral for MMR, whereas 8 showed an intermediate capacity to restore MMR and therefore could not be definitively classified. The latter may correspond to weak variants with attenuated (but not abrogated) DNA damage response capacity that confer only a moderate risk of cancer, as suggested for other genes 37,38 or pathologies 39 . This hypothesis remains to be tested in the context of LS.…”

Variable population prevalence estimates of germline TP53 variants: A gnomAD-based analysis

“…This variant remains within our exploratory dataset as we only restricted to variants with a MAF less than 1% to allow for broad capture of potentially deleterious variants. The p.R290H variant is often considered as a VUS in clinical practice primarily due to the lack of LFS phenotypic evidence (Arcand et al., ), its “supertrans” classification in yeast functional assays (Kato et al., ), in vitro studies showing apparently normal functional activity (Wang, Niu, Lam, Xiao, & Ren, ; Zerdoumi et al., ), and evidence of potential lack of segregation with disease (Quesnel et al., ). However, its presence in some LFS families reported in IARC, and its current LP classification by some clinical laboratories, warrant further evaluation to determine its true pathogenic potential.…”

O 6 -methylguanine–induced transcriptional mutagenesis reduces p53 tumor-suppressor function

“…The protein product of the CDKN1A gene is p21, which is a cyclin-dependent kinase (CDK) inhibitor that interacts with CDK2, preventing progression through the G1 phase of the cell cycle ( 19 ). Among the well-established effects of mutated p53 protein is its reduced ability to transactivate the CDKN1A gene, resulting in limited production of p21 protein and ensuing failed G1 arrest ( 20 , 21 ). This is particularly problematic when DNA damage occurs in the genome, since lack of G1 arrest in such cases allows the cell to progress to S phase, resulting in an increased risk of mutagenesis and subsequent tumor development.…”