Background: Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome associated with TP53 germline mutations and characterized by increased risk to multiple early-onset cancers. Studies in families from Southern and Southeastern Brazil have identified a germline founder mutation in the TP53 gene, the p.R337H, in high population prevalence (∼0.3%). Unlike the majority of the mutations in TP53, which are missense mutations located in the DNA-binding domain (DBD) of the protein, the TP53 p.R337H is located in the oligomerization domain (OD). Recently, we identified a new rare germline variant, the rs78378222 (A>C), in the 3′ UTR of the gene in 7/130 of the patients who tested negative for coding germline TP53 mutations. Based on the broad spectrum of p53 functions, which include metabolism and DNA damage response regulation, in the present study we aimed to characterize the functional impact of germline TP53 mutations identified in Brazilian families. Methods: Primary fibroblasts from p.R337H/WT (n = 2), p.R337H/p.R337H (n = 1), DBD mutations (n = 2), rs78378222[C] patients and from WT p53 controls (n = 2) were included. The DNA damage induction was performed using UVB (0.2W/m2) and ionizing irradiation (IR) (1 Gy). Results: First, we observed that all p53-mutated fibroblasts appeared to have lower p53 levels but higher levels of reactive oxygen species (ROS) and mitochondrial biomass compared to controls. Fibroblasts homozygous to p.R337H mutation presented more pronounced ROS production and, notably, higher expression of two antioxidant proteins, SOD2 and GPX1. On the other hand, the results of high-resolution respirometry showed higher oxygen consume in DBD-mutated fibroblasts. Second, using a specific multiplexed enzymatic DNA repair assay on biochip, we simultaneously investigated several DNA repair pathways. Overall, we observed a high DNA repair activation profile in p.R337H/p.R337H and poor DNA repair capacity in DBD-mutated fibroblasts. Interestingly, all p53-mutated fibroblasts showed higher repair activity of apurinic/pyrimidinic (AP) sites, a DNA lesion caused mainly by ROS attacks. Conclusions: Our data indicate important alterations in both metabolism and DNA damage response in normal cells (fibroblasts) from LFS patients, suggesting haploinsufficiency mechanism associated to these mutations. We were also able to demonstrate distinct phenotypes according to mutational status, which may explain, at least in part, the clinical variability in LFS.

Citation Format: Gabriel Macedo, Sylvie Sauvaigo, Michele Silva Alves, Sylvain Caillat, Igor Araujo Vieira, Fernanda Timm, Cristina Brinckmann Oliveira Netto, Mauro Antonio Alves Castro, Ivi Bristot, Angela Fachel, Fabio Klamt, Patricia Ashton-Prolla. Energetic metabolism and DNA damage response in fibroblasts from Li-Fraumeni syndrome patients: new insights into the molecular mechanisms of the disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2015-804