Abstract 785: Identification of TP53 splicing mutations in colorectal tumors.

Background & Aim: The tumor suppressor TP53 gene is one of the most frequently mutated in different types of human cancer. Particularly in colorectal cancer (CRC), it is believed that TP53 mutations play a role in the adenoma-carcinoma transition of tumors during pathological process. Alternative splicing expands the diversity of RNA transcripts and plays an important role in tissue-specific differentiation, what can be misregulated in diseases. The high value of silent mutations found in TP53 databases is much more than what would be expected if synonymous mutations were effectively neutral. Previous in silico studies suggested that p53 synonymous mutations colocalized with splicing sites. The purpose of this study was to determine the correct splicing patterns and to understand if synonymous mutations may affect TP53 function in tumoral processes by analyzing the complete coding region of TP53 mRNA searching for mutations involved with splicing mechanisms. Methods: Tumor samples from 101 patients with sporadic CRC from AC Camargo Hospital in Brazil were evaluated. RNA and DNA were isolated from frozen tumor tissue using Trizol and phenol/chloroform based protocol. TP53 sequences were evaluated by Sanger sequencing. Results: There were detected four deletions and four insertions. Of these, two deletions (c.159del1, c.792_796del5) and two insertions (c.672_673ins5, c.398_399ins4) were detected, to our knowledge, for the first time and submitted to GenBank. In addition, two deletions and one insertion lead to splicing variants or splicing errors, as the region deleted or inserted contains sequences that could be used as alternative splicing sites. The insertion results from a synonymous mutation. The other samples do not contain another mutation in RNA sequence that could be related to splicing errors. Considering the total number of alterations found, of the 42 types of alterations, 3 (7.1%) of them represent splicing variants, rate seven times higher than what was shown on TP53 IARC Database. The higher frequency could be explained by the strategy adopted for the screening, as the use of cDNA sequencing facilitated the detection of possible splicing variants that might have been underestimated in previous analyses using genomic approaches. Conclusion: The analysis of the whole transcript sequence of TP53 mRNA is important to determine the specific contribution of the alterations found. Synonymous mutations may alter p53 function. The findings strongly suggest that particular splicing modifications are associated with CRC and that there is considerably more transcript complexity than previously appreciated. Citation Format: Ligia P. Oliveira, Bianca Garcia Lisboa, Ignacio Lopez, Monica Marin, Dirce Maria Carraro, Renata de Almeida Coudry. Identification of TP53 splicing mutations in colorectal tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 785. doi:10.1158/1538-7445.AM2013-785