Abstract A29: Identification of somatic and de novo mutations by exome sequencing in sporadic Wilms tumor
Authors: Torrezan Giovana T. , Ferreira Elisa N. , Castro Mayra T M , Nakahata Adriana M. , Galante Pedro A. , Ohara Daniel T. , Barros Bruna D. , Maschietto Mariana , Cunha Isabela W. , Costa Cecilia M L , Camargo Beatriz D. , Carraro Dirce M. 
Published: May 2015

Abstract
Background: Wilms Tumor (WT) is the most common pediatric kidney cancer and in approximately 2% of cases presents a familial predisposition. In both sporadic and inherited WT cases, several genes have been identified as being somatically mutated. The most frequent WT mutated genes are WT1, CTNNB1 and WTX, that together account for approximately 30% of cases. In addition, mutation in other genes such as TP53, IGF2, DIS3L2, FBXW7, MYCN, and DICER have also been described in few cases. However, up to 60-70% of Wilms tumors remain without any associated driver mutation. In this sense, the aim of this project was to identify novel somatic and de novo alterations possibly associated with WT tumorigenesis through exome sequencing. Methods: The study design consisted of performing exome sequencing of 4 samples related to a sporadic WT patient: patient blood and tumor samples, in addition to mother's and father's blood samples. Coding regions of genomic DNA were enriched using SureSelect Human All Exon 50Mb Kit (Agilent). Libraries construction and sequencing at SOLiD 4 and 5500xl were performed following the manufacturer's instructions. Resulting sequences were mapped to a reference genome with Bioscope and NovoalignCS software. Sequence variants were identified with SAMtools and filtered if present on dbSNP database. Initially a small set of de novo and somatic mutations were selected to be validated by sanger sequencing. Results: From 84 unknown de novo alterations, we selected 11 for validation according to the following criteria: all loss of function variants (1 splice site and 2 nonsenses; 8 missense variants that were classified as pathogenic in the prediction programs SIFT and/or Polyphen2 and were not at highly repetitive or homologous regions of the genome. From these 11 candidates, only one was confirmed to be a de novo mutation, while the remaining ten were either false positive or inherited variants. Regarding somatic alterations, 8 candidate mutations were identified and selected for validation. After sanger sequencing, only one was validated as a somatic mutation, one variant was also present in patient's blood and 6 alterations were not confirmed in the tumor, suggesting it to be sequencing artifacts. The gene affected by the validated somatic mutation encodes an important protein involved in RNA regulation and cell differentiation, and was not described to be mutated in any tumor type until now. Remarkably, this somatic mutation affects a highly conserved amino acid of this gene and this residue is a known binding site for a magnesium cofactor that is essential for the protein activity. The presence of this mutation was further evaluated in a cohort of 97 Wilms tumor samples and found to be present in 10% of cases. Preliminary functional studies of HEK293 cells transfected with an expression vector of this mutation demonstrated an impaired activity of the mutated protein. Conclusion: In this study we were able to identify one de novo and one somatic novel mutations in Wilms tumor. Initial results from our group suggest that this somatic mutation represents a driver and frequent event in this type of tumor.
Citation Format: Giovana T. Torrezan, Elisa N. Ferreira, Mayra T M Castro, Adriana M. Nakahata, Pedro A. Galante, Daniel T. Ohara, Bruna D. Barros, Mariana Maschietto, Isabela W. Cunha, Cecilia M L Costa, Beatriz D. Camargo, Dirce M. Carraro. Identification of somatic and de novo mutations by exome sequencing in sporadic Wilms tumor. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A29.