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Wilms tumor genomic re-sequencing for evaluating gene loci of APC, CTNNB1, WT1, WTX and PLCG2

Grant number: 10/14841-9
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): November 01, 2010
Effective date (End): July 31, 2012
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Dirce Maria Carraro
Grantee:Bruna Durães de Figueiredo Barros
Home Institution: Hospital A C Camargo. Fundação Antonio Prudente (FAP). São Paulo , SP, Brazil

Abstract

Normal kidney differentiation is a result of several successive molecular events that are coordinately regulated. Mutation or disruption of any crucial step of this morphogenetic progression may lead to kidney malformation and disease, including cancer. Wilms Tumor (WT) originates from pluripotent embryonic kidney precursor cells and analyses of the molecular and cellular biology of this malignancy have provided important clues about the relationship of early renal development and WT biology. There are some molecular evidences that cellular pathways effector genes are involved in both kidney diferenciation and WT, suggesting that these genes may control both processes. The WT1 gene encodes a zinc finger transcription factor expressed in a highly controlled manner during kidney development, and mutations in this gene are related to approximately 10% of WT cases. Modulation in level expression in other genes such as CTNNB1, also play a crucial role in nephrogenesis and tumorigenesis. Recently, despite no evidence of association of the WTX with kidney differentiation, mutations in WTX were found in approximately 30% of WT cases. However, latter studies suggested that this number is over-estimated, and that mutations in WTX, CTNNB1 and WT1 together are associated to 30% of the WT cases, remaining the majority of the cases without associated genes. Results from a project developed in this laboratory demonstrated that the level expression is modulated during nephrogenesis, and it´s altered in WTs, recapitulating the first steps of kidney development, pointing the APC and PLCG2 genes as possible candidates to be altered in WT.The APC gene is associated with other cancers, such as colorectal carcinoma, where mutations appeared in more than 80% of the cases while PLCG2 was associated to a disease for the first time. Although its physiological function is not well described, it is kwon to be involved with increase of intracellular calcium. Our group has been establishing libraries where patients are individually marked allowing the evaluation of several patients in one run of 454 Sequencer FLX System (Roche), becoming viable to perform this methodology in this project. Using the next generation technology of sequencing it is interesting the evaluation of genomic regions in a reasonable time and cost. Therefore the goal of thisstudy is to verify genomic sequence alterations, including introns and exons, of the APC, CTNNB1, WT1, WTX and PLCG2 genes in WT samples. The five genes of this study are mapped in a 430 Kb long region. Up until now, we were able to prepare the fragments concerning the WTX, WT1 and CTNNB1 genes in 40 patients, and established all methodology steps of library confection for parallel sequencing, thus our proposal is to completed the amplification for the 5 genes in all 54 patients, and submit a library set to parallel sequencing using the 454-Roche platform, and analyze the data to identified the alterations. Therefore, our proposal is to transform this project, that initially was for master's degree, to a doctorate program, including technical validation and biological steps, besides correlates the DNA alterations data with qualitative and quantitative transcripts expresiion. The data generated with this nucleotide resolution of a 430 Kb genomic region which these genes are mapped, among detailed expression analyses, will contribute to defining the spectrum of mutation in this tumor. (AU)