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Doctoral Thesis
DOI
10.11606/T.17.2018.tde-24072018-151829
Document
Author
Full name
Cláudia Tarcila Gomes Sares
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2018
Supervisor
Committee
Reis, Rodolfo Borges dos (President)
Tirapelli, Daniela Pretti da Cunha
Faria, Sabrina Thalita dos Reis
Rodrigues Junior, Antônio Antunes
Trindade Filho, José Carlos Souza
Title in Portuguese
Avaliação da contaminação do sequenciamento do genoma mitocondrial por inserções nucleares de origem mitocondrial (NUMTs) no carcinoma renal de células claras
Keywords in Portuguese
Carcinoma renal de células claras
DNA mitocondrial
Pseudogenes nucleares de origem mitocondrial (NUMTs)
Sequenciamento genético
Abstract in Portuguese
O carcinoma renal de células claras é o tumor renal maligno mais frequentemente diagnosticado nos adultos. Uma série de defeitos genéticos tem sido observada no tecido tumoral renal e tais achados podem estar envolvidos na gênese ou progressão desses tumores. Alterações metabólicas e genéticas da mitocôndria são fatores que contribuem para muitas doenças humanas, incluindo o câncer. Objetivo: Estabelecer método para extração mitocondrial sem contaminação pelo DNA nuclear; verificar se existe contaminação por inserções nucleares de origem mitocondrial (NUMTs) no sequenciamento do genoma mitocondrial no carcinoma renal de células claras. Métodos: Para o estudo foram selecionados quatro pacientes portadores de carcinoma renal de células claras. Após a cirurgia obtivemos de cada paciente um fragmento de tumor e um fragmento de parênquima renal sem comprometimento neoplásico, as amostras de cada paciente foram extraídas de forma que ao final pudéssemos obter quatro amostras de DNA, sendo duas com isolamento da mitocôndria e duas sem o isolamento da mitocôndria. As amostras obtidas foram submetidas às seguintes análises genéticas: Sequenciamento completo do mtDNA; Reação em cadeia da polimerase para avaliação da contaminação do mtDNA obtido com isolamento da organela por DNA nuclear; avaliação do número de cópias do mtDNA (depleção) e patogenicidade das mutações. Resultados: Com os resultados obtidos neste estudo podemos afirmar que é possível a realização da extração do DNA mitocondrial sem a contaminação do genoma nuclear; e que o DNA mitocondrial extraído de maneira clássica do DNA total não apresentou contaminação por inserções nucleares de origem mitocondrial (NUMTs).
Title in English
Evaluation of the mitochondrial genome sequencing contamination by nuclear inserts of mitochondrial origin (NUMTs) in clear cell renal carcinoma
Keywords in English
Clear cell renal carcinoma
Genetic sequencing
Mitochondrial DNA
Nuclear mitochondrial pseudogenes (NUMTs)
Abstract in English
Clear cell renal carcinoma is the most frequently diagnosed malignant renal tumor in adults. A number of genetic defects have been observed in renal tumor tissue and such findings may be involved in the genesis or progression of these tumors. Metabolic and genetic changes in mitochondria are contributing factors to many human diseases, including cancer. Objective: To establish a method for mitochondrial extraction without nuclear DNA contamination; check for contamination by nuclear inserts of mitochondrial origin (NUMTs) in the sequencing of the mitochondrial genome in clear cell renal carcinoma. Methods: Four patients with clear cell renal carcinoma were selected for the study. After surgery, we obtained from each patient a tumor fragment and a renal parenchyma fragment without neoplastic involvement, the samples from each patient were extracted so that in the end we could obtain four DNA samples, two with mitochondrial isolation and two without the mitochondria isolation of mitochondria. The obtained samples were submitted to the following genetic analyzes: Complete sequencing of mtDNA; Polymerase chain reaction to evaluate the contamination of mtDNA obtained with organelle isolation by nuclear DNA; evaluation of mtDNA copy number (depletion) and pathogenicity of mutations. Results: With the results obtained in this study we can affirm that it is possible to perform mitochondrial DNA extraction without contamination of the nuclear genome; and that the mitochondrial DNA extracted from the classical DNA of the total DNA was not contaminated by nuclear inserts of mitochondrial origin (NUMTs).
 
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Publishing Date
2018-07-25
 
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