ANALYSIS OF THE MITOCHONDRIAL GENOME SEQUENCING CONTAMINATION BY NUCLEAR INSERTS OF MITOCHONDRIAL ORIGIN (NUMTS) IN CLEAR CELL RENAL CARCINOMA.
Clear cell renal carcinoma is the most frequently diagnosed malignant renal tumor in adults. Several genetic defects 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 selected for the study. After surgery, we obtained from each patient a tumor fragment, and a renal parenchyma fragment without neoplasm involvement, the samples from each patient, 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 collected samples submitted to the subsequent genetic analyses: 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 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 without contaminated by nuclear inserts of mitochondrial origin (NUMTs).
Clear cell renal carcinoma; Mitochondrial DNA; Nuclear mitochondrial pseudogenes (NUMTs); Genetic sequencing.
Faculdade de Medicina de Ribeirão Preto-FMRP-USP - Sao Paulo - Brasil
Cláudia Tarcila Gomes Sares, Claudia Ferreira Rosa Sobreira, Silvia Helena Andrião Escarso, Renato Mendes Rossi De Lucca, Kelly Gomes Duarte, Rodolfo Borges dos Reis