Contribution of respiratory chain protein studies in the molecular characterization of mitochondrial diseases

• Grant number: 07/04312-6
• Support Opportunities: Regular Research Grants
• Start date: November 01, 2007
• End date: May 31, 2010
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Célia Harumi Tengan
• Grantee: Célia Harumi Tengan
• Host Institution: Departamento de Neurologia e Neurocirurgia. Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

There is great difficulty in the diagnosis of mitochondrial encephalopathies, mainly due to the fact that muscle biopsy is normal in these forms of mitochondrial diseases. The main causes of mitochondrial encephalopathies include complex I deficiency and molecular investigation is very difficult because there are many genes involved. The most important diagnostic method in this form of mitochondrial disease is based on spectrophotometric analysis of respiratory complexes activities. However, this method is not easily available in Brazil and not even in other countries, where it is performed only in specialysed centers. Furthermore, there are several technical problems which make the large scale use of this method very difficult. Thus, the objective of this project is to evaluate the contribution of protein studies in the identification and characterization of the molecular defects involved in mitochondrial diseases. We will compare different methods of protein analyses in patients with mitochondrial diseases with different molecular defects and verify whether the protein expression pattern can help in the diagnosis and direct the investigation of the molecular defect. We will compare the following methods: multiplex Western blotting (using 5 panels of antibodies); analysis after immunocapture of respiratory complexes (immunoprecipitation of proteins of each complex , followed by electroporesis and visualization with silver nitrate staining); BN-PAGE (blue native polyacrylamide gel electrophoresis), where the respiratory complexes are in the native form, with detection by immunoblotting, and also detection of in gel activity of each complex. We will perform the comparison of all these methods with the results obtained from the spectrophotometric method. The samples used for these comparison will be obtained from cybrid cell lines (control and with mtDNA mutations) and fibroblasts (control, and from patients with confirmed or high suspicion of mitochondrial disease). After the comparison, we will identify which is the method that could help in the diagnosis of mitochondrial diseases and its applicability for small samples, such as those obtained from muscle biopsies. (AU)