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Title: (Re)defining the concept of "pathogenic mutation" : translational analysis of an unusual lhon case
Authors: Guiomar, Pedro Filipe Rebelo 
Orientador: Grazina, Maria Manuela Monteiro
Girão, Henrique Manuel Paixão dos Santos
Keywords: Atrofia óptica hereditária de Leber; DNA mitocondrial
Issue Date: 2014
Abstract: Mitochondrial cytopathies are a heterogeneous group of diseases that are characterized by a primary loss of the homeostatic energetic function of the mitochondria. Leber hereditary optic neuropathy (LHON) is a mitochondrial disease resulting from the degeneration of retinal ganglion cells, and represents a major cause of blindness in young males. Besides ocular manifestations, patients may also present neurological findings, being this condition known as LHON-plus. So far, three main sequence variations of the mitochondrial genome (m.11778G>A, m.3460G>A and m.14484T>C) are known to be a primary cause of LHON. These alterations cause single amino acid substitutions at the protein level, affecting three of the seven mitochondrial-encoded subunits of the mitochondrial respiratory chain complex I (ND4 p.R340H, ND1 p.A52T and ND6 p.M64V). However, the mechanisms underlying the pathogenicity of the above mentioned mutations remain unclear, in addition to the fact that there are families where incomplete “penetrance” is observed. In 2007, an unusual case was reported by Grazina et al.. A 3 years old female patient presented clinical manifestations of LHON-plus associated with the m.11778G>A sequence variation, homoplasmic in muscle, lymphocytes and skin-derived fibroblasts. Moreover, 72% of individuals of the maternal lineage are homoplasmic for the same genetic variation but lack the expression of the phenotype. The aim of this study was to understand the role of the m.11778G>A sequence variation in the molecular mechanism underlying LHON. For that, skin-derived cultured fibroblasts and blood-derived lymphocytes of the aforementioned patient and her mother were studied using a translational approach, resorting to several functional assays aiming to clarify the intracellular impact produced by the presence of that sequence variation. A preliminary in silico study indicated that m.11778G>A is probably a pathogenic mutation, impacting on the function and stability of the complex I subunit ND4. Quantification of the mitochondrial DNA presents reduced number values in the patient under study. Assessment of the mitochondrial respiratory chain (MRC) presented a mild decrease in the activity of complex I during the acute phase of the disease. Also, when cells were subjected to metabolic, complex I failed to maintain its activity. Protein quantification by western blot and native electrophoresis provided insight on the assembly state of the MRC components, presenting a considerable decrease in the amount of assembled complex III in the patient and her parents’ lymphocytes, and a greater decrease in the amount of complex IV in the patient than that observed in the parents. The affected subunit ND4 was also analysed in primary skin fibroblasts, presenting a decreased quantity in the patient and her mother, compared to controls. Fluorescence microscopy was used to evaluate the autophagy status in primary skin fibroblasts. An increase on the basal rate of protein ubiquitination was observed by the accumulation of p62 upon pharmacological inhibition of autophagy. Mitochondrial morphology was assessed using transmission electron microscopy and no major modifications were observed. Also, there was a remarkable presence of dictiosomes in the patient. Posterior study of the Golgi apparatus confirmed the abnormal morphology of this intracellular structure in the patient and time lapse imaging suggests defects on the pinching off of vesicles. This study represents a contribution for the understanding of the role of the m.11778G>A sequence variation in the context of LHON, also providing molecular evidences that this genetic alteration may not be sufficient per se to cause the expression of clinical manifestations associated to LHON.
Description: Dissertação de mestrado em Investigação Biomédica, apresentada à Faculdade de Medicina da Universidade de Coimbra
Rights: openAccess
Appears in Collections:FMUC Medicina - Teses de Mestrado
UC - Dissertações de Mestrado

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