Izzo, Antonella (2010) Functional and molecular effects of chromosome 21 trisomy. [Tesi di dottorato] (Inedito)

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Abstract

Down Syndrome (DS) is the most frequent autosomal aneuploidy that is compatible with post-natal life. The DS phenotype has been attributed to overexpression of chromosome 21 (Hsa21) genes. It is unknown which, and how many, chromosome 21 genes are responsible for each DS phenotypic sign such as mental retardation, cardiac defects, muscle hypotonia, immunological disorders, etc. Hsa21 trisomy has been associated to mitochondrial dysfunction in several DS cells and mouse models suggesting that a mitochondrial dysfunction contributes to DS phenotype. We demonstrated, by microarray analysis and by qRT-PCR, the global upregulation of Hsa21 genes and the dysregulation of genes located on other chromosomes in trisomic fetal hearts at 18-22 weeks of gestation. Downregulation of genes encoding mitochondrial enzymes was a hallmark of trisomic fetal samples. Molecular, functional and morphological studies of mitochondria in primary lines of fetal fibroblasts were performed in order to evaluate the mitochondrial dysfunction associated to the dysregulation of mitochondrial gene expression in DS. Molecular analysis of trisomic fibroblasts demonstrated that the upregulation of chromosome 21 genes and the dysregulation of mitochondrial genes also occur in these cells, and that it is not identical to the one observed in fetal hearts. Functional studies demonstrated a significant reduction of the oxygen consumption rate and of respiratory chain complex I activity in trisomic fibroblasts, a decrease of mtDNA copy number and an increased production of reactive oxygen species. Furthermore, the mitochondria ultrastructure of trisomic fibroblasts, assessed by electron microscopy, revealed morphological abnormalities like giant mitochondria with irregular shape, evident breaks of both inner and outer membranes and an altered cristae pattern. These results are indicative of a widespread mitochondrial dysfunction in DS. To detect the earliest changes in gene expression profile, a new in vitro cell culture model was set up. Human stem cells from euploid as well as from Hsa21 trisomic fetuses were obtained. Both fresh and frozen amniotic fluid cultures were successfully used and the lines obtained appear to be suitable to study the differentiation processes in trisomic vs. euploid cells.

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