• ataxia
• FGF14
• fibroblast

Autosomal dominant spinocerebellar ataxia due to intronic GAA repeat expansion (RE) in the FGF14 gene (SCA27B) located in human chromosome 13 is a recently identified, relatively common form of late-onset ataxia.
Evidence of impaired balance and gait, vertigo, and difficult speaking are frequent clinical features whereas cerebellar ocular motor signs are also frequent in SCA27B.. The blurriness around the phenotypic spectrum leads to late diagnosis and unadapt treatments. Severity of the disease has been suggested to correlate with different extent of the RE, yet no exhaustive data are available to date to validate this hypothesis. Several research groups are currently investigating this hypothesis. The present work of this thesis aims at elucidating the link between the size of GAA repeat-expansion and clinical features. This requires a comprehensive study that spans from DNA analysis to biomolecular assays in cultured cells.
First, blood samples were collected, processed to extract DNA, using two in-house implemented protocols. Samples were analyzed with a Long-PCR to determine the exact number of GAA triplet-repeat expansion. A confirmative assay was performed using a TP-PCR (Triplet Repeat Primed PCR) followed by a capillary sequencing with the 3500 Dx Series Genetic Analyzer.
Patients with GAA repetitions ≥300 underwent a skin biopsy to pursue the research on fibroblasts obtained from this procedure. Cells were cultured to determine their survival and to focus on protein expression and redox metabolism.
Redox metabolism was evaluated by quantitative measurement of ROS (Reactive Oxygen Species) production in cell cultures. ROS production is knowingly intertwined with oxidative stress in cells, and can increase dramatically in several pathologic states, including neurodegenerative disorders. ROS level in cultured fibroblasts was determined using dichlorodihydrofluorescein assay (H2DCFDA). Additionally, mitochondrial ROS generation was evaluated with a more specific approach (MitoSOX™ Mitochondrial Superoxide Indicators for live-cell imaging).
Overall, in this work we tested a connection between biomolecular parameters at cytosolic and mitochondrial level and genetic traits of SCA27B patients, with the aim of shedding new light on mechanistic aspects of this condition.