Abstract
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that leads to motoneuron degeneration. Genetics is considered a risk factor for the development of the disease so patients are advised to undergo molecular diagnosis in research for a disease causing variant. The molecular diagnosis is performed on a panel of ALS associated genes. All variants are classified according to an international classification system defining five pathogenic classes: Benign (B), Probably Benign (PB), Variant of unknown Significance (VUS), Probably Pathogenic (PP) and Pathogenic (P). The presence of rare variants in patients’ sequences represents a major challenge for molecular diagnosis because there are not enough elements allowing to classify them as either pathogenic or benign. Often they are absent from databases and there is no functional evidence about their involvement in the disease. In consequence, they are classified as VUS or PP. Functional analysis of the variants is a strong classification element and in some cases it allows to redefine the pathogenic class of variants.In the current thesis work, we analysed the functional effect induced by overexpression of 4 candidate variants from SOD1 gene and 1 candidate variant OPTN gene in zebrafish (Danio Rerio) embryos. SOD1 codes for the enzyme superoxyde dismutase 1 and is responsible for 20% of familial ALS cases. There are mainly gain of function mutations in that gene, provoking the aberrant aggregation of the proteins which is toxic for the cells. Lastly, SOD1 is one of the major targets for antisense gene therapy. The candidate variants that we selected in that gene are: D126N, K137*, E134del et I150M. The effect of their overexpression is compared with the known pathogenic variants A5V and G94A, as well as with the WT variant. The gene OPTN is a minor ALS gene that codes for a protein implicated in the cellular trafficking, in Golgi apparatus maintenance as well as in the inflammatory reaction by inhibiting NF- kappaβ. The mutated variants of this gene alternate its function and also lead to formation of toxic aggregates. The candidate variant of this gene is L429R, its effect is compared with the known pathogenic E478G and with the OPTN WT variant. Overexpression of candidate and of pathogenic variants in zebrafish embryos induces locomotor impairments in 2-days post fertilization (dpf) Reduction of locomotor activity is seen for some larvae at 5dpf. In addition, locomotor phenotype from 2dpf is associated with shortening of the axonal projection of motoneurons and defects of the neuromuscular junction. This first phenotypical characterisation show that candidate variants have a pathogenic effect and suggest to reclassify them in the Pathogenic class. Lastly, this study shows that zebrafish can be used as a rapid tool for pathogenicity screening of rare variants and underlies its potential for ALS molecular diagnosis.