This diploma thesis provides an overview of gene conversion, its role in the pathogenesis of human diseases and the use of methods based on next-generation sequencing (NGS) for detection rare variants of DNA sequence. Labeling of target DNA molecules by random nucleotides in primer and NGS were used for detection point mutations arising de novo in the β-glucocerebrosidase gene by gene conversion between it and its pseudogene in meiotic and mitotic cells of control subjects. Primers specific for the active gene were used to selectively amplify the ninth and tenth exon of the gene where "recombinant" variants occur most frequently. Sequences generated from 20 genomic DNA samples on Illumina MiSeq platform were quality filtered, sorted by unique labels and consensus sequences were created from alignments of sequences carrying the same DNA tag. The number of potential point mutations in the samples ranged between 12 and 48. The mutations were manually re-evaluated from the alignments. The number of alignments with unique labeling was in the range of 7-15 thousand per sample. Only three samples carried possible recombinant mutations, suggesting a lower frequency of conversion in the region than reported by other techniques. Analysis of unique sequences in primer indicated possible ways to improve the...

Dans un contexte où les iminosucres multivalents représentent, en tant qu’inhibiteurs puissants de glycosidases, des structures privilégiées pour le développement de nouveaux agents thérapeutiques, nous nous sommes intéressés à ce type de composés pour le traitement de deux maladies génétiques rares. Le premier axe de recherche a consisté à synthétiser des iminosucres di- à tétravalents en série 1-désoxymannojirimycine dans le but d’inhiber l’α1,2-mannosidase I du réticulum endoplasmique qui est impliquée dans la destruction de la protéine delF508-CFTR chez les malades atteints de la mucoviscidose. Un effet multivalent fort sur la correction de cette protéine mutée a alors été mis en évidence avec un composé trivalent basé sur le pentaérythritol. Efficace à des concentrations submicromolaires, ce dernier s’est montré 140 fois plus efficace que le modèle monovalent correspondant. Le second axe de recherche a consisté à identifier de nouveaux chaperons pharmacologiques de la β-glucocérébrosidase, l’enzyme lysosomale impliquée dans la maladie de Gaucher. Pour cela, nous avons préparé une série d’iminosucres hétérodivalents conçus pour cibler simultanément le site actif et un site secondaire de cette enzyme. Même si cet objectif n’a pas encore été atteint, nous avons malgré tout mis en évidence des chaperons monovalents capables de quasiment quadrupler l’activité de la β-glucocérébrosidase portant la mutation G202R. En marge de ces deux axes principaux, une sonde mécanistique basée sur un C-glycoside multivalent a également été développée dans le but de préciser les mécanismes à l’origine des effets multivalents puissants observés pour l’inhibition des glycosidases. / Because multivalent iminosugars represent, as potent glycosidase inhibitors, privileged structures for the design of novel drugs, we took a particular interest in this class of compounds for the treatment of two rare genetic diseases. The first research topic was dedicated to the synthesis of di- to tetravalent iminosugars in the 1-deoxymannojirimycin series in order to inhibit the endoplasmic reticulum α1,2-mannosidase I involved in the destruction of delF508-CFTR, the mutant protein responsible of cystic fibrosis. A strong multivalent effect for restoring its activity in cells was reported with a trivalent analogue based on pentaerythritol. This submicromolar corrector was found to be 140-fold more potent than the corresponding monovalent model. The second research topic focused on the identification of novel pharmacological chaperones of the β-glucocerebrosidase, the lysosomal enzyme involved in Gaucher’s disease. For this purpose, we developed a series of heterodivalent iminosugars designed to both bind to the active site and a secondary site of the enzyme. This goal could not be reached yet, nevertheless we identified monovalent chaperones which were able to fourfold increase β-glucocerebrosidase activity in G202R cell lines. Next to these main research topics, a mechanistic probe based on a multivalent C-glycoside was also developed to investigate the multivalent effect of iminosugar clusters in glycosidase inhibition.