UNRAVELING THE MOLECULAR COMPLEXITY OF PARKINSON�S DISEASE: FROM GENETIC RISK FACTORS TO MENDELIAN CAUSATIVE GENES
Creators: name: STRANIERO, LETIZIA
Publisher: name: Universit� degli Studi di Milano
Year: 2017

description: Parkinson?s disease (PD) is a neurodegenerative disorder characterized by the progressive death of dopaminergic neurons in the substantia nigra. PD is a complex disease caused by the combination of environmental factors and genetic components. In recent years, considerable progresses have been made in the identification of the genetic determinants of PD: several genes were shown to cause rare monogenic forms of the disease. Moreover, a larger number of predisposing genetic variants have been associated with sporadic PD by genome-wide association studies. Among them, GBA seems to be the main genetic risk factor for PD. However, despite these advancements, a large fraction of the expected PD heritability is still missing. In this frame, the main aims of my PhD project were, on one hand, the study of the possible mechanisms of regulation of GBA, in particular the assessment of the role of GBAP1, its pseudogene, with the view to identify novel strategies to augment glucocerebrosidase activity and, on the other hand, the identification of novel genetic determinants for the disease by whole-exome sequencing (WES) of selected PD families. Concerning GBA we demonstrated that mir-22-3p can modulate the levels of GBA transcripts down-regulating GCase expression and that GBAP1 might work as competing endogenous RNA acting as sponge and decreasing the miRNA mediated control on GBA. We then characterized the splicing pattern of the pseudogene and we showed that GBAP1 transcripts are down-regulated by the nonsense-mediated mRNA decay mechanism. On the basis of these results, we propose the existence of an RNA-based complex regulatory network involving GBA, GBAP1 and miR-22-3p. Regarding the identification of novel PD genes, 24 PD families with a dominant or a recessive inheritance pattern were selected and whole-exome sequencing was performed on the proband of each family and on affected cousins or uncles, when available. Six of these families resulted carriers of mutations in genes already associated with parkinsonism (4 GBA, 1 LRRK2, and 1 ATP7B). Moreover, we found 2 homozygous mutations in novel candidate genes in 2 consanguineous families: a splicing mutation in a gene involved in the unfolded protein response (DNAJC12) and a missense variant in a gene coding for a lysosomal enzyme (HGSNAT). We characterized the splicing mutation and we demonstrated that this variation causes the skipping of the downstream exon and the introduction of the premature stop codon. Interestingly, we demonstrated that the silencing of the DNAJC12 gene increases the ?-synuclein protein levels in SH-SY5Y neuroblastoma cells. Concerning the missense mutation, we measured the activity of the HGSNAT enzyme in the patient?s fibroblasts, which resulted below the lower limit of the normal range. We hypothesize that the HGSNAT activity is not reduced enough to cause a lysosomal disorder, but may predispose to PD. Moreover, the pathogenic role of the two novel PD genes identified in my PhD project is strongly supported by the identification, by us and by our collaborators in Vancouver, of one additional unrelated PD family with mutations in the same gene for both HGSNAT and DNAJC12. Ongoing analyses will characterize the functional role of the novel candidate genes in PD pathogenesis.