Next Generation Sequencing in Early-Onset Parkinson’s Disease: Insights into the Role of Endo-Lysosomal Pathway from Genetic Knowledge

Abstract

[eng] Parkinson’s Disease (PD) is the second most common neurodegenerative disorder, with an etiology remaining unknown in the majority of patients. From a genetic perspective, PD is classified into Mendelian cases (15%) and sporadic cases (85%). The genetic architecture of sporadic cases is complex due to risk factors with incomplete penetrance that could facilitate the expression of PD, alongside gene-environment interactions. PD exhibits considerable heterogeneity, and the etiological diagnosis of the disease remains elusive in many cases. In the study of Mendelian forms of PD, it has been possible to identify genes involved in diverse biological pathways, including vesicular trafficking, autophagy, and dysfunction of organelles such as lysosomes and mitochondria. The aim of this study was to identify new variants that could potentially cause or increase the risk of PD through genomic studies. These variants may follow a Mendelian inheritance pattern or contribute to an oligogenic inheritance, affecting one or several pathways involved in the pathogenic mechanisms of PD. Notably, there is evidence of a greater contribution of genetic factors in early- onset Parkinson's Disease (EOPD); hence, our investigation was conducted in this group of patients. One approach implemented in this study was the analysis of variants in specific regulatory regions of PD-associated genes. Among the five non-coding candidate variants identified, the c.-253C>T variant in the 5'UTR of the DNAJC13 showed an allele-specific impact. Functional studies indicated a significant effect on gene expression and basal autophagy activity in the patient's fibroblasts. This variant affects the regulation of DNAJC13 expression, suggesting possible dysfunctions in vesicular transport and autophagic processes. The application of whole exome sequencing (WES) in a cohort of 49 patients enabled the genetic diagnosis of 9 patients (18.36%) through a knowledge-driven analysis designed in our laboratory that prioritises filtered variants. Additionally, the WES data allowed us to develop a new biological approach, considering aspects such as the molecular mechanisms underlying PD and the subcellular location of proteins to subgroup patients based on their most affected pathways. This multifaceted approach highlighted the relevance of variants in lysosomal genes, such as GLA:p.Asp313Tyr and GLB1:p.Arg419Gln. Functional studies on the patients’ fibroblasts showed deficiencies in lysosomal enzymes and alterations in the morphology of the Golgi apparatus and lysosomal network, suggesting that the lysosomal dysfunction could contribute to the etiology of EOPD patients. The identified variants through WES suggest an oligogenic mode of inheritance for EOPD, where the combination of variants can be unique for each EOPD patient. We also evaluated a new allele, LRRK2:p.Leu119Pro;p.Leu488Pro in a PD-affected family. This new allele is proposed as the cause of the disease in the family. Although the change did not affect the function, expression and stability of the protein, the increased interaction of the mutated LRRK2 with Rab8a suggests an impairment in vesicular trafficking. These findings emphasize the importance of endo-lysosomal pathway dysfunction in the etiology of EOPD, and highlight the need for new perspectives in genomic data interpretation to understand the genetic architecture of this heterogeneous disease.