Genetics and genomics of LRRK2 linked-disease

Abstract

Genetic variants in Leucine Rich Repeat Kinase 2 (LRRK2) are implicated in multiple diseases, most notably Parkinson’s disease (PD). These variants range in effect from protecting from disease to causing disease. Of the thousands of possible disease-associated genetic variants in the LRRK2 gene, function has only been assigned to a few. Exploiting new functional and bioinformatic approaches, herein, we developed an unbiased pipeline working from the genetic variant back to function, rather than starting with disease status and working back to a candidate variant. We interrogated thousands of missense variants for potential to impact LRRK2 function from both a PD-enriched cohort and broader non-disease related cohort utilizing the combinatorial, computational prediction tool REVEL tailored to predict classifications of rare missense variants. Using newly minted whole-genome sequencing libraries, we identified hundreds of novel LRRK2 variants across a range of ethnicities with high probability of damaging LRRK2-protein function. We performed structural modeling to predict outcome of the altered function of the missense variant on LRRK2 function, and identified top candidates to evaluate biochemically. Additionally, we identified two novel LRRK2 haplotypes with important implications in PD. These findings anchored on two novel assays that were developed to further investigate LRRK2 protein function and intricacies of variant-driven alterations. The first, a high-throughput biomarker-based assay to screen novel LRRK2 variants for functional impact, and the second, a proximity-labeling proteomics assay to uncover network changes associated with functional genetic variation. We developed three novel single-molecule immunoassays (SiMoA) on the Quanterix SR-X platform to evaluate LRRK2 variant functional consequences, particularly centered around the trans-phosphorylation of pT73-Rab10. We also screened our novel variant candidates for another main readout of LRRK2 enzymatic function, autophosphorylation of pS1292-LRRK2. We identified novel variants in LRRK2 that impact kinase function in one or both of the biochemical screens. Collectively, we observed that these two LRRK2 kinase events do not correlate with one another and have the potential to be uncoupled in the context of some LRRK2 coding variants. We also tested our novel SiMoA assays for use in a biomarker context by evaluating them across various biological sample matrices including rodent serum, and human cerebrospinal fluid and urine. The second proximity-labeling proteomics approach was adapted from a previous publication, modified for LRRK2 fusion and optimized for quantitative assessment of LRRK2 proximity network changes under various conditions of the enzyme. Herein we used the ascorbate peroxidase (APEX2) proximity labeling enzyme fused to our protein of interest, LRRK2. We performed appropriate quality control experiments and optimized the labeling and lysate processing approaches to improve the yield and reduce variability of the mass spectrometry results. We tested our APEX2-LRRK2 proximity-labeling fusion construct across a spectrum of LRRK2 kinase functional states: basal, pharmacological kinase inhibition and genetic activation states in vitro. We found that the active conformation of LRRK2 may be the most important in determining proximity networks independent of LRRK2s ability to phosphorylate. In all we developed a novel LRRK2 proximity-labeling tool optimized for quantitative experimental readouts. As LRRK2-targeted therapies enter efficacy stages in clinical trials, the identification of novel variant carriers that could benefit from LRRK2-targeted therapeutics combined with new efficacious biomarker strategies could be transformative to enhance precision approaches in genetically-defined disease. Moreover, the impacts of novel variants on kinase function combined with advanced proteomic evidence surrounding the molecular function of LRRK2 and disruptions that arise in disease states will be crucial in better understanding LRRK2’s typical function and role in disease pathogenesis.