Functional effect sizes correlated with pathway activation in patient-derived immune cells, altogether providing a framework for ACMG-based variant interpretation in which kinase activation can support PS3 functional evidence for reclassification of variants.
Abstract
Pathogenic variants in leucine-rich repeat kinase 2 (LRRK2) 1 are among the most frequent monogenic causes of Parkinson's disease (PD) and act through a gain-of-function mechanism of increased kinase activity. LRRK2-targeted therapies are in clinical development, but interpretation of the rapidly expanding catalogue of rare LRRK2 variants remains a barrier to translation. Here, we present functionally annotated data on more than 350 LRRK2 variants using a standardized cellular assay with Rab10 phosphorylation as a readout of kinase activity and integrated these data with curated genetic and clinical annotations from the Movement Disorders Society Genetic Mutation Database (MDSGene). Variants differed in activation magnitude, ranging from modest increases (e.g., p.G2019S) to strongly activating substitutions such as p.Y1699C or p.L1795F. Activating variants occurred across the full length of LRRK2, although the largest effects clustered within the ROC-COR regulatory hub, where structural analysis identified subdomains forming an allosteric scaffold controlling kinase output. All known/established pathogenic variants showed increased activity, whereas benign and likely benign variants remained within the wild-type range. Functional effect sizes correlated with pathway activation in patient-derived immune cells, altogether providing a framework for ACMG-based variant interpretation in which kinase activation can support PS3 functional evidence for reclassification of variants.
Background: LRRK2 variants are major contributors to Parkinsons disease (PD). Many pathogenic variants increase kinase activity, underscoring the value of functional assays in nominating therapeutic targets and kinase inhibitors as potential disease-modifying therapies. Objective: To develop an interactive resource that provides functional context and ancestry-specific variant frequencies. Methods: Genotyping and short-read sequencing data were analyzed for 101,678 individuals (61,709 PD, 39,969 controls) from the Global Parkinsons Genetics Program (GP2) and integrated with clinical and in-vitro biochemical kinase activity information. Results: The LRRK2 Browser (http://gp2.org/lrrk2browser) displays ancestry-specific genetic data for 19,596 LRRK2 variants (968 exonic, 14 disease-associated) across 11 populations, and functional data for 171 variants. Clinical annotations include age, age at onset, and family history of PD. Discussion: The publicly available LRRK2 Browser represents an open-access, multi-ancestry resource to support LRRK2 variant interpretation. It aims to enhance the translational potential of genetic and functional data for precision medicine and the implementation of gene-targeted therapies in diverse populations.
S. Grant, V. van Midden, Elias Fernandez-Toledo et al.· medRxiv· 0 citations
Long-read sequencing (LRS) and diploid genome assembly have enabled nearly complete structural variant (SV) discovery. Using 293 nearly complete genomes, we characterize the full spectrum of genetic variation and show that while 99% of the variants between any two genomes are single base-pair substitutions, 88% of the euchromatic variant base pairs are SVs, including insertions, deletions, duplications, and inversions. We identify 24 gene-rich regions subject to megabase-scale variation, 2,293 potentially unstable tandem repeats, and 890 novel expression quantitative trait loci associated with SVs in humans. Expanding to 1,218 LRS samples from the 1000 Genomes Project and applying a newly developed cross-platform breakpoint evaluation tool, BoostSV, we construct a nonredundant callset comprising 614,522 SVs. We demonstrate the utility of this population-level SV reference callset by filtering >99% of the common variation from 44 unsolved LRS probands from the Undiagnosed Diseases Network to discover likely disease-causing SVs. Second, we genotype 1,053 high-impact biallelic SVs from the pangenome callset in 232,090 samples from All of Us and discover 105 SVs with significant associations, including 26% where the SV is the lead variant. This publicly available pangenome SV resource will drive new disease associations and further our understanding of the missing heritability of human genetic disease.
J. Lin, J. Gustafson, J. Wertz et al.· medRxiv· 0 citations
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the second most common cause of autosomal-dominant Parkinson's disease (PD), and increased LRRK2 kinase activity is also observed in idiopathic PD, making LRRK2 a major actionable therapeutic target. LRRK2 is a 286-kDa multidomain enzyme containing a Ras-like GTPase (ROC) and a kinase domain. Using cryo-electron microscopy (cryo-EM), biochemical reconstitution, and cell-based assays, we show that the ROC GTPase governs switching between autoinhibited and active states: GTP binding promotes activation, whereas GDP binding enforces autoinhibition. Two common PD-linked mutations, G2019S and R1441C/G/H, activate LRRK2 through distinct structural mechanisms, revealing genotype-specific routes to dysregulation. These findings provide a unified framework for understanding LRRK2 regulation with broad therapeutic implications. Stabilizing the guanosine diphosphate (GDP)-bound state may inhibit LRRK2 by maintaining autoinhibition, whereas promoting the GTP-bound state could be advantageous in specific cellular contexts, such as the lung, where increased LRRK2 kinase activity may play protective or regulatory roles.
Amalia Villagran Suarez, Kathryn S. Hatch, Tatyana Bodrug et al.· Cell· 0 citations
Introduction Autosomal recessive Parkinson’s disease (ARPD) arises from impaired mitophagy due to dysfunction of the PINK1/Parkin pathway, where PINK1-mediated phosphorylation of ubiquitin at Ser65 is essential for pathway activation. However, experimental limitations obscure the effects of disease-associated mutations on intrinsically disordered regions (IDRs) and post-translational modification (PTM) dynamics. Methods An integrated computational pipeline was employed to screen 825 PINK1 missense variants, identifying two high-confidence deleterious mutations, T313M and L347P, within the kinase domain. Variant prioritization was complemented by conserved residue, IDR, and PTM analyses, followed by protein–protein docking, molecular dynamics simulations, MM/PBSA binding free-energy calculations, principal component analysis (PCA), and dynamic cross-correlation matrix (DCCM) analysis. Results T313M overlapped a conserved phosphorylation site, whereas L347P mapped to conserved active-site residues, with complementary support from IDR analysis. Docking analysis revealed a progressive reduction in binding affinity from the wild type (–88.4 ± 8.2) to L347P (–81.9 ± 3.4) and T313M (–77.4 ± 4.9), accompanied by decreased electrostatic stabilization (–357.6 → –260.6 → –235.5 kcal/mol) and buried surface area (1741.6 → 1624.1 → 1547.4 Å2). Molecular dynamics simulations demonstrated that T313M produced the greatest structural and dynamic perturbations, whereas L347P induced moderate destabilization with increased solvent exposure. Although MM/PBSA analysis indicated broadly comparable binding energetics across all systems, PCA and DCCM analyses revealed increased conformational flexibility and altered residue communication in the mutant complexes, particularly T313M. Discussion These findings establish a mechanistic link between mutation-induced structural dynamics and impaired PINK1–ubiquitin recognition at Ser65, providing a mutation-specific framework for understanding early mitophagy impairment in ARPD and supporting future molecular assessment and targeted therapeutic development.
Zhiguang Jia, B. Malik, Deborah Vincent et al.· Frontiers in Molecular Neuro...· 0 citations
Leucine-rich repeat kinase 2 (LRRK2) has emerged as a central molecular node linking genetic risk, membrane trafficking, lysosomal homeostasis, and immune signalling in Parkinson’s disease (PD). Rather than functioning as a conventional protein kinase, LRRK2 operates as a conformationally regulated, Rab-directed signalling machine whose activity is governed by long-range interdomain communication, membrane recruitment, and cooperative interactions with small GTPases. Converging advances in cryo-electron microscopy, quantitative phosphoproteomics, and human genetics indicate that pathogenic mutations, lysosomal stress, and pharmacological inhibitors do not simply alter catalytic output, but reshape the conformational landscape of LRRK2, biasing it toward distinct structural states with divergent cellular consequences. A defining feature of this system is the selective phosphorylation of Rab GTPases at low stoichiometry—most prominently Rab8 and Rab10—yet with disproportionate functional impact on vesicle trafficking, ciliogenesis, autophagy, and organelle positioning. The identification of Rab-directed phosphatases, particularly PPM1H, further establishes that LRRK2 signalling is governed by a dynamically balanced kinase–phosphatase circuit operating in space and time. These observations, together with emerging evidence linking LRRK2 activation to lysosomal damage and immune pathways, support a unifying hypothesis: PD-associated LRRK2 dysfunction arises from maladaptive stabilization of specific conformational and spatial states within a membrane-responsive signalling network, leading to persistent misregulation of Rab-dependent trafficking and organelle homeostasis, rather than from kinase hyperactivity alone. In this review, we integrate structural, biochemical, and cellular evidence to advance this framework and discuss its implications for disease mechanisms and therapy. We highlight key unresolved challenges—including conformation-selective drug targeting, spatial control of Rab phosphorylation, and context-dependent immune–neuronal crosstalk—and propose that restoring physiological regulation of LRRK2, rather than simply inhibiting its activity, will be essential for achieving mechanism-based disease modification in Parkinson’s disease.
Oscar Arias-Carrión, Magdalena Guerra-Crespo, Daniel Ortuño-Sahagún et al.· International Journal of Mol...· 0 citations
Purpose
As sequencing improves, identifying variants causing inherited retinal diseases (IRDs) is essential for gene therapy. Structure-based network analysis (SBNA) predicts missense variant impact based entirely on structural first principles rather than historical phenotypic or clinical outcome data, distinguishing it among contemporary missense prediction tools. Here, we expanded the application of SBNA to artificial intelligence (AI)-generated protein structures, facilitating application to all known IRD-associated proteins.
Methods
We first calculated SBNA scores for structures from the Protein Data Bank (PDB) and AI-generated structures from AlphaFold2, comparing scores for pathogenic and benign ClinVar variants. We then used these results to identify the putative genetic basis of disease for patients with IRDs, demonstrating the clinical applicability of this approach.
Results
We found a significant difference between SBNA scores for known benign and pathogenic variants across all human protein structures from the PDB (median, -0.6 vs. 1.8; P < 0.0001; AUC = 0.763) and across the corresponding AlphaFold2 structures (median, -0.2 vs. 1.9; P < 0.0001; AUC = 0.755). This difference was also significant for AlphaFold2 structures from 374 IRD-associated proteins (median, -0.4 vs. 1.9; P < 0.0001; AUC = 0.779), including 185 without available structural data. This model identified likely causative disease variants in 56% of IRD patients without a known genetic basis for disease.
Conclusions
SBNA can identify variants in human proteins that are likely to cause disease, and it can help predict variants causative of IRDs in an unbiased fashion using both AlphaFold2-generated structural models and experimental structural data.
Blake Hauser, E. Place, Yuyang Luo et al.· Investigative Ophthalmology...· 0 citations