Functional pathway phenotyping reveals increased LRRK2 activity beyond recognised genetic causes of Parkinson’s disease

Abstract Pathogenic LRRK2 variants cause Parkinson’s disease (PD) through increased kinase activity, with VPS35 p.D620N and other emerging genetic factors also increasing LRRK2 signalling. Findings in idiopathic PD also suggest broader LRRK2 pathway involvement. As LRRK2-targeted therapies advance, identifying patients with elevated LRRK2 kinase activity beyond known mutations is essential. Here, we quantified LRRK2-dependent Rab10 phosphorylation at threonine 73 in blood neutrophils from 181 genetically enriched PD patients and 71 controls. Whole-exome sequencing identified pathogenic and rare variants. VPS35 p.D620N carriers showed marked LRRK2 pathway activation, defining a biological reference threshold. Fourteen percent of PD patients without recognised pathogenic variants exhibited similar or greater pathway activation. In a subset of PD patients with increased LRRK2 pathway activity, genes harbouring rare variants were selected for exploratory functional assessment using siRNA knockdown in A549 cells. The screen identified several candidate genetic modifiers of LRRK2 signalling, including NECAP2. Overall, functional assessment of LRRK2 pathway activity identified a subset of PD patients with convergent pathway activation despite the absence of recognised pathogenic variants, supporting the integration of functional pathway phenotyping with genomic analysis for patient stratification.