Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease

Abstract

Crohn's disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of nonsynonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2066 CD cases and 3633 healthy controls. We detected association signals in the LRRK2 gene that conferred risk for CD (N2081D variant, P = 9.5 × 10^-10) or protection from CD (N551K variant, tagging R1398H-associated haplotype, P = 3.3 × 10^-8). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson's disease (PD), and healthy controls revealed extensive pleiotropy, with shared genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant was associated with increased kinase activity, whereas neither N551K nor R1398H variants on the protective haplotype altered kinase activity. We also confirmed that R1398H, but not N551K, increased guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases.

Figure 1. Crohn’s disease association within the LRRK2 locus

(A)Single-point association without covariates, using Exome chip-genotyped variants only. (B)Association conditioned on N2081D genotypes, using Exome chip-genotyped variants only.

Figure 2. Odds ratios for Crohn’s disease (CD) and Parkinson’s disease (PD) analysis

(A)Ashkenazi Jewish cohort odds ratios: 23 of 26 independent variants (88%) exhibited effects in the same direction for both diseases (binomial test P=5.2×10⁻⁶). (B)Non-Jewish cohort odds ratios: 25 of 29 variants (86%) exhibited effects in the same direction for both diseases (P=7.6×10⁻⁶). Red indicates LRRK2 variants for which both diseases have the same direction of effect; blue indicates opposite-direction effects. Only the variants with at least nominal significance (P<0.05) in both CD and PD analysis after linkage disequilibrium pruning are shown. Circle sizes correspond inversely to the significance (P-value) of CD association at each variant.

Figure 3. A LRRK2-focused sub-network within the inflammatory bowel disease-associated gene network

The full intestinal Bayesian network was comprised of 8,382 genes, 551 (6.6%) of them were IBD-associated. From the intestinal network, the largest connected sub-network of genes that were within a path length of two IBD-associated genes was identified; this portion of the network that includes LRRK2 is shown.

Figure 4. Effect of LRRK2 mutations on protein kinase activity and GTPase activity

(A)Schematic representation of LRRK2 domain structure and the respective locations of the N551K, R1398H, and N2081D amino acid substitutions relative to the previously reported PD-associated G2019S mutation and CD-associated M2397T mutation. (B) Representative immunoblot (left panel) and quantification (right panel) of Rab10 phosphorylation by wild-type (WT) and LRRK2 variants in patient macrophages in vitro. (C) GTPase activity of WT and LRRK2 variants. Representative GTP hydrolysis assay (left) and the fraction of hydrolyzed GTP (GDP) over bound GTP (right panel). All values represent the mean of 3 independent experiments ± standard error, and significance was calculated by ANOVA. *P≤0.05, **P≤0.01.ARM, armadillo; ANK, ankyrin repeat region; LRR, leucine-rich repeat; Roc, Ras in complex protein; COR, C terminal of Roc; MAPKKK, MAP kinase kinase kinase, WD40, WD40 protein-protein interaction domain.

Figure 5. Effects of CD-associated LRRK2 mutations on human monocyte-derived macrophages

(A) Representative immunoblot showing expression of acetylated α-tubulin, p62, and LC3B (forms I-II) under control (culture medium, Med) or starvation (saline, PBS) conditions in macrophages from patients with different LRRK2 genotypes (left panel). Bar graphs depicting normalized protein expression ratio of acetylatedα-tubulin to total α-tubulin and the ratio during autophagy-inducing starvation. Ratio of protein expression during control (Med) and starvation (PBS) for p62 and LC3-II are also shown. Studies were performed in macrophages from non-carriers (n=4), and carriers of the N551K (n=4) or N2081D variants (n=2). Three independent technical replicates were performed for each sample.(B) Representative flow cytometry data presented as histograms illustrating lysosensor fluorescence after starvation (saline PBS, top), culture medium control (Med, middle) or isotype antibody control (bottom). Flow cytometry was performed on macrophages from non-carriers (n=4), and N551K (n=5) or N2081D variant carriers (n=4) (left panel). The mean Lysosensor fluorescence ratio for PBS versus culture medium control are shown. All values represent mean ± standard error, and significance was calculated by mixed model ANOVA with a random effect of a biological sample (panel A) or order-constrained ANOVA (57) (panel B). *P≤0.05, **P≤0.01.