Research ArticleCROHN’S DISEASE

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

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Science Translational Medicine  10 Jan 2018:
Vol. 10, Issue 423, eaai7795
DOI: 10.1126/scitranslmed.aai7795
  • Fig. 1 Crohn’s disease association within the LRRK2 locus.

    Association P values were calculated using logistic regression for each protein-coding variant. Color represents correlation [linkage disequilibrium (LD)] with the most highly associated variant in each plot. (A) Single-point association without covariates using Exome chip–genotyped variants only. (B) Association with N2081D genotypes included as covariates in logistic regression using Exome chip–genotyped variants only.

  • Fig. 2 Odds ratios for Crohn’s disease and Parkinson’s disease 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). CD, Crohn’s disease; PD, Parkinson’s disease. 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 LD pruning are shown. Circle sizes correspond inversely to the significance (P value) of CD association at each variant.

  • Fig. 3 An LRRK2-focused subnetwork within the inflammatory bowel disease–associated gene network.

    A coexpression-based Bayesian network was constructed using gene expression data from intestinal biopsies in inflammatory bowel disease (IBD) patients to elucidate IBD-relevant genes within the chromosome 12q12 region. The full network comprised 8382 genes, of which 551 (6.6%) were IBD-associated. The largest connected subnetwork of genes defined within a distance that is two genes away from a known IBD-associated gene is shown. These IBD-associated genes were delineated in a previous immunochip-based study (1). Red nodes indicate established IBD-associated genes, and the cyan node represents LRRK2. Dark blue nodes indicate all other genes.

  • Fig. 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) and quantification (right) of Rab10 phosphorylation (pRab10) by wild type (WT) and LRRK2 variants in patient macrophages in vitro. (C) Guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity of WT and LRRK2 variants. Representative guanosine triphosphate (GTP) hydrolysis assay (left) and the fraction of hydrolyzed GTP (guanosine diphosphate, GDP) over bound GTP (right). All values represent the mean of three independent experiments ± SE, and significance was calculated by analysis of variance (ANOVA). *P ≤ 0.05 and **P ≤ 0.01. ARM, armadillo; ANK, ankyrin repeat region; LRR, leucine-rich repeat; ROC, Ras of complex proteins; COR, C terminal of ROC; MAPKKK, mitogen-activated protein (MAP) kinase kinase kinase; WD40, WD40 protein-protein interaction domain.

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

    (A) Representative immunoblot showing expression of acetylated α-tubulin, p62, and autophagosome-bound form of the microtubule-associated protein 1 light chain 3β (LC3B; forms I and II) under control (medium) or starvation [phosphate-buffered saline (PBS)] conditions in macrophages from patients with different LRRK2 genotypes (left). Bar graphs depicting normalized protein expression ratio of acetylated α-tubulin to total α-tubulin under autophagy-inducing starvation and normal (Medium) conditions (right). Ratio of protein expression during control (medium) and starvation (PBS) conditions for p62 and LC3-II is also shown. Studies were performed in macrophages from noncarriers (n = 4) and carriers of the N551K (n = 4) or N2081D LRRK2 variants (n = 2). Three independent technical replicates were performed for each sample. (B) Representative flow cytometry data presented as histograms illustrating lysosomal acidity as measured by LysoSensor assay fluorescence after starvation (PBS; top), culture medium control (Medium; middle), or isotype antibody control (Isotype; bottom). Flow cytometry was performed on macrophages from noncarriers (n = 4) and N551K + R1398H (n = 5) or N2081D variant carriers (n = 4) (left). The mean LysoSensor assay fluorescence ratio for PBS versus culture medium control is shown. All values represent mean ± SE, and significance was calculated by mixed model ANOVA with a random effect of a biological sample (A) or order-constrained ANOVA (B) (58). *P ≤ 0.05 and **P ≤ 0.01.

  • Table 1 List of the top variants that reached genome-wide significance in meta-analysis.

    SNP, single nucleotide polymorphism; MAFCD, minor allele frequency in CD cases; MAFCTRL, MAF in controls; OR, odds ratio. P values for discovery and replication cohorts calculated using χ2 test. Meta-analysis performed using METAL default method. Chr, chromosome.

    Ref SNP IDChrCoordinateGeneSubstitutionDiscovery (N = 1477 cases,
    2614 controls)
    Replication (N = 589 cases,
    1019 controls)
    Meta-
    analysis
    MAFCD
    (%)
    MAFCTRL
    (%)
    PORMAFCD
    (%)
    MAFCTRL
    (%)
    PORP
    rs11209026167705958IL23RR381Q3.228.036.79 × 10−180.383.158.053.36 × 10−80.371.38 × 10−24
    rs1395188631240499594SLC2A13S6N8.104.842.68 × 10−91.737.655.369.58 × 10−31.461.39 × 10−10
    rs73087201240657700LRRK2N551K6.649.857.06 × 10−70.657.7810.451.27 × 10−20.723.28 × 10−8
    rs339958831240740686LRRK2N2081D8.134.862.56 × 10−91.737.405.614.40 × 10−21.349.51 × 10−10
    rs1413267331650138853HEATR3R642S2.781.033.16 × 10−92.741.870.932.29 × 10−22.024.76 × 10−10
    rs20668421650744624NOD2P268S32.4223.032.25 × 10−201.6032.4420.074.21 × 10−151.913.31 × 10−33
    rs20668441650745926NOD2R702W3.631.881.19 × 10−61.973.822.114.25 × 10−31.841.76 × 10−8
    rs1048954471650750842NOD2M863V4.061.051.57 × 10−193.983.571.081.15 × 10−63.391.28 × 10−24
    rs20668451650756540NOD2G908R8.734.215.14 × 10−172.187.993.294.12 × 10−92.551.52 × 10−24
    rs20668471650763781NOD2L1007fs8.332.756.27 × 10−303.217.472.407.09 × 10−123.283.43 × 10−40
  • Table 2 Allele frequencies and association statistics for LRRK2 nonsynonymous variants in imputed data sets.

    CI, confidence interval.

    MAFCD (%)MAFPD (%)MAFCTRL(%)* CD versus control associationPD versus control association
    OR (95% CI) POR (95% CI)P
    N551K variant
    Ashkenazi Jewish6.87.79.80.67 (0.57–0.79)1.4 × 10−60.77 (0.67–0.90)3.9 × 10−4
    Non-Jewish6.06.26.90.89 (0.79–1.0)5.1 × 10−20.87 (0.77–1.0)4.4 × 10−2
    R1398H variant
    Ashkenazi Jewish6.87.69.10.71 (0.60–0.84)5.0 × 10−50.84 (0.72–0.98)1.6 × 10−2
    Non-Jewish6.16.26.90.88 (0.78–0.99)4.0 × 10−20.88 (0.77–1.0)5.6 × 10−2
    N2081D variant
    Ashkenazi Jewish8.05.95.41.7 (1.4–2.0)4.3 × 10−81.1 (1.0–1.4)3.6 × 10−2
    Non-Jewish2.92.41.81.6 (1.3–2.0)2.1 × 10−61.3 (1.0–1.6)1.7 × 10−2

    *Combined control MAF. Each healthy control was randomly assigned to only one disease association analysis to ensure independence. P values calculated using logistic regression.

    • Table 3 Subphenotypic values by LRRK2 N2081D and R1398H genotype in pooled Ashkenazi Jewish and non-Jewish CD cohorts.

      Similar results were found for the N551K variant (in strong LD with R1398H; r2 = 0.81). P values were calculated using simple linear regression. SD, standard deviation; N, group sample size; ns, not significant.

      N2081D genotypeAge of CD onset (SD)
      [N]
      Disease location in
      ileum [N]
      AA26.5 (14.0) [5601]80.5% [5311]
      GA24.6 (13.1) [482]86.1% [453]
      GG20.8 (9.0) [12]90.9% [11]
      P = 0.002P = 0.01
      R1398H genotype
      GG26.3 (13.9) [5365]81.1%[5095]
      GA26.4 (14.1) [701]80.7% [652]
      AA27.2 (19.4) [29]71.4% [28]
      nsns

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/423/eaai7795/DC1

      Materials and Methods

      Fig. S1. Schematic workflow of genetic analysis by analytic stages.

      Fig. S2. Variants identified through exome sequencing by MAF and imputation quality.

      Fig. S3. Principal components analysis.

      Fig. S4. Q-Q plot of CD association results shows enrichment of true-positive signals below 10−3.

      Fig. S5. Single-point association with CD and PD in the Ashkenazi Jewish cohort using imputed genotypes within the LRRK2 locus, conditioned and unconditioned on the CD-associated coding variant N2081D.

      Fig. S6. Log odds ratio–weighted additive risk allele burden scores.

      Table S1. Power calculations.

      Table S2. Ashkenazi Jewish enriched exome variants genotyped as custom content.

      Table S3. Sample cohort description.

      Table S4. All variants with Ashkenazi Jewish CD discovery P < 2 × 10−5 (Excel file).

      Table S5. LRRK2-phased haplotype association.

      Table S6. All imputed variants with nominal CD or PD association (P < 0.05) within the LRRK2 locus (Excel file).

    • Supplementary Material for:

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

      Ken Y. Hui, Heriberto Fernandez-Hernandez, Jianzhong Hu, Adam Schaffner, Nathan Pankratz, Nai-Yun Hsu, Ling-Shiang Chuang, Shai Carmi, Nicole Villaverde, Xianting Li, Manual Rivas, Adam P. Levine, Xiuliang Bao, Philippe R. Labrias, Talin Haritunians, Darren Ruane, Kyle Gettler, Ernie Chen, Dalin Li, Elena R. Schiff, Nikolas Pontikos, Nir Barzilai, Steven R. Brant, Susan Bressman, Adam S. Cheifetz, Lorraine N. Clark, Mark J. Daly, Robert J. Desnick, Richard H. Duerr, Seymour Katz, Todd Lencz, Richard H. Myers, Harry Ostrer, Laurie Ozelius, Haydeh Payami, Yakov Peter, John D. Rioux, Anthony W. Segal, William K. Scott, Mark S. Silverberg, Jeffery M. Vance, Iban Ubarretxena-Belandia, Tatiana Foroud, Gil Atzmon, Itsik Pe'er, Yiannis Ioannou, Dermot P. B. McGovern, Zhenyu Yue, Eric E. Schadt, Judy H. Cho, Inga Peter*

      *Corresponding author. Email: inga.peter{at}mssm.edu

      Published 10 January 2018, Sci. Transl. Med. 10, eaai7795 (2018)
      DOI: 10.1126/scitranslmed.aai7795

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. Schematic workflow of genetic analysis by analytic stages.
      • Fig. S2. Variants identified through exome sequencing by MAF and imputation quality.
      • Fig. S3. Principal components analysis.
      • Fig. S4. Q-Q plot of CD association results shows enrichment of true-positive signals below 10−3.
      • Fig. S5. Single-point association with CD and PD in the Ashkenazi Jewish cohort using imputed genotypes within the LRRK2 locus, conditioned and unconditioned on the CD-associated coding variant N2081D.
      • Fig. S6. Log odds ratio–weighted additive risk allele burden scores.
      • Table S1. Power calculations.
      • Table S2. Ashkenazi Jewish enriched exome variants genotyped as custom content.
      • Table S3. Sample cohort description.
      • Table S5. LRRK2-phased haplotype association.
      • Legends for tables S4 and S6

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S4. All variants with Ashkenazi Jewish CD discovery P < 2 × 10−5 (Excel file).
      • Table S6. All imputed variants with nominal CD or PD association (P < 0.05) within the LRRK2 locus (Excel file).

      [Download Tables S4 and S6]

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