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Genome-wide association analysis of eosinophilic esophagitis provides insight into the tissue specificity of this allergic disease

Nature Genetics volume 46, pages 895–900 (2014)Cite this article

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Abstract

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder associated with allergic hypersensitivity to food. We interrogated >1.5 million genetic variants in EoE cases of European ancestry and subsequently in a multi-site cohort with local and out-of-study control subjects. In addition to replicating association of the 5q22 locus (meta-analysis P = 1.9 × 10−16), we identified an association at 2p23 spanning CAPN14 (P = 2.5 × 10−10). CAPN14 was specifically expressed in the esophagus, was dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to interleukin (IL)-13, and was located in an epigenetic hotspot modified by IL-13. Genes neighboring the top 208 EoE-associated sequence variants were enriched for esophageal expression, and multiple loci for allergic sensitization were associated with EoE susceptibility (4.8 × 10−2 < P < 5.1 × 10−11). We propose a model to explain the tissue-specific nature of EoE that involves the interplay of allergic sensitization with an EoE-specific, IL-13–inducible esophageal response involving CAPN14.

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Figure 1: Manhattan plot of the P values obtained from the genome-wide association analysis.
Figure 2: CAPN14 is specifically expressed in esophageal epithelium and is dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to IL-13.
Figure 3: IL-13 induces CAPN14 expression and calpain activity.
Figure 4: Genes at EoE risk loci with differential expression in biopsies from EoE cases relative to controls.

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Acknowledgements

We kindly thank the participants and their families. This study would not be possible without the CoFAR site investigators: S.H. Sicherer, S.M. Jones, T.T. Perry, A. Scurlock, E. Kim, E. Dellon, A.W. Burks, G.T. Furuta, D.Y.M. Leung and D. Stablein. Other people who contributed to this study include J. Gau, J. Fronda, D. Ananos, K. Mudd, T. Fitzgerald, P. Chandler, E. O'Brien, S. Carlisle, A. Hiegel, P. Steele, J. Gebhart, D. Hamilton, W. Moore, J. Newton, S. Leung, D. Brown, M. Plaut and J. Poyser.

Funding for this project was provided by the US NIH, National Institute of Allergy and Infectious Diseases (NIAID), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Heart, Lung, and Blood Institute (NHLBI) and National Human Genome Research Institute (NHGRI) (U19 AI066738, U01 HG006828, U01 HG006828-S1, U01 HG006828-S2, U01 AI066560, R37 AI024717, P01 AI083194, T32 HL7752-19, K23 AI099083 and P01 AR049084). The CoFAR arm of the study was supported by US NIH grant U19 AI066738 from NIAID and NIDDK. The project was also supported by several grants from the National Center for Research Resources (NCRR), a component of the US NIH: UL1 TR001082 (National Jewish), UL1 TR-000067 (Mount Sinai), UL1 TR-000039 (Arkansas), UL1 TR-000083 (University of North Carolina) and UL1 TR-000424 (Johns Hopkins). Support was also received from the US Department of Veteran Affairs (IMMA 9) and the US Department of Defense (PR094002). This research was supported in part by the Cincinnati Children's Research Foundation and its Cincinnati Genomic Control Cohort. Other support was derived from the Campaign Urging Research for Eosinophilic Diseases (CURED), the Buckeye Foundation, the Food Allergy Research Education (FARE) Foundation and the Foundation of the American College of Allergy, Asthma and Immunology.

The paper's contents are solely the responsibility of the authors and do not necessarily represent the official view of the NCRR or NIH. We also thank S. Hottinger for editorial assistance.

Author information

Author notes

  1. Leah C Kottyan and Benjamin P Davis: These authors contributed equally to this work.

  2. John B Harley and Marc E Rothenberg: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Pediatrics, Division of Rheumatology, Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA

    Leah C Kottyan, Kenneth Kaufman, Matthew T Weirauch, Samuel Vaughn, Sara Lazaro, Albert Magnusen & John B Harley

  2. US Department of Veterans Affairs Medical Center, Cincinnati, Ohio, USA

    Leah C Kottyan, Kenneth Kaufman, Sara Lazaro, Albert Magnusen & John B Harley

  3. Department of Pediatrics, Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA

    Leah C Kottyan, Benjamin P Davis, Joseph D Sherrill, Kan Liu, Mark Rochman, Emily M Stucke, Katherine A Kemme, J Pablo Abonia & Marc E Rothenberg

  4. Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA

    Matthew T Weirauch, Andrew M Rupert, Mojtaba Kohram & Phillip Dexheimer

  5. Department of Pediatrics, Division of Human Genetics, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA

    Hua He & Lisa J Martin

  6. Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Mirna Chehade & Hugh A Sampson

  7. Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Robert A Wood

  8. Department of Pediatrics, University of Arkansas for Medical Sciences and Arkansas Children's Hospital, Little Rock, Arkansas, USA

    Robbie D Pesek

  9. Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina, USA

    Brian P Vickery

  10. Department of Pediatrics, National Jewish Health, Denver, Colorado, USA

    David M Fleischer

  11. The EMMES Corporation, Rockville, Maryland, USA

    Robert Lindbad

  12. Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA

    Vincent A Mukkada & Phil E Putnam

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  1. Leah C Kottyan
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  2. Benjamin P Davis
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  7. Matthew T Weirauch
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Contributions

M.E.R. and J.B.H. conceived the project and supervised the study. L.C.K. and M.E.R. wrote the manuscript. L.C.K. led and performed the genetic analysis. H.H. and L.J.M. contributed statistical consultation and support. B.P.D., J.D.S. and M.R. performed the experiments with assistance from E.M.S. and K.A.K., K.L., K.K., M.T.W., A.M.R., M.K., P.D. and S.V. provided analytical and bioinformatics support. S.L. genotyped samples with assistance from A.M., M.C., R.A.W., R.D.P., B.P.V., D.M.F., R.L. and H.A.S. provided samples through the CoFAR consortium. V.A.M., P.E.P., J.P.A. and M.E.R. provided samples for the CCHMC cohort. All of the authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Marc E Rothenberg.

Ethics declarations

Competing interests

M.E.R., J.B.H. and L.C.K. are co-inventors on a Cincinnati Children's Hospital-owned patent application concerning some of the content in this manuscript.

Integrated supplementary information

Supplementary Figure 1 Manhattan plot of the P values obtained from the genome-wide association analysis.

Data are from 1,468,075 genetic variants with minor allele frequencies greater than 1% in the subjects with EoE in 514 EoE cases and 6,758 controls (a) and 222 cases and 2,488 controls (b). The –log value of the probability is shown as a function of genomic position on the autosomes. Genome-wide significance (red dotted line, P ≤ 5 × 10–8) and suggestive significance (blue line, P ≤ 10–7) are indicated.

Supplementary Figure 2 Expression of XKR6.

(Data collected from http://www.biogps.org/). The colors of the bars correspond to the tissue source.

Supplementary Figure 3 Residual association at 5q22 after adjusting for rs1438672.

After adjusting for any of the most highly associated variants in the region (rs1438672), a group of 12 variants between TSLP and WDR36 demonstrate residual association (0.001 < P < 0.05). After accounting for multiple testing, we cannot reject the null hypothesis that there is one independent genetic effect in the region. P values (–log10) for the genetic association analysis are plotted as a function of the genomic positions of each imputed variant (minor allele frequency (MAF) > 0.01) on chromosome 5 (Chr5) using a logistic regression model adjusting for the genotype of rs1438672. Genes in the region are shown below. The blue lines indicate the recombination rates in cM per Mb using HapMap controls.

Supplementary Figure 4 Expression of CAPN14.

(Data collected from http://www.biogps.org/). The colors of the bars correspond to the tissue source.

Supplementary Figure 5 Fine mapping of loci associated (replicated and 5 × 10–8 < P < 10–6) with EoE risk.

P values (–log10) for the genetic association analysis of imputed variants on the y axis are plotted as a function of the genomic position of each genotyped and imputed SNP (MAF > 0.05). Genes in the region are shown below. The LD values (r2) between the lead SNP and the other SNPs as assessed in the March 2012 release of the 1000 Genomes Project are indicated in different colors. The blue lines indicate the recombination rates in cM per Mb using HapMap controls.

Supplementary Figure 6 Expression of genes near EoE-associated genetic variants (P < 10–4) in the esophageal biopsies of EoE cases versus controls.

In this data set, 9 of the 215 transcripts (representing 208 genes) have an average difference of ≥2 FPKM between esophagi from active EoE cases and controls. The differences in expression were sufficient to segregate EoE cases from controls. The eight transcripts with differences in expression of greater than twofold can be found in Figure 4. Subjects with EoE are noted with blue bars, and subjects without EoE are indicated with red bars. All expression is normalized to the average expression in esophageal biopsies without EoE.

Supplementary Figure 7 Two genes including CAPN14 are replicated in EoE genetic, expression and epigenetic analyses.

We assessed overlap from analyses of genes within 25 kb of genetic variants with P < 10–4, genes with increased H3K27ac levels after 6-h exposure to IL-13 (P < 0.01, M < –1 at 5 kb from TSS) and genes with increased esophageal expression in EoE (twofold increase in expression in esophageal biopsy in EoE compared to control, corrected P < 0.05).

Supplementary Figure 8 Proposed model of increased EoE risk at the CAPN14 locus.

CAPN14 is expressed specifically in the esophagus (Fig. 2a). Allergic inflammatory mediators including IL-13 and IL-4 induce CAPN14 expression and activity (Figs. 2h and 3a and refs. 36, 37), and regulation of increased expression is mediated in part through the acetylation of histones (Fig. 2i). The abundance of IL-13 and IL-4 in the esophagus of patients with EoE5–8results in increased CAPN14 expression (Fig. 2b–d) and activity (Fig. 3b), and the calpain activity of CAPN14 potentially attenuates further inflammation by digesting endogenous proteins. The genetic variants associated with EoE risk at the CAPN14 locus lead to decreased CAPN14 expression (Fig. 2e) feasibly through the binding of a protein (Fig. 2j), a transcription factor that potentially acts as a transcriptional repressor. Together, our data are consistent with a model (a) in which CAPN14 is induced along with other anti-inflammatory agents such as microRNAs, cell surface receptors and T regulatory cells. (b) When exposed to IL-13 and IL-4, chromosomes in epithelial cells with the CAPN14 EoE risk allele are unable to induce CAPN14 expression to the extent of chromosomes with the non-risk allele. We propose a model in which the allelic change in CAPN14 expression dysregulates a critical negative feedback loop in the esophagus resulting in increased risk of pathology and EoE.

Supplementary Figure 9 Association of variants in loci previously reported to be associated with allergic sensitization with EoE risk.

The variant that was most highly associated in the allergic sensitization study is indicated by a purple diamond and is given in each panel. The LD values (r2) between the lead SNP and the other SNPs as assessed in the March 2012 release of the 1000 Genomes Project are indicated in different colors. The blue lines behind the genetic variants indicate the recombination rates in cM per Mb using HapMap controls.

Supplementary Figure 10 Flow chart of the EoE GWAS analytical pipeline.

External controls from the University of Michigan (acquired through dbGaP) were randomly assigned to the CCHMC and CoFAR cohorts. CoFAR, NIH Consortium of Food Allergy Research; EoE, eosinophilic esophagitis; GWAS, genome-wide association study; MAF, minor allele frequency; SNP, single-nucleotide polymorphism.

Supplementary Figure 11 The Genome Studio cluster plot for rs8041227 (also known as kgp344467) at 15q13 from the OMNI5 analysis (all EoE cases and local controls).

The normalized theta (Norm Theta) value is the ratio of the signal intensities assayed for the A and B forms of the rs8041227 allele against normalized R (signal intensity; Norm R). Subjects in this graph are homozygous for the risk allele (red), heterozygous (purple) or homozygous for the non-risk allele (blue).

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Kottyan, L., Davis, B., Sherrill, J. et al. Genome-wide association analysis of eosinophilic esophagitis provides insight into the tissue specificity of this allergic disease. Nat Genet 46, 895–900 (2014). https://doi.org/10.1038/ng.3033

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