Accepted Manuscript

Variants in TRIM22 that Affect NOD2 Signaling Are Associated With Very Early Onset Inflammatory Bowel Disease

Qi Li, Cheng Hiang Lee, Lauren A. Peters, Lucas A. Mastropaolo, Cornelia Thoeni, Abdul Elkadri, Tobias Schwerd, Jun Zhu, Bin Zhang, Yongzhong Zhao, Ke Hao, Antonio Dinarzo, Gabriel Hoffman, Brian A. Kidd, Ryan Murchie, Ziad Al Adham, Conghui Guo, Daniel Kotlarz, Ernest Cutz, Thomas D. Walters, Dror S. Shouval, Mark Curran, Radu Dobrin, Carrie Brodmerkel, Scott B. Snapper, Christoph Klein, John H. Brumell, Mingjing Hu, Ralph Nanan, Brigitte Snanter-Nanan, Melanie Wong, Francoise Le Deist, Elie Haddad, Chaim M. Roifman, Colette Deslandres, Anne M. Griffiths, Kevin J. Gaskin, Holm H. Uhlig, Eric E. Schadt, Aleixo M. Muise

PII: S0016-5085(16)00123-2 DOI: 10.1053/j.gastro.2016.01.031 Reference: YGAST 60258

To appear in: Gastroenterology Accepted Date: 22 January 2016

Please cite this article as: Li Q, Lee CH, Peters LA, Mastropaolo LA, Thoeni C, Elkadri A, Schwerd T, Zhu J, Zhang B, Zhao Y, Hao K, Dinarzo A, Hoffman G, Kidd BA, Murchie R, Al Adham Z, Guo C, Kotlarz D, Cutz E, Walters TD, Shouval DS, Curran M, Dobrin R, Brodmerkel C, Snapper SB, Klein C, Brumell JH, Hu M, Nanan R, Snanter-Nanan B, Wong M, Le Deist F, Haddad E, Roifman CM, Deslandres C, Griffiths AM, Gaskin KJ, Uhlig HH, Schadt EE, Muise AM, Variants in TRIM22 that Affect NOD2 Signaling Are Associated With Very Early Onset Inflammatory Bowel Disease, Gastroenterology (2016), doi: 10.1053/j.gastro.2016.01.031.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT

Variants in TRIM22 that Affect NOD2 Signaling Are Associated With Very Early Onset Inflammatory Bowel Disease

Qi Li 1,2,§,*, Cheng Hiang Lee 3,4,§,*, Lauren A Peters 5,6,*, Lucas A Mastropaolo 2,§,# , Cornelia Thoeni 1,2,§,#, Abdul Elkadri 1,2,7,#,§, Tobias Schwerd 8,# , Jun Zhu 6, Bin Zhang 6, Yongzhong Zhao 6, Ke Hao 6, Antonio Dinarzo 6, Gabriel Hoffman 6, Brian A Kidd 6, Ryan Murchie 1,2,§ , Ziad Al Adham 1,2,7,§, Conghui Guo 2,§* , Daniel Kotlarz 9, Ernest Cutz 10, Thomas D Walters 1,2,§ , Dror S Shouval 11,§, Mark Curran 12 , Radu Dobrin 12 , Carrie Brodmerkel 12 , Scott B Snapper 11,13, Christoph Klein 9, John H Brumell 1,7,14§, Mingjing Hu 3,4 , Ralph Nanan 3,4 , Brigitte Snanter-Nanan 3,4 , Melanie Wong 15, Francoise Le Deist 16, Elie Haddad 17, Chaim M Roifman 18, Colette Deslandres 19, Anne M Griffiths 1,2,§ , Kevin J Gaskin 3,4 , Holm H Uhlig 8, Eric E Schadt 6,% , Aleixo M Muise 1,2,7,§, % ^

Institutions 1. SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada. 2. Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Toronto, Hospital for Sick Children, Toronto, ON, Canada 3. Gastroenterology Department, The Children’s Hospital at Westmead, Westmead, 2145, New South Wales, Australia 4. The James Fairfax Institute of Paediatric Nutrition, the University of Sydney, New South Wales, Australia. 5. Icahn School of Medicine at Mount Sinai, New York, New York, USA. Graduate School of Biomedical Sciences, New York, New York, USA 6. Icahn School of Medicine at Mount Sinai, Department of Genetics and Genomic Sciences and the Icahn Institute for Genomics and Multiscale Biology, New York, NY 10029 MANUSCRIPT 7. Institute of Medical Science, University of Toronto, Toronto, ON, Canada. 8. Translational Gastroenterology Unit, Nuffield Department Clinical Medicine, Experimental Medicine Division, University of Oxford, and Department of Pediatrics, John Radcliffe Hospital, Oxford, UK 9. Department of Pediatrics, Dr. von Hauner Children’s Hospital, Ludwig-Maximilians-University, Munich, Germany 10. Division of Pathology, The Hospital for Sick Children, Toronto, Canada 11. Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, USA 12. Janssen R&D, LLC, 1400 McKean Road, Spring House, PA 19477 13. Division of Gastroenterology and Hepatology, Brigham & Women’s Hospital, Department of Medicine, Boston, USA 14. Molecular Genetics, University of Toronto 15. Immunology Department, The Children’s Hospital at Westmead, Westmead, 2145, New South Wales, Australia 16. Department of Microbiology and Immunology, CHU Sainte Justine and Department of Microbiology, Infectiology and Immunology, University of Montreal, QC, Canada 17. CHU Sainte-Justine, Department of Pediatrics, Department of Microbiology, Infectiology and Immunology, University of Montreal, QC, Canada 18. Division of Immunology, Department of Pediatrics, University of Toronto, The Hospital for Sick Children, Toronto, Canada ACCEPTED 19. Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, CHU Sainte-Justine, Montreal, QC, Canada

§ interNational Early Onset Pediatrics IBD Cohort Study (www.NEOPICS.org) *Contributed equally and should be considered aequo loco #Contributed equally and should be considered aequo loco

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%Contributed equally and should be considered aequo loco

^ Address Correspondence to:

Aleixo Muise MD, PhD 555 University Ave. The Hospital for Sick Children Toronto, ON, Canada, M5G 1X8 Email: [email protected] Phone: 416-813-7735 Fax: 416-813-6531

QL, CHL, LAP, LAM, CT, AE, TS, DRS, JZ, BZ, YZ, KH, AD, GH, BK, RM, ZAA, CG, DK carried out all the experiments under supervision of MC, RD, CB, SBS, CK, MH, RN, JB, BSN, MW, KJG, HHU, EES, and AMM. EC, TDW, FLD, EH CMR, CD, AMG, KJG provided clinical care or clinical testing. All authors provided critical insight and interpretation of the experimental data. The manuscript was written by LAP, QL, CHL, and EES and AMM with contributions from all authors. MANUSCRIPT

Grant Support: The authors thank the patients and their families described here from Canada and Australia. The authors thank Karoline Fielder and Dr. Amanda Charlton for assistance with patient related materials. Thanks to Neil Warner for critical reading of the manuscript. CK/DK were supported by the Deutsche Forschungsgemeinschaft (SFB1054) and BioSysNe. HHU is supported by the Crohn’s & Colitis Foundation of America (CCFA). TS is supported by the Deutsche Forschungsgemeinschaft (SCHW1730/1-1). QL and AE are supported by a Crohn’s and Colitis Canada (CCC), Canadian Association of Gastroenterology (CAG), and Canadian Institute of Health Research (CIHR) Fellowship. AMM is funded by a CIHR – Operating Grant (MOP119457) and AMM, CK, SBS, ES, and HHU are funded by the Leona M. and Harry B. Helmsley Charitable Trust to study VEOIBD.

Conflict of InterestACCEPTED: none

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Title : Variants in TRIM22 that Affect NOD2 Signaling Are Associated With Very Early Onset Inflammatory Bowel Disease

Abstract : Background & Aims : Severe forms of inflammatory bowel disease (IBD) that develop in very young children can be caused by variants in a single gene. We performed whole-exome sequence (WES) analysis to identify genetic factors that might cause granulomatous colitis and severe perianal disease, with recurrent bacterial and viral infections, in an infant of consanguineous parents.

Methods : We performed targeted WES analysis of DNA collected from the patient and her parents. We validated our findings by a similar analysis of DNA from 150 patients with very early onset IBD not associated with known genetic factors analyzed in Toronto, Oxford, and Munich. We compared gene expression signatures in inflamed vs. non-inflamed intestinal and rectal tissues collected from patients with treatment-resistant Crohn's disease who participated in a trial of ustekinumab. We performed functional studies of identified variants in primary cells from patients and cell culture.

Results : We identified a homozygous variant in the tripartite motif containing 22 gene ( TRIM22 ) of the patient, as well as in 2 patients with a disease similar phenotype. Functional studies showed that the variant disrupted the ability of TRIM22 to regulate nucleotide binding oligomerization domain containing 2 (NOD2)-dependenMANUSCRIPTt activation of interferon-beta signaling and NF-κB. Computational studies demonstrated a correlation between the TRIM22–NOD2 network and signaling pathways and genetic factors associated very early onset and adult-onset IBD. The network also associated with antiviral and mycobacterial effectors and markers of inflammation such as fecal calprotectin, c-reactive protein, and Crohn’s disease activity index scores.

Conclusion : In WES and targeted exome sequence analyses of an infant with severe IBD, characterized by granulomatous colitis and severe perianal disease, we identified a homozygous variant of TRIM22 that affect the ability of its product to regulate NOD2. Combined computational and functional studies showed that the TRIM22–NOD2 network regulates antiviral and anti-bacterial signaling pathways that contribute to inflammation. Further study of this network could lead to new disease markers and therapeutic targets for patients with very early and adult-onset IBD. ACCEPTED KEY WORDS: VEOIBD, CDAI, genetics, risk, susceptibility

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Background

Very early onset inflammatory bowel diseases (VEOIBD) often presents with severe multi-systemic disease that is difficult to treat with conventional therapies. These young patients frequently have novel causal 1-3 and risk variants 4-8 and common networks are now being established (reviewed in 9). For example, mutations in IL10RA/B genes cause a Mendelian form of VEOIBD with severe colitis and perianal disease 10 and mutations in TTC7A cause a severe form of apoptotic enterocolitis 1.

Tripartite motif-containing 22 (TRIM22; also known as STAF50 ) is a RING finger E3 ubiquitin ligase 11 that is expressed in the intestine 12 and in macrophages 13 , and has a role in lineage-specific differentiation of lymphocytes 14 . TRIM22 was originally identified as an interferon inducible protein that possesses antiviral activity 15-17 and activates NF-κB signaling 13 . Here we identify TRIM22 functional variants associated with a distinct VEOIBD phenotype characterized by granulomatous colitis and severe perianal disease and show the TRIM22-NOD2 network as a key antiviral and mycobacterial regulator.

Methods: Abbreviated Methods (see Supplemental MANUSCRIPT Material for Full Description). Subjects: All experiments were carried out with the approval of the Research Ethics Board (REB) at the Hospital for Sick Children. Informed consent to participate in research was obtained. A copy of the consent is available on the interNational Early Onset Pediatric IBD Cohort Study (NEOPICS) website at http://www.neopics.org/NEOPICS_Documents.html.

Whole exome sequencing: For Patient 1 and her parents (trio), whole exome sequencing (WES) was performed using the Agilent SureSelect Human All Exon 50Mb kit with high-throughput sequencing conducted using the SOLiD 4 System at The Center for Applied Genomics (TCAG) through the Hospital for Sick Children (Toronto, ON). Sanger sequencing was used to verify variant genotypes in the Family 1 and infantile patients from the collaborating institutions were screened for TRIM22ACCEPTED variants.

Validation: In order to validate these findings, we examined WES results from 150 infantile international VEOIBD patients without a genetic diagnosis who were previously sequenced in

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Toronto (NEOPICS), Oxford, and Munich (Care-For-Rare) and targeted exome sequencing of the TRIM22 gene in ten IL10RA/B, and IL10 negative patients without previous WES.

Computational Analysis

Datasets: Biopsy data were collected at baseline from anti-TNF resistant Crohn's patients enrolled in the ustekinumab trial previously described 18 . The ustekinumab trial expression data were used for construction of the adult IBD network. RNA-seq from the RISK cohort as previously described 19 was used for generation of the pediatric IBD Bayesian network.

Differential expression: To ascertain tissue-specific signatures, we examined inflamed versus non-inflamed tissue from various anatomical regions of the small intestine, colon, and rectum. To identify differential expression signatures, we used an unbiased univariate filter to select top- varying genes and then applied significance analysis of microarrays (SAM) 20 to whole-genome expression data collected from biopsy tissue samples and whole blood of individuals in the ustekinumab trial. To control for multiple hypothesis testing, we used the Benjamini & Hochberg adjustment on the raw p-values to control the family-wise error rate and set a false discovery rate threshold of 1% or 5%. All analyses were performed MANUSCRIPT using the R statistical package, version 2.15.24.1

Bayesian Network: We employed Monte Carlo Markov Chain (MCMC)21 simulation to identify potentially thousands of different plausible networks that are then combined to obtain a consensus network. eSNP data were used as priors as follows: genes with cis-eSNP 22 are allowed to be parent nodes of genes without cis-eSNPs, but genes without cis-eSNPs are not allowed to be parents of genes with cis-eSNPs.

Key Driver Analysis: Key driver analysis (KDA) takes as input a set of genes (G) and a directed gene network N (e.g. Bayesian network)19, 23-26 . The objective is to identify the key regulators for the gene sets with respect to the given network. KDA first generates a sub-network N G, defined as the set of nodesACCEPTED in N that are no more than h-layers away from the nodes in G, and then searches the h-layer neighborhood (h=1,..,H) for each gene in N G (HLN g,h ) for the optimal h*, such that where ES h,g is the computed enrichment statistic for HLN g,h . A node becomes a candidate driver if its HLN is significantly enriched for the nodes in G.

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Candidate drivers without any parent node (i.e., root nodes in directed networks) are designated as global drivers while the remaining are designated as local drivers.

Pathway Enrichment: eQTL analysis was performed on IBD, UC, and CD GWAS hits from the NHGRI catalog, plus all WTCCC IBD GWAS SNPs with pvalue 0.001. 10% FDR eGenes were thus extracted, and tested for enrichment of pathways from the Metacore database.

Clinical variable correlation: We used Spearman correlation to examine the relationship between the mRNA expression of TRIM22 in intestine and blood and several key IBD traits. The correlation test was performed with the R function “corr.test”. We applied a simple Bonferroni correction to a 0.01 significance level across all gene-trait correlation tests performed.

Transfection and Luciferase Reporter Assays: NF-κB luciferase reporter plasmids (Promega) and ISRE promoter and IFN β promoter luciferase reporter plasmids were obtained from Dr. Hong-bing Shu (Wuhan University). A total of HEK 293 cells (1 × 10 5) were seeded on 24-well dishes and transfected the following day using Lipofectamine 2000 method. Empty control plasmid was added to ensure that each transfection received the same amount of total DNA. To normalize for transfection efficiency, 0.01 g of pRL-TK (Renilla luciferase) reporter plasmid was added to each transfection. Approximately 24 MANUSCRIPT hr. after transfection, luciferase assays were performed using a dual-specific luciferase assay kit (Promega). Firefly luciferase activities were normalized on the basis of Renilla luciferase activities. Cells were treated with recombinant TNF α, RSV, or L18-MDP (InvivoGen). Statistical analysis of luciferase assay data consisted of two-way ANOVA followed by Bonferroni post-hoc testing to compare across conditions. Statistical significance was assigned to tests with p<0.05 after Bonferroni correction.

Coimmunoprecipitation and Immunoblot Analysis: For transient transfection and coimmunoprecipitation experiments, HEK 293 cells (1 × 10 6) were seeded for 24 hr. then transiently transfected for 18–24 hr. using Lipofectamine 2000 according to the manufacturer’s protocols. Transfected cells were lysed in 1 mL of lysis buffer (150mM NaCl, 50mM HEPES, 1% Triton X-100, 10%ACCEPTED glycerol, 1.5mM MgCl 2, 1.0mM EGTA) supplemented with protease and phosphatase inhibitors (aprotinin (1:1000), leupeptin (1:1000), pepsin (1:1000) and, PMSF (1:100). For each immunoprecipitation, a 0.9mL aliquot of the lysate was incubated with 0.5 μg of the indicated antibody and 40 μL of a 1:1 slurry of Protein G Sepharose (Bioshop) for 1 hr. Sepharose beads were washed 3X with 1 mL of high salt lysis buffer containing 0.5 M NaCl. The

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precipitates were analyzed by standard immunoblot procedures using the following antibodies: mouse monoclonal anti-FLAG (Sigma), anti-HA (Origene), anti-NOD2 (Novus), anti-GFP (Invitrogen), rabbit polyclonal anti-TRIM22 (Abnova) and goat polyclonal anti-NOD2 (Ingenix). For endogenous coimmunoprecipitation experiments, HT29 cells (5 × 10 7) were stimulated with MDP (10 g/mL) for the indicated times or left untreated. The coimmunoprecipitation and immunoblot experiments were performed as described above.

Results Case History: Patient 1 was born in Australia to consanguineous parents. She initially presented on day 8 of life with fever, oral ulcers, diarrhea and failure to thrive and later developed severe fistulising perianal disease (Figure 1A) and granulomatous colitis (Figure 1B). She deteriorated despite treatment with corticosteroid and biologic therapy. Symptomatic control was only achieved by faecal steam diversion with combined ileostomy and subtotal colectomy. Patients 2 and 3 had a very similar clinical course with granulomatous colitis and severe perianal disease (Figure 1C-D) requiring ileostomy followed by total proctocolectomy. Both Patients 2 and 3 presented with severe anemia and severe hypoalbuminMANUSCRIPTemia secondary to colitis. Patients 1 and 2 had numerous bacterial and viral infections. The three patients did not have mutations in the IL10RA/B, IL10 , XIAP, or NOD2 genes and were all very difficult to treat medically and surgically with their disease non-responsive to anti-TNF α biologic therapy (Table S1A; Supplemental Material for full clinical description). Whole Exome Sequencing: Whole exome sequencing of Patient 1 (and parents) resulted in greater than 100 times mean exome coverage and identified 152,020 variants including 16,097 rare variants (minor allelic frequency MAF < 0.01) predicted to be damaging (Supplemental Figure 1). From this list we identified 12 non-synonymous homozygous variants (Supplemental Table 2) that were inherited in an autosomal recessive manner. Based on known protein function, expression profiles, animal models, mutation conservation, and network analysis (discussed below), this list ACCEPTED was narrowed down to a single candidate gene – TRIM22 . Patient 1 had a homozygous non-synonymous variant in exon 7 of the TRIM22 gene inherited from both parents (Figure 1E). This missense variant resulted in an arginine to cysteine substitution (c.1324C>T; p.Arg442Cys) predicted to be highly deleterious (Figure 1F and Table S1B). It is highly

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conserved in a functionally significant region of the exposed beta sheet around the variable loop 3 (V3) in the B30.2 (SPRY) domain thought to be responsible for subcellular localization 27, 28 and the 3-dimensional structure suggest it results in an elongated or additional beta strand, which in the wild type, forms a relaxed loop segment. To validate, we examined WES from 150 VEOIBD patients but did not identify damaging variants in TRIM22 . Therefore we performed targeted exome sequencing in ten patients with a similar severe phenotype and identified two additional patients with TRIM22 variants (Patients 2 and 3). Patient 2 had compound heterozygous TRIM22 variants (Figure 1E) including a novel arginine to threonine substitution (c.449G>C, p.Arg150Thr) inherited paternally; and a serine to leucine substitution (c.731C>T; p.Ser244Leu) inherited maternally. Both variants were predicted to be damaging and disrupt the highly conserved coiled-coil region of TRIM22 that facilitates homodimer/heterodimer multimers (Figures 1F, S2, and Table S1B). R150T is in an exposed region and S244L is buried and resides in the spacer-2 region that contains a bipartite nuclear localization sequence and was previously predicted to be a serine phosphorylation site 27 .

Patient 3 had a homozygous TRIM22 variant inherited from both parents (Figure 1E). This non-synonymous variant resulted in an arginine to MANUSCRIPTlysine substitution (p.Arg321Lys; c.962G>A). The R321K variant was predicted to be deleterious and located in the highly conserved SPRY domain on an exposed residue of a beta sheet that has a functional role in the antiviral activity patch of the SPRY domain 27 (Figure 1F; Table S1B).

Integrative Analysis Supports TRIM22 as a Key Regulator in IBD Associated Networks Constructing a VEOIBD-informed network . To gain insight into the role of TRIM22 in the pathogenesis of IBD, we constructed a probabilistic causal network model by integrating 7,568 RNA-sequence derived gene expression traits from ileal biopsies from 322 treatment naïve pediatric Crohn’s patients in the RISK cohort 19 and expression quantitative trait loci (eQTL). To identify the VEOIBD component of this molecular network, we projected all genes known to be causally associatedACCEPTED with VEOIBD 9 (Table S3) onto this network, and then identified the most connected subnetwork containing these VEOIBD genes within the full RISK network, resulting in the VEOIBD network comprised of 665 genes, which included TRIM22 (Figure 2A).

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To understand the regulation of this VEOIBD network, we employed a previously established procedure 26 to identify key regulator genes that are predicted to modulate the state of the network (Table S4). Interestingly, the most significant key regulators identified in the VEOIBD network included SNX10, STX11, CYBB and DRAM1, which directly connect TRIM22 to the IBD gene NOD2 via two equidistant paths. The first pathway, TRIM22
SNX10
STX11
DRAM1
NOD2, is linked to the known VEOIBD gene STAT1 via STX11 and GBP5, while the second pathway, TRIM22
SNX10
CYBB
DRAM1
NOD2, contains the known VEOIBD gene CYBB (Figure 2B). Both STAT1 and CYBB, in addition to IRF8, which is directly connected to TRIM22, are associated with Mendelian susceptibility to mycobacteria deficiency 29 . As expected, we did not identify any common DNA variants from the IIBDGC GWAS 30 within the TRIM22 locus (Tables S5-6), however, the VEOIBD subnetwork is well represented in genes involved in TRIM22-relevant pathways, including p53, ubiquitin response, and antiviral signaling, which were among the top pathways enriched for genes associated with intestine and blood expression quantitative trait loci (eQTL) that were also associated with adult IBD loci identified in the IIBDGC cohort (Figure S3, Table S7). Further, we found that the VEOIBD network was 2.1 fold enriched (p = 2.3 e-5) for Crohn’s genes annot MANUSCRIPTated from adult IBD genetic studies that also give rise to blood eQTLs 31 (Table S8).

Adult-onset IBD informed by a TRIM22-centered network . Given preliminary support for adult IBD in the VEOIBD network, we explored whether the TRIM22 component of the VEOIBD network was associated with adult IBD. We constructed an adult IBD causal gene network from gene expression and genotype data generated on 203 intestinal biopsies that included inflamed and non-inflamed tissues from ileum, ascending colon, descending colon, transverse colon, sigmoid and rectum. All of these tissues, in addition to blood, were collected at baseline from 54 anti-TNF α resistant Crohn’s patients enrolled in the ustekinumab (IL12/IL23 p40 monoclonal antibody) clinical trial 18 (Figure S4). We found that TRIM22 was a significantly up-regulated (1.33,ACCEPTED p=1.37e-5), key driver (p = 4.0e-6) gene in inflamed sigmoid and inflamed rectum (1.24, p=1.83e-6), (p = 1.0e-4) compared to the non-inflamed samples of these same tissues.

We identified the homologous TRIM22 component of the above adult IBD network by first identifying all genes within a path length of 5 to TRIM22 in the VEOIBD network, which

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included NOD2, leaving a network comprised of 179 genes (referred to here as the TRIM22 network; Table S9). This TRIM22 network contained many macrophage/monocyte expressed genes associated with mycobacterial and anti-viral responses (including genes involved in autophagy, NF-κB pathway and apoptosis; Figure S5; Table S10). Strikingly, of the 480 key driver genes we identified in the adult IBD network, 62 of these genes, including STX11, CYBB, and DRAM1 were represented in the pediatric TRIM22 network, 7.2-fold enrichment over what is expected by chance (p = 6.5 e-38) (Table S11). Further, the TRIM22 containing co-expression module from the blood of ustekinumab patients, which was 12.7-fold enriched for response to virus (p = 6.3e-14), was 9-fold enriched in the pediatric TRIM22 network (p < 1e-16). Further supporting the pediatric TRIM22 network’s association to viral response was a 3.2-fold enrichment (p = 2.5e-5) of eQTLs associated with dendritic cell response to stimulation with flu or interferon beta 32 and an 8.3-fold enrichment (p = 1.1e-16) of genes in a tuberculosis infection signature in dendritic cells 30 .

The homologous TRIM22 network in the adult IBD network was then identified by projecting nodes from the TRIM22 network onto the adult IBD network, identifying those nodes that were either in the TRIM22 network or directlyMANUSCRIPT connected to such nodes (Figure 2C). In this TRIM22 pediatric oriented adult IBD network, TRIM22 is in close proximity to several drug targets that are either approved or in clinical development for IBD, including TNF α, the target of adalimumab (humira), certolizumab (cimzia), infliximab (remicade), and colimumab (simponi); IL12R β, a chain shared between the receptors that bind IL12/23, the molecular target of ustekinumab; JAK3, the target of tofacitinib; and IL17RA, the target of brodalumab.

Since S100A8 (calprotectin), a stool biomarker for inflammation, is also co-localized with TRIM22 in this network 33-35 (Figure 2C), we decided to further investigate the role of TRIM22 in treatment refractory patients, exploring associations between TRIM22 and IBD- associated clinical traits in the ustekinumab clinical trial (Tables S12). We found that expression levels of TRIM22 in the colon were positively correlated with fecal calprotectin regardless of biopsy inflammationACCEPTED status. Overall, both inflamed and non-inflamed baseline intestine levels of TRIM22 revealed a correlation to blood c-reactive protein (CRP) of which consistently high levels is associated with subsequent development of perianal fistulae in Crohn’s patients 36 and

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response to anti-TNF α37 . TRIM22 expression levels in blood were positively correlated with

Crohn’s disease activity index (CDAI) (Tables S13).

Taken together, the intestinal network models provide causal support for the TRIM22 regulation of NOD2 and the correlation of the TRIM22-NOD2 pathway with predicted disease regulators, functionally relevant VEO and adult onset IBD genetic loci, effectors of anti-viral and mycobacterial function, correlation with inflammatory clinical variables and validated IBD drug targets.

Identification of TRIM22 as a NOD2 Interacting Protein: As NOD2 signaling plays a critical role in Crohn’s disease susceptibility 38, 39 ; we further investigated this in silico TRIM22-NOD2 interaction. Immunofluorescence showed that NOD2 and TRIM22 colocalized in colonic biopsies and in the cytoplasm of transient transfected HEK293 cells (Figure S6A-C). This NOD2 immunostaining was blocked by a NOD2-specific peptide and TRIM22 did not colocalize with NOD1 (Figure S6D-F) another member of the NOD family confirming the specificity of this TRIM22-NOD2 interaction. Immunostaining of colonic biopsy samples from Patients 1-3 with TRIM22 variants showed altered expression and localization of NOD2 and TRIM22 (Figure S6A-B). MANUSCRIPT TRIM22 Influences NOD2 Signaling Pathways: NOD2 senses muramyl dipeptide (MDP), a conserved structure in peptidoglycan present in most Gram-negative and Gram-positive bacteria. We found that TRIM22 was weakly associated with endogenous NOD2 in co- immunoprecipitation experiments in unstimulated HT29 cells, a human intestinal epithelial cell line, and this association increased after stimulation with MDP (Figure 3A). Furthermore, transiently transfected TRIM22 and NOD2 were found to reciprocally co-immunoprecipitate and these interactions were reduced with the TRIM22 variants (Figure 3B-C and S7A).

Since TRIM22 co-localized and co-immunoprecipitated with NOD2, we next determined if TRIM22 influenced MDP-induced NF-κB signaling via NOD2. In reporter assays, co- expression of TRIM22ACCEPTED and NOD2 enhanced MDP-induced activation of the NF-κB (Figure 3D) in HEK293 cells. As TRIM22 regulates viral activity15-17 and NOD2 functions as cytoplasmic viral pathogen recognition receptor by triggering interferon-β (IFN β) activation in response to Respiratory Syncytial Virus (RSV) 40, 41 , we next determined whether TRIM22 also regulated NOD2-dependent RSV-induced IFN β signaling. As shown in Figure 3D, overexpression of

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TRIM22 and NOD2 both enhanced RSV-induced activation of the IFN β promoter and the Interferon Stimulated Response Element (ISRE). Neither wildtype (WT)-TRIM22 nor the TRIM22-R442C variant enhanced TNF α gene expression in response to TNF α (Figure S7B). As TNFα activates NF-κB target genes independent of NOD2 signaling 42 , this suggested that TRIM22 regulation of MDP-induced NF-κB activation is NOD2-dependent.

TRIM22 Variants Influence NOD2 Signaling: We next examined the role of TRIM22 to regulate NOD2-dependent NF-κB and IFN β signaling. Luciferase assays using HEK293 cells and real-Time PCR experiments using HCT116 showed that expression of wild type TRIM22 potentiated NOD2 signaling in response to both MDP-induced NF-κB and RSV-induced IFN β activation (Figures 3D and S7C-F). However, the TRIM22 variants (Figures 3D and S7C-E) and TRIM22 knockdown using short hairpin RNA (shRNA; Figure S8A) showed impaired NOD2- dependent NF-κB and IFN β signaling (Figure S8B). MDP-mediated NOD2-dependent signaling was also significantly reduced in only Patient 2 with the heterozygous TRIM22 variants in the coiled-coil domain (Figure S9 and Supp. Material). Together this data suggests that TRIM22 is a positive regulator of antiviral and antibacterial NOD2-dependent signaling pathways. TRIM22 mediates K63-linked PolyubiquitinationMANUSCRIPT of NOD2: As TRIM22 is a RING finger E3 ubiquitin ligase 11 and NOD2 has high confidence predicted ubiquitination sites at K436 and K445 (CKSAAP_UbSITE 43 ), we next determined if TRIM22 mediated ubiquitination of NOD2. Immunoprecipitation experiments using a HEK293 cell line co-expressing HA-NOD2 and Flag- TRIM22 showed that overexpression of TRIM22 enhanced NOD2 polyubiquitination (Figure 4A). The TRIM22 variants (Figure 4A and S10A) were unable to mediate this NOD2 polyubiquitination. TRIM22 did not increase polyubiquitination of other components (MAVS or RIPK2) of the NOD2 signaling pathways known to be ubiquitinated 44, 45 (Figure S10B). Using the TRIM22 shRNA HT29 stable cell line, we found that only wildtype TRIM22 rescued NOD2 polyubiquitination and not the TRIM22-R442C variant (Figure 4B). Taken together, this suggested that TRIM22ACCEPTED specifically mediates NOD2 polyubiquitination. K63-linked polyubiquitination regulates the localization and signaling activity whereas K48-linked polyubiquitination often mediates degradation 46 . NOD2 has been previously shown to be K48-linked ubiquitinated by TRIM27 and results in NOD2 degradation 47 . Using ubiquitin mutants, we showed that overexpression of TRIM22 mediated K63-linked but not K48-linked

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polyubiquitination of NOD2 while the TRIM22 variants were unable to mediate K63- or K48- linked NOD2 polyubiquitination (Figure 4C and S11A). We next determined that TRIM22 is required for MDP- and RSV-induced K63-linked NOD2 polyubiquitination and knockdown of TRIM22 reduced this MDP- and RSV–induced K63-linked (Figure 4D-E), but not K48-linked NOD2 polyubiquitination (Figures S11B-C).

Discussion

TRIM proteins are involved in a number of biological processes, including cell proliferation, apoptosis, innate immunity, autoimmunity, and inflammatory response 48, 49 . Variants in TRIM genes result in monogenic diseases including familial Mediterranean fever (FMF) and Opitz syndrome type 1, and are implicated in a number of cancers and autoimmune diseases including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus 48, 49 . TRIM22 belongs to a subfamily of TRIM proteins that contain a SPRY domain and are thought to have evolved to limit innate immune responses to viruses 20 . Mutations in the SPRY domain of TRIM20 (pyrin) result in autoimmune diseases including FMF 50 . Overexpression of TRIM22 is known to activate NF-κB in a dose-dependent mannerMANUSCRIPT and both the N-terminal RING domain and C-terminal SPRY domain are crucial for TRIM22-media ted NF-κB activation 13 and antiviral activity 15-17 , and are implicated in the pathogenesis of autoimmune diseases 51 . Patients 1 and 3 both had homozygous variants in the SPRY domain and Patient 2 had compound heterozygous variants both predicted to disrupt the coiled-coil domain and had loss of NOD2-dependent MDP signaling. The characterized TRIM22 variants resulted in impaired NOD2 binding and K63- linked NOD2 polyubiquitination. The predicted NOD2 polyubiquitination sites described earlier are in the NACHT domain, a conserved domain required for NOD2 conformational change in response to MDP sensing through the LRR domain or interaction with binding adaptor proteins for NOD2 52 . Therefore, as all identified TRIM22 variants disrupt both NOD2-dependent NF-κB and IFN β signaling,ACCEPTED this suggests that K63-linked polyubiquitination of NOD2 meditated by TRIM22 is critical for NOD2 function (Figure 4F).

NOD2 has long been recognized as a critical player in Crohn’s disease pathogenesis 38, 39 where it is proposed to regulate innate immunity through NF-κB-induced pro-inflammatory responses triggered by PGN (reviewed in 53 ). Numerous studies have shown that the loss of

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function variants in NOD2 associated with Crohn’s disease result in the loss of NF-κB-induced pro-inflammatory cytokine response to MDP (reviewed in 54, 55 ) and therefore mirror the defect we observe in Patient 2 with TRIM22 mutations in the coiled-coil domain. Similarly, mutations in XIAP (also an E3 ubiquitin ligase) are associated with IBD with granulomatous colitis and perianal disease 56, 57 and are associated with loss of NOD2-dependent mediated NF-κB signalling 58-60 and XIAP is also regulated by TRIM proteins 61 . XIAP deficiency usually presents in early childhood but there are cases of patients presenting with IBD at over 40 years of age 9 with and without hemophagocytic lymphohistiocytosis (HLH) (reviewed in 62 ). Furthermore, XIAP not only regulates NOD2-dependent innate immunity responses but also is proposed to regulate inflammasome and adaptive immunity 62 . Loss of function NOD2 variants associated with IBD have low penetrance and do not cause the severe early onset disease observed in this group with either TRIM22- or XIAP-deficiency. Therefore it is most likely that in Patients 1 and 3, with TRIM22 SPRY domain variants, defects in other pathways regulated by TRIM22 result in the early onset and severity of disease. Alternatively hypomorphic TRIM22 variants act in an oligogenic manner with mutations in other genes similar to what has been observed with hypomorphic XIAP mutations 63 and as observed in Patient 3 who is also a carrier of a XIAP mutation. Together, this suggests that loss of NOD2 MANUSCRIPT-dependent mediated signaling in response to MDP contributes to a severe form of IBD and that other yet defined pathways must also be involved in disease pathogenesis. With regards to TRIM22 specifically, we anticipate that other pathways not identified in these studies play a role in disease pathogenesis. Our computational studies further support our functional studies demonstrating that TRIM22 influences both NOD2-dependent IFN β and NF-κB signaling pathways. While the loss of function TRIM22 variants in our VEOIBD patients may manifest as an immunodeficiency, the inflammation and tissue damage that ensues from a dysregulated response to microbial burden, may resemble the profile of adult-onset IBD. The functional outcome of pro-inflammatory NF- κB activation may differ depending on the cell population in which TRIM22 is expressed, with circulating plasmacytoidACCEPTED dendritic cells (DCs) and monocytes predominating in the blood and resident intestinal DCs, macrophages and enterocytes in the intestine. We see from the intestine network that the regulation of causal TRIM22 results in modulation of a mycobacterial and viral response subnetwork that may also result in feedback to TRIM22 expression. Thus, either due to immunodeficiency arising from the TRIM22 variants or excessive inflammation in non-TRIM22

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variant carried by anti-TNF α refractory patients, TRIM22 driven NOD2 activation of NF-κB in response to MDP or viral stimuli, contributes to dysregulation of NF-KB and IFN β driven pathways as reflected in the networks. Together, this demonstrates that the TRIM22-NOD2 network plays a critical role in the development of IBD. Investigation of VEO and adult onset IBD in treatment refractory disease will elucidate new treatment opportunities by revealing complexity of disease network dysregulation. Causal and correlative based network analysis allows for consideration of the synergistic effects between genes in disease, which can be a greater determinant than evaluation of single target genes, to inform disease drivers and opportunities for combinatorial therapy. Thus network analysis can be focused by novel variants, as seen with TRIM22 identified through WES in VEOIBD patients to generate hypothesis and potentially identify biomarkers and guide drug discovery and repositioning in both VEO and adult-onset IBD patient subsets.

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References

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Author names in bold designate shared co-first authors.

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Figure Legends Figure 1: Clinical Features and Pedigrees of Patients with TRIM22 Variants. A, B) Clinical presentation and histopathologic findings in colonic biopsy from Patient 1. Panel A shows severe perianal fistulization with seton placement. Recto-vaginal fistulae were also present. Panel B shows non-caseating granulomas on a background of chronic inflammatory cell infiltrates (H&E stain). C, D) Clinical presentation and histopathologic findings in colonic biopsy from Patient 2. Panel C shows multiple, severe cavitating perianal fistulae. Panel D shows a colonic biopsy showing chronic inflammation with non-caseating granuloma (black arrow) (H&E stain, scale bar represents 75 µm). E) Pedigree analysis of the genetic inheritance pattern for each family trio. F) Schematic view of the protein domain architecture of TRIM22 with the relative position of each identified variant depicted. Number indicates amino acid position.

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Figure 2: TRIM22 - NOD2 Network. A) VEOIBD loci focused subnetwork within RISK Pediatric Crohn’s ileum network. To define the context of VEOIBD in the ileum network, genes known to be causally associated with VEOIBD (not including TRIM22) were projected onto the RISK network and extended out a path length of two. The resulting VEOIBD focused sub network is comprised of 665 genes and contains TRIM22 and NOD2. The TRIM22-NOD2 paths are highlighted in bold and are connected to the known VEOIBD loci CYBB, and STAT1, as well as the GWAS locus IRF8. B) Local network structure surrounding the NOD2/TRIM22 paths in the 665-node VEOIBD focused subnetwork. Representative nodes from the 179-gene TRIM22 network within the 665 node VEOIBD loci focused RISK subnetwork. C) TRIM22-NOD2 interaction is conserved in the adult IBD intestine. This network provides the context of the TRIM22/NOD2 interaction in the adult IBD intestine. The 179-gene TRIM22 network was projected and extended out an additional path length on the adult IBD pan intestine Bayesian network. IBD GWAS (green), VEOIBD loci (orange), IBD drug targets approved or in clinical development (red), and calprotectin (S100A8) (purple) are all represented in this conserved subnetwork.

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Figure 3: TRIM22 Interacts with and Activates NOD2 Signaling. A) Endogenous NOD2 and TRIM22 co-immunoprecipitate following MDP stimulation. HT29 cells were stimulated with MDP (1 g/mL) for various time periods. Following treatment, cells were lysed and endogenous TRIM22 was immunoprecipitated using anti-TRIM22 antibody conjugated to Protein G-Sepharose. Bound levels of endogenous NOD2 were evaluated using Western blotting. As stimulation time with MDP increased, the binding of endogenous NOD2 to TRIM22 increased. Immunoprecipitation with IgG bound to Protein G-Sepharose was used as a negative IP control. B) TRIM22 interacts with NOD2. HEK293T cells were transiently co-transfected with HA-NOD2 and either wildtype (WT) or variant (R150T, S244L, R321K, and R442C) FLAG-epitope tagged TRIM22. Twenty-four hours post-transfection, cells were lysed followed by immunoprecipitation of the FLAG-TRIM22 by anti-FLAG-agarose. Reduced binding of NOD2 to TRIM22 was observed in varying degrees for each variant compared to the wildtype protein. HA-NOD1 and an empty vector construct were used as negative controls. C) Quantification of NOD2 co-immunoprecipitation with wild-type or mutant TRIM22 . Densitometry analysis was performed on Western blots performed in 3B. Each TRIM22 point mutation significantly reduced NOD2 co-immunoprecipitation (Student’s t-test, *p<0.05, **p<0.01, n=3). D) TRIM22 mutants fail to activate NF-κB and IFN β downstream of NOD2. HCT116 cells were transiently co-transfected with (or without) NOD2, MANUSCRIPTTRIM22-WT, or TRIM22 point mutants (R150T, S244L, R321K, R442C). Twenty-four hours post transfection, cells were stimulated with either MDP (10 µg/mL) or respiratory syncytial virus (RSV) (0.1 g) for 18 hr before NF- κB or IFN β promoter activation was evaluated by luciferase assay. Cells transfected with TRIM22 mutants showed significantly reduced NF-κB and IFN β promoter activation following stimulation compared to wild type TRIM22 transfected controls (*p < 0.001 after Bonferroni post-hoc testing, n=3).

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Figure 4: TRIM22 Mediates K63-linked NOD2 Polyubiquitination. A) TRIM22 enhances NOD2 polyubiquitination in HEK293 cells. HEK293T cells were transiently co-transfected with HA-NOD2, GFP-ubiquitin, and either wildtype (WT) or variant (R442C) FLAG-TRIM22. Twenty-four hours post-transfection, cells were lysed followed by immunoprecipitation of HA-NOD2. Ubiquitination of NOD2 was evaluated by immunoblotting for GFP (ubiquitin). Co-transfection with wildtype TRIM22 enhanced ubiquitination of NOD2, which was abrogated with the R442C variant TRIM22. All experiments were carried out in triplicate, independent experiments. B) Transgenic TRIM22 rescues NOD2 polyubiquitination in TRIM22-shRNA-HT29 cells. HT29 cells stably transduced with control (scramble) or TRIM22 shRNA were transiently co- transfected with HA-NOD2 and either wildtype (WT) or variant (R442C) FLAG-TRIM22. Twenty-four hours post-transfection, cells were lysed followed by immunoprecipitation of the HA-NOD2. Evaluation of NOD2 poly-ubiquitination by GFP immunoblotting demonstrated rescued ubiquitination following transfection of WT-TRIM22 in the TRIM22 shRNA cell line that was not observed following transfection of the variant R442C. All experiments were carried out in triplicate, independent experiments. C) Variants in TRIM22 abrogates K63-linked NOD2 polyubiquitination. HEK293T cells were transiently co-transfected with FLAG-NOD2, HA-(K63-specific) ubiquitin, and either wildtype (WT) or variant (R150T, S244L, R321K, or R442C) V5-TRIM22. Twenty- four hours post-transfection, cells were lysed followed by immunoprecipitation of the FLAG- NOD2. Evaluation of NOD2 K63 specific-ubiquitinatioMANUSCRIPTn by HA immunoblotting demonstrated significantly reduced ubiquitination in cells co-tr ansfected with TRIM22 variants compared to wildtype. All experiments were carried out in triplicate, independent experiments. D and E) TRIM22-shRNA reduces MDP- and RSV-induced K63-linked NOD2 polyubiquitination. HEK293T cells stably transduced with control (scramble) or TRIM22 shRNA were transfected with HA-tagged K63-specific ubiquitin and FLAG-NOD2. Forty-eight hours after transfection, cell lysates were stimulated by MDP (D) or RSV (E) for indicated the time points followed by lysis and immunoprecipitation using anti-FLAG-agarose. K63-poly-ubiquination of NOD2 was evaluated by immunoblotting using anti-HA antibody. Following stimulation by either MDP or RSV, NOD2 K63-specific ubiquitination increased over treatment time. However, in TRIM22 knockdown cells, this ubiquitination was significantly reduced or absent. All experiments were carried out in triplicate independent experiments and blots are representative. F) Schematic overview of the proposed impact of TRIM22 and its identified variants on the NOD2 signaling ACCEPTEDaxis. Wildtype TRIM22 binds and mediates the K63-specific polyubiquitination of NOD2, potentiating downstream signaling through the MAVS and RIPK2 signaling axes depending on antigen. TRIM22 variants disrupt binding to NOD2, abrogating this polyubiquination thus attenuating downstream pro-inflammatory signaling.

23 Table S3: VEO IBD Gene List: Genes PreviouslyACCEPTED Reported to hav eMANUSCRIPT a Causal Association with VEO IBD PMID:25058236

COL7A1 FERMT1 IKBKG TTC7A ADAM17 GUCY2C CYBB CYBA NCF1 NCF2 NCF4 SLC37A4 G6PC3 ITGB2 MVK PLCG2 MEFV STXBP2 XIAP SH2D1A HPS1 HPS4 HPS6 ICOS LRBA IL21 BTK PIK3R1 CD40LG AICDA WAS MANUSCRIPT DCLRE1C ZAP70 RAG2 IL2RG LIG4 ADA CD3g DKC1 RTEL1 DOCK8 FOXP3 IL2RA STAT1 IL10RA IL10RB IL10 MASP2 SKIV2L ACCEPTED TTC37 ACCEPTED MANUSCRIPT

Table S4: VEO Top Key Drivers keydrivers is_signature hits downstream signature_in_subnetworksubnetwork_sizesignature_in_networknetwork_size signature optimal_layer fold_change_wholepvalue_whole fold_change_subnetpvalue_subnetpvalue_corrected_subnetkeydriver

NFAM1 1 201 615 664 7528 664 7528 665 6 3.70537761 6.13E-71 3.70537761 6.13E-71 4.62E-67 1

GBP5 1 145 395 664 7528 664 7528 665 5 4.1618118 5.19E-57 4.1618118 5.19E-57 3.90E-53 1

STAT1 1 31 31 664 7528 664 7528 665 2 11.3373494 1.07E-33 11.3373494 1.07E-33 8.03E-30 1

STX11 1 70 164 664 7528 664 7528 665 4 4.83911255 5.39E-32 4.83911255 5.39E-32 4.05E-28 1

ARHGDIB 1 154 671 664 7528 664 7528 665 8 2.60201462 7.61E-32 2.60201462 7.61E-32 5.73E-28 1

VAV1 1 30 34 664 7528 664 7528 665 3 10.0035436 4.16E-28 10.0035436 4.16E-28 3.13E-24 1

ARHGAP30 1 125 537 664 7528 664 7528 665 5 2.63904781 3.77E-26 2.63904781 3.77E-26 2.84E-22 1

CASZ1 1 24 25 664 7528 664 7528 665 3 10.8838554 7.69E-25 10.8838554 7.69E-25 5.79E-21 1

C1ORF228 1 30 39 664 7528 664 7528 665 5 8.721038 1.24E-24 8.721038 1.24E-24 9.33E-21 1

CYBB 1 22 22 664 7528 664 7528 665 2 11.3373494 4.59E-24 11.3373494 4.59E-24 3.45E-20 1 SLAMF8 1 26 30 664 7528 664 7528MANUSCRIPT 665 3 9.82570281 4.74E-24 9.82570281 4.74E-24 3.57E-20 1 DRAM1 1 53 127 664 7528 664 7528 665 5 4.73133479 7.86E-24 4.73133479 7.86E-24 5.92E-20 1

SNX10 1 26 60 664 7528 664 7528 665 3 4.91285141 9.31E-13 4.91285141 9.31E-13 7.01E-09 1

ACCEPTED Table S5: ACCEPTED MANUSCRIPT IIBDGC GWAS ENRICHMENT In TRIM22 locus 0.01 cM: chr position IBD: p-value IBD: n_markers CD: p-value CD: n_markers UC: p-value UC: n_markers TRIM22 11 5734619 0.08913 163 0.006027 157 0.03147 165 0.1 cM: chr position IBD: p-value IBD: n_markers CD: p-value CD: n_markers UC: p-value UC: n_markers TRIM22 11 5734619 0.003694 332 0.006027 319 0.02398 337 1cM: chr position IBD: p-value IBD: n_markers CD: p-value CD: n_markers UC: p-value UC: n_markers TRIM22 11 5734619 0.001465 5682 0.0003264 5640 0.0002493 5731

Table S6: IIBDGC GWAS ENRICHMENT TRIM22 eQTL (NCBI) enrichment IBD CD UC chr11:5077653 0.1452 0.4114 0.04264 chr11:5060791 0.1603 0.3978 0.04048 chr11:5059064 0.1559 0.4066 0.03872 chr11:5020799 0.3528 0.3084 0.07128 chr11:5020832 0.3478 0.3139 0.07045 chr11:5019217 0.3495 0.3058 0.07004 chr11:5019357 0.3503 0.3076 0.07054 chr11:5020189 0.3513 0.3084 0.07105 chr11:5020509 0.3538 0.3081 0.07163 chr11:5024139 0.346 0.317 0.07096 chr11:5073486 0.05608 0.5467 0.08671 chr11:5016553 0.4019 0.4234 0.1344 chr11:5719667 0.7601 0.657 0.5903

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Table S7 Genes in eQTL Enriched Pathways Enrichment analysis report Enrichment by Pathway Maps WTCCC_IBD_GWAS_1e-3_union_NHGRI_IBD_UC_CD_blood_genelist WTCCC_IBD_GWAS_1e-3_union_NHGRI_IBD_UC_CD_intestine_genelist Network Objects from Network Objects from # Maps Total min(pValue) Min FDR p-value FDR In Data p-value FDR In Data Active Data Active Data 1 Transcription_P53 signaling pathway 39 2.441E-04 4.171E-02 8.248E-02 3.406E-01 2 MKP-7, VEGF-A 2.441E-04 4.171E-02 3 VEGF-A, c-Fos, MKP-7 2 Signal transduction_PKA signaling 51 3.812E-04 1.403E-01 3.812E-04 1.403E-01 5 AKAP12, Adenylate cyclase, PP2A regulatory, DARPP-32, G-protein alpha-12 family 1.502E-01 1.783E-01 1 Adenylate cyclase 3 Immune response_C3a signaling 48 4.532E-04 4.171E-02 2.760E-03 1.410E-01 4 RhoA, Adenylate cyclase, CD40(TNFRSF5), VEGF-A 4.532E-04 4.171E-02 3 Adenylate cyclase, VEGF-A, CD40(TNFRSF5) 4 Immune response_Antiviral actions of Interferons 52 5.741E-04 4.171E-02 2.573E-02 2.192E-01 3 IRF1, MHC class I, HLA-A 5.741E-04 4.171E-02 3 CIITA, HLA-A, MHC class I 5 Immune response_HSP60 and HSP70/ TLR signaling pathway 54 6.416E-04 4.171E-02 4.240E-03 1.410E-01 4 TLR4, MHC class I, CD40(TNFRSF5), TPL2(MAP3K8) 6.416E-04 4.171E-02 3 HSP70, CD40(TNFRSF5), MHC class I 6 Regulation of CFTR activity (normal and CF) 62 9.436E-04 1.403E-01 9.436E-04 1.403E-01 5 Syk, Annexin V, Adenylate cyclase, COMMD1 (MURR1), PP2A regulatory 1.796E-01 1.938E-01 1 Adenylate cyclase 7 Neuroprotective action of lithium 63 1.008E-03 5.240E-02 1.808E-01 4.428E-01 2 VEGF-A, PP1-cat 1.008E-03 5.240E-02 3 VEGF-A, HSP70, WNT 8 Immune response_PGE2 signaling in immune response 45 2.173E-03 1.410E-01 2.173E-03 1.410E-01 4 Adenylate cyclase, IL-2, CREM (activators), CREM (repressors) 1.337E-01 1.783E-01 1 Adenylate cyclase 9 Proteolysis_Putative ubiquitin pathway 23 2.366E-03 7.636E-02 2.475E-01 5.017E-01 1 UBCH7 2.366E-03 7.636E-02 2 HSP70, UBCH7 10 Proteolysis_Role of Parkin in the Ubiquitin-Proteasomal Pathway 24 2.576E-03 7.636E-02 2.567E-01 5.112E-01 1 UBCH7 2.576E-03 7.636E-02 2 HSP70, UBCH7

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Table S8 Crohn's GWAS Enriched Blood eQTL Overlap with the VEO Network snp snp_chr snp_pos_hg19 gene ensembl_id gene_symbol cis_trans_indicatorfdr pvalue tissue IBD_META_P rs1872691 16 50350210 ENSG00000121281ADCY7 cis 0 0 Blood 5.59E-18 rs3813755 16 50348577 ENSG00000121281ADCY7 cis 0 0 Blood 7.89E-18 rs3826173 16 50345012 ENSG00000121281ADCY7 cis 0 0 Blood 1.88E-17 rs4785210 16 50340154 ENSG00000121281ADCY7 cis 0 0 Blood 3.44E-11 rs4785403 16 50340217 ENSG00000121281ADCY7 cis 0 0 Blood 3.42E-11 rs4785412 16 50385468 ENSG00000121281ADCY7 cis 0 0 Blood 1.12E-17 rs7184802 16 50355996 ENSG00000121281ADCY7 cis 0 0 Blood 7.30E-18 rs8047812 16 50389485 ENSG00000121281ADCY7 cis 0 0 Blood 1.04E-19 rs4643314 16 50375955 ENSG00000121281ADCY7 cis 0 1.00E-13 Blood 1.11E-20 rs12325247 16 50363559 ENSG00000121281ADCY7 cis 0 1.03E-13 Blood 5.90E-21 rs9525625 13 43018030 ENSG00000023516AKAP11 cis 0 1.06E-06 Blood 1.00E-10 rs3116494 2 204592021 ENSG00000178562CD28 cis 0 6.11E-14 Blood 1.00E-10 rs1569723 20 44742064 LRG_40 CD40 cis 0 0 Blood 6.55E-11 rs2425752 20 44702120 LRG_40 CD40 cis 0 0 Blood 3.09E-08 rs4810485 20 44747947 LRG_40 CD40 cisMANUSCRIPT 0 0 Blood 3.89E-10 rs6131010 20 44724305 LRG_40 CD40 cis 0 0 Blood 3.89E-08 rs12624433 20 44680853 LRG_40 CD40 cis 0 2.00E-20 Blood 3.69 E-09 rs1569723 20 44742064 LRG_40 CD40 cis 0.03 6.13E-05 Blood 6. 55E-11 rs11230556 11 60765984 ENSG00000013725ENSG00000013725CD6 Cis 0.1 1.46E-06 Blood 6.43E-07 rs11230555 11 60763134 ENSG00000013725ENSG00000013725CD6 Cis 0.1 1.86E-06 Blood 8.19E-07 rs12421176 11 60805385 ENSG00000013725ENSG00000013725CD6 Cis 0.1 3.39E-06 Blood 4.70E-07 rs12293191 11 60806747 ENSG00000013725ENSG00000013725CD6 Cis 0.1 3.51E-06 Blood 3.95E-07 rs11230556 11 60765984 ENSG00000013725ENSG00000013725CD6 Cis 0.1 9.63E-06 Blood 6.43E-07 rs11085735 19 10602180 ENSG00000129355CDKN2D cis 0.01 1.48E-05 Blood 1.36E-10 rs913678 20 48955424 ENSG00000172216CEBPB cis 0 8.55E-18 Blood 4.59E-08 rs1199096 10 59950234 ENSG00000122873ACCEPTEDCISD1 Cis NA 0 Whole blood 5.78E-07 rs1199096 10 59950234 ENSG00000122873CISD1 Cis NA 0 Whole blood 5.78E-07 rs1199096 10 59950234 ENSG00000122873CISD1 Cis NA 0 Whole blood 5.78E-07 rs1199096 10 59950234 ENSG00000122873CISD1 cis 0 0 Blood 5.78E-07 rs1199103 10 59947231 ENSG00000122873CISD1 cis 0 0 Blood 1.88E-07 ACCEPTED MANUSCRIPT

rs1416764 10 60040351 ENSG00000122873CISD1 cis 0 0 Blood 2.56E-07 rs1698402 10 59936444 ENSG00000122873CISD1 cis 0 0 Blood 2.57E-07 rs1769044 10 59958956 ENSG00000122873CISD1 cis 0 0 Blood 4.54E-07 rs1867571 10 60038850 ENSG00000122873CISD1 cis 0 0 Blood 3.00E-07 rs1867573 10 60042972 ENSG00000122873CISD1 cis 0 0 Blood 3.55E-07 rs2153281 10 59972453 ENSG00000122873CISD1 cis 0 0 Blood 2.71E-07 rs2251039 10 60028894 ENSG00000122873CISD1 cis 0 0 Blood 2.46E-07 rs2253192 10 59999323 ENSG00000122873CISD1 cis 0 0 Blood 1.93E-07 rs2254775 10 60036815 ENSG00000122873CISD1 cis 0 0 Blood 2.20E-07 rs2486491 10 59970391 ENSG00000122873CISD1 cis 0 0 Blood 2.97E-07 rs2590307 10 59977161 ENSG00000122873CISD1 cis 0 0 Blood 5.12E-07 rs2590313 10 59986383 ENSG00000122873CISD1 cis 0 0 Blood 3.31E-07 rs2590339 10 60012231 ENSG00000122873CISD1 cis 0 0 Blood 2.78E-07 rs2790155 10 59974744 ENSG00000122873CISD1 cis 0 0 Blood 2.92E-07 rs2790170 10 60028507 ENSG00000122873CISD1 cis 0 0 Blood 1.87E-07 rs2790179 10 60035372 ENSG00000122873CISD1 Cis NA 0 Whole blood 2.38E-07 rs2790179 10 60035372 ENSG00000122873CISD1 CisMANUSCRIPT NA 0 Whole blood 2.38E-07 rs2790179 10 60035372 ENSG00000122873CISD1 Cis NA 0 Whole blood 2.38E-07 rs2790179 10 60035372 ENSG00000122873CISD1 cis 0 0 Blood 2.38E-07 rs2790182 10 60039877 ENSG00000122873CISD1 cis 0 0 Blood 2.25E-07 rs2790189 10 60042576 ENSG00000122873CISD1 cis 0 0 Blood 1.09E-07 rs2790216 10 59997926 ENSG00000122873CISD1 cis 0 0 Blood 1.60E-07 rs2790236 10 59985102 ENSG00000122873CISD1 cis 0 0 Blood 2.79E-07 rs2790241 10 60015313 ENSG00000122873CISD1 cis 0 0 Blood 2.28E-07 rs2790242 10 59980652 ENSG00000122873CISD1 cis 0 0 Blood 5.78E-07 rs1199096 10 59950234 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 2.81E-18 Blood 5.78E-07 rs1199103 10 59947231 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 2.91E-18 Blood 1.88E-07 rs2486491 10 59970391 ENSG00000122873ENSG00000122873ACCEPTEDCISD1 Cis 0.1 3.03E-18 Blood 2.97E-07 rs2153281 10 59972453 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 3.05E-18 Blood 2.71E-07 rs1769044 10 59958956 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 3.07E-18 Blood 4.54E-07 rs2790242 10 59980652 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 3.09E-18 Blood 5.78E-07 rs2790155 10 59974744 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 3.11E-18 Blood 2.92E-07 ACCEPTED MANUSCRIPT

rs2590313 10 59986383 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 3.15E-18 Blood 3.31E-07 rs2790236 10 59985102 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 3.16E-18 Blood 2.79E-07 rs2790216 10 59997926 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 4.26E-18 Blood 1.60E-07 rs1698402 10 59936444 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 4.34E-18 Blood 2.57E-07 rs2590307 10 59977161 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 4.78E-18 Blood 5.12E-07 rs2253192 10 59999323 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 5.19E-18 Blood 1.93E-07 rs2590339 10 60012231 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 1.28E-17 Blood 2.78E-07 rs2790189 10 60042576 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 1.87E-17 Blood 1.09E-07 rs2790170 10 60028507 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 2.02E-17 Blood 1.87E-07 rs2251039 10 60028894 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 2.19E-17 Blood 2.46E-07 rs1867571 10 60038850 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 2.41E-17 Blood 3.00E-07 rs2790179 10 60035372 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 2.60E-17 Blood 2.38E-07 rs2254775 10 60036815 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 2.63E-17 Blood 2.20E-07 rs1416764 10 60040351 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 2.88E-17 Blood 2.56E-07 rs1867573 10 60042972 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 3.01E-17 Blood 3.55E-07 rs1416763 10 60029163 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 4.00E-17 Blood 1.80E-07 rs2790182 10 60039877 ENSG00000122873ENSG00000122873CISD1 CisMANUSCRIPT 0.1 4.05E-17 Blood 2.25E-07 rs2790241 10 60015313 ENSG00000122873ENSG00000122873CISD1 Cis 0.1 5.42E-17 Blood 2.28E-07 rs740495 19 1124835 ENSG00000064666CNN2 cis 0 1.06E-06 Blood 1.20E-14 rs2472649 4 74857708 ENSG00000163735CXCL5 cis 0 0 Blood 4.15E-07 rs7512140 1 161463601 ENSG00000072694FCGR2B cis 0 0 Blood 5.90E-07 rs7103915 11 63944370 LRG_180 FERMT3 cis 0.01 1.39E-05 Bloo d 9.57E-09 rs11231713 11 63931849 LRG_180 FERMT3 cis 0.02 3.75E-05 Blo od 5.27E-09 rs2096706 11 63924446 LRG_180 FERMT3 cis 0.02 4.46E-05 Bloo d 1.32E-08 rs11601686 11 64189110 LRG_180 FERMT3 cis 0.02 4.83E-05 Blo od 1.19E-08 rs947939 11 63885287 LRG_180 FERMT3 cis 0.03 6.18E-05 Blood 2.54E-08 rs1205843 20 30742996 ENSG00000101336HCK cis 0 1.06E-05 Blood 9.38E-07 rs7911264 10 94436851 ENSG00000152804ACCEPTEDHHEX cis 0 1.50E-19 Blood 2.98E-08 rs2497306 10 94485211 ENSG00000152804HHEX cis 0 2.93E-15 Blood 7.38E-08 rs2497304 10 94492716 ENSG00000152804HHEX cis 0 6.22E-15 Blood 6.26E-08 rs10882091 10 94374377 ENSG00000152804HHEX cis 0 2.76E-14 Blood 1.01E-07 rs6583830 10 94398118 ENSG00000152804HHEX cis 0 4.64E-14 Blood 8.86E-08 ACCEPTED MANUSCRIPT

rs7914814 10 94382950 ENSG00000152804HHEX cis 0 4.64E-14 Blood 8.66E-08 rs947591 10 94495753 ENSG00000152804HHEX cis 0 1.03E-12 Blood 1.06E-07 rs12778642 10 94464307 ENSG00000152804HHEX cis 0 2.32E-11 Blood 3.67E-08 rs5015480 10 94465559 ENSG00000152804HHEX cis 0 3.31E-08 Blood 3.29E-07 rs7924271 10 94542446 ENSG00000152804HHEX cis 0.02 5.96E-05 Blood 7.07E-07 rs1007654 17 38111354 ENSG00000161405IKZF3 cis 0 0 Blood 2.80E-12 rs11869286 17 37813856 ENSG00000161405IKZF3 cis 0 0 Blood 1.80E-09 rs1476278 17 37836243 ENSG00000161405IKZF3 cis 0 0 Blood 2.60E-12 rs1877031 17 37814080 ENSG00000161405IKZF3 cis 0 0 Blood 1.84E-09 rs2271308 17 37817482 ENSG00000161405IKZF3 cis 0 0 Blood 1.33E-07 rs2517955 17 37843681 ENSG00000161405IKZF3 cis 0 0 Blood 3.66E-13 rs8065126 17 38099035 ENSG00000161405IKZF3 cis 0 0 Blood 3.36E-13 s903502 17 37829604 ENSG00000161405IKZF3 cis 0 0 Blood 5.21E-15 rs9303274 17 37836353 ENSG00000161405IKZF3 cis 0 0 Blood 2.24E-12 rs9303277 17 37976469 ENSG00000161405IKZF3 Cis NA 0 Whole blood 7.90E-28 rs9303277 17 37976469 ENSG00000161405IKZF3 Cis NA 0 Whole blood 7.90E-28 rs9303277 17 37976469 ENSG00000161405IKZF3 CisMANUSCRIPT NA 0 Whole blood 7.90E-28 rs3859192 17 38128648 ENSG00000161405IKZF3 cis 0 4.41E-16 Blood 2.38E-14 rs12453334 17 38153473 ENSG00000161405IKZF3 cis 0 2.28E-13 Blood 5.25E-09 rs2227319 17 38170845 ENSG00000161405IKZF3 cis 0 3.49E-13 Blood 5.01E-09 rs907091 17 37921742 ENSG00000161405IKZF3 cis 0 3.39E-12 Blood 4.87E-29 rs8077456 17 38128765 ENSG00000161405IKZF3 cis 0 1.32E-11 Blood 5.30E-07 rs10445308 17 37938047 ENSG00000161405IKZF3 cis 0 2.53E-11 Blood 6.67E-36 rs907092 17 37922259 ENSG00000161405IKZF3 cis 0 6.65E-11 Blood 1.77E-37 rs8075668 17 38137623 ENSG00000161405IKZF3 cis 0 2.98E-08 Blood 6.32E-11 rs6503525 17 38095174 ENSG00000161405IKZF3 cis 0 4.14E-06 Blood 2.66E-15 rs8079416 17 38092713 ENSG00000161405IKZF3 cis 0 5.04E-06 Blood 2.71E-15 rs7212938 17 38122680 ENSG00000161405ACCEPTEDIKZF3 cis 0 6.90E-06 Blood 1.07E-13 rs1008723 17 38066267 ENSG00000161405IKZF3 cis 0.01 1.89E-05 Blood 1.18E-25 rs869402 17 38068043 ENSG00000161405IKZF3 cis 0.01 1.97E-05 Blood 8.09E-24 rs2305479 17 38062217 ENSG00000161405IKZF3 cis 0.01 1.99E-05 Blood 1.34E-29 rs3816470 17 37985801 ENSG00000161405IKZF3 cis 0.01 2.32E-05 Blood 4.69E-27 ACCEPTED MANUSCRIPT

rs7224129 17 38075426 ENSG00000161405IKZF3 cis 0.02 3.60E-05 Blood 4.74E-24 rs7219923 17 38074518 ENSG00000161405IKZF3 cis 0.02 3.72E-05 Blood 6.98E-24 rs9303281 17 38074046 ENSG00000161405IKZF3 cis 0.02 3.78E-05 Blood 5.34E-24 rs9901146 17 38043343 ENSG00000161405IKZF3 cis 0.02 3.82E-05 Blood 1.74E-29 rs907091 17 37921742 ENSG00000161405IKZF3 cis 0.02 3.92E-05 Blood 4.87E-29 rs9303277 17 37976469 ENSG00000161405IKZF3 cis 0.02 5.35E-05 Blood 7.90E-28 rs8070454 17 38160754 ENSG00000161405IKZF3 cis 0.03 6.08E-05 Blood 4.05E-10 rs8078723 17 38166879 ENSG00000161405IKZF3 cis 0.03 6.88E-05 Blood 3.62E-10 rs2070729 5 131819921 ENSG00000125347IRF1 cis 0 4.51E-10 Blood 7.57E-23 rs10077785 5 131801158 ENSG00000125347IRF1 cis 0 4.58E-09 Blood 6.64E-19 rs10039559 5 131804507 ENSG00000125347IRF1 cis 0 4.66E-09 Blood 6.95E-18 rs2248116 5 131804347 ENSG00000125347IRF1 cis 0 5.18E-09 Blood 1.77E-37 rs2522057 5 131801947 ENSG00000125347IRF1 cis 0 7.83E-09 Blood 4.61E-37 rs2188962 5 131770805 ENSG00000125347IRF1 cis 0 8.50E-09 Blood 3.74E-37 rs11745587 5 131796922 ENSG00000125347IRF1 cis 0 9.96E-09 Blood 4.27E-29 rs12521868 5 131784393 ENSG00000125347IRF1 cis 0 1.23E-08 Blood 3.05E-37 rs3857440 5 131794069 ENSG00000125347IRF1 cisMANUSCRIPT 0 1.66E-08 Blood 1.99E-18 rs2548993 5 131808869 ENSG00000125347IRF1 cis 0 2.10E-08 Blood 3.84E-19 rs11748193 5 131725329 ENSG00000125347IRF1 cis 0 3.24E-08 Blood 7.93E-36 rs11746555 5 131727033 ENSG00000125347IRF1 cis 0 3.41E-08 Blood 5.24E-36 rs4540166 5 131779857 ENSG00000125347IRF1 cis 0 3.50E-08 Blood 2.74E-19 rs11744116 5 131779760 ENSG00000125347IRF1 cis 0 3.51E-08 Blood 1.31E-19 rs11739135 5 131733397 ENSG00000125347IRF1 cis 0 4.07E-08 Blood 4.50E-36 rs4235801 5 131782444 ENSG00000125347IRF1 cis 0 6.04E-08 Blood 4.30E-19 rs17622208 5 131717050 ENSG00000125347IRF1 cis 0 3.43E-07 Blood 1.69E-25 rs11950562 5 131652529 ENSG00000125347IRF1 cis 0 3.58E-07 Blood 7.94E-25 rs10058074 5 131686146 ENSG00000125347IRF1 cis 0 5.12E-07 Blood 1.72E-25 rs4705938 5 131694077 ENSG00000125347ACCEPTEDIRF1 cis 0 5.41E-07 Blood 1.82E-25 rs10071051 5 131752620 ENSG00000125347IRF1 cis 0 7.87E-07 Blood 1.31E-15 rs17622656 5 131820997 ENSG00000125347IRF1 cis 0 8.81E-07 Blood 3.10E-37 rs2285673 5 131755969 ENSG00000125347IRF1 cis 0 1.06E-06 Blood 5.66E-16 rs17715481 5 131815384 ENSG00000125347IRF1 cis 0 5.80E-06 Blood 5.41E-09 ACCEPTED MANUSCRIPT

rs7705189 5 131623358 ENSG00000125347IRF1 cis 0.01 1.15E-05 Blood 7.89E-17 rs2089855 5 131573529 ENSG00000125347IRF1 cis 0.03 8.77E-05 Blood 2.17E-20 rs7701414 5 131585958 ENSG00000125347IRF1 cis 0.04 9.12E-05 Blood 7.08E-20 rs4594848 5 131586598 ENSG00000125347IRF1 cis 0.04 9.61E-05 Blood 6.65E-20 rs10758677 9 5188078 ENSG00000096968JAK2 cis 0 0 Blood 2.10E-14 rs10815144 9 5010192 ENSG00000096968JAK2 cis 0 0 Blood 9.17E-17 rs1328917 9 5049065 ENSG00000096968JAK2 cis 0 0 Blood 6.31E-18 rs2149556 9 5059440 ENSG00000096968JAK2 cis 0 0 Blood 2.42E-15 rs2230724 9 5081780 ENSG00000096968JAK2 cis 0 0 Blood 5.57E-17 rs7023146 9 5040163 ENSG00000096968JAK2 cis 0 0 Blood 4.64E-18 rs7847294 9 5097281 ENSG00000096968JAK2 cis 0 0 Blood 1.18E-14 rs7861755 9 5273180 ENSG00000096968JAK2 cis 0 0 Blood 1.25E-09 rs7034753 9 5021514 ENSG00000096968JAK2 cis 0 4.00E-20 Blood 4.86E-14 rs7850484 9 5147817 ENSG00000096968JAK2 cis 0 6.90E-19 Blood 1.27E-26 rs11793659 9 5109707 ENSG00000096968JAK2 cis 0 3.90E-18 Blood 3.34E-27 rs17425637 9 5110000 ENSG00000096968JAK2 cis 0 3.90E-18 Blood 4.86E-28 rs10815157 9 5108771 ENSG00000096968JAK2 cisMANUSCRIPT 0 4.19E-18 Blood 8.07E-27 rs3824433 9 5113577 ENSG00000096968JAK2 cis 0 5.15E-18 Blood 4.62E-27 rs3780374 9 5099677 ENSG00000096968JAK2 cis 0 5.39E-18 Blood 1.03E-27 rs17425819 9 5114773 ENSG00000096968JAK2 cis 0 5.48E-18 Blood 5.42E-28 rs3780373 9 5098223 ENSG00000096968JAK2 cis 0 7.61E-18 Blood 3.00E-26 rs7047795 9 5181467 ENSG00000096968JAK2 cis 0 9.65E-18 Blood 9.30E-26 rs10119004 9 5071049 ENSG00000096968JAK2 cis 0 9.84E-18 Blood 9.93E-17 rs3780381 9 5114523 ENSG00000096968JAK2 cis 0 1.52E-17 Blood 8.73E-28 rs7038687 9 5134065 ENSG00000096968JAK2 cis 0 2.48E-17 Blood 1.07E-26 rs10121316 9 5147539 ENSG00000096968JAK2 cis 0 2.54E-17 Blood 1.32E-26 rs10758674 9 5163645 ENSG00000096968JAK2 cis 0 2.57E-17 Blood 3.16E-26 rs7029084 9 5174638 ENSG00000096968ACCEPTEDJAK2 cis 0 2.58E-17 Blood 5.71E-26 rs7871515 9 5175288 ENSG00000096968JAK2 cis 0 2.76E-17 Blood 7.51E-26 rs7040922 9 5174829 ENSG00000096968JAK2 cis 0 3.00E-17 Blood 1.47E-25 rs3780382 9 5232299 ENSG00000096968JAK2 cis 0 3.21E-17 Blood 2.38E-22 rs16922779 9 5200127 ENSG00000096968JAK2 cis 0 3.25E-17 Blood 6.92E-23 ACCEPTED MANUSCRIPT

rs10491650 9 5203054 ENSG00000096968JAK2 cis 0 3.47E-17 Blood 3.56E-22 rs12343038 9 5178579 ENSG00000096968JAK2 cis 0 3.61E-17 Blood 7.37E-26 rs10491651 9 5200060 ENSG00000096968JAK2 cis 0 3.64E-17 Blood 2.27E-23 rs16922786 9 5200714 ENSG00000096968JAK2 cis 0 3.64E-17 Blood 3.58E-23 rs6476948 9 5204404 ENSG00000096968JAK2 cis 0 3.84E-17 Blood 5.25E-23 rs10974993 9 5182159 ENSG00000096968JAK2 cis 0 4.00E-17 Blood 3.77E-26 rs10122037 9 5192874 ENSG00000096968JAK2 cis 0 4.69E-17 Blood 2.48E-24 rs1322222 9 5190444 ENSG00000096968JAK2 cis 0 4.74E-17 Blood 1.56E-23 rs10118930 9 5148278 ENSG00000096968JAK2 cis 0 4.80E-17 Blood 4.57E-27 rs7850294 9 5191128 ENSG00000096968JAK2 cis 0 4.97E-17 Blood 1.51E-22 rs12115301 9 5180548 ENSG00000096968JAK2 cis 0 5.74E-17 Blood 7.05E-26 rs2146040 9 5260039 ENSG00000096968JAK2 cis 0 6.34E-17 Blood 1.35E-21 rs7870536 9 5245493 ENSG00000096968JAK2 cis 0 6.46E-17 Blood 9.41E-23 rs10283467 9 5244356 ENSG00000096968JAK2 cis 0 6.55E-17 Blood 2.10E-23 rs7850228 9 5210671 ENSG00000096968JAK2 cis 0 9.97E-17 Blood 6.59E-19 rs12347727 9 5000811 ENSG00000096968JAK2 cis 0 2.75E-16 Blood 1.01E-35 rs3780367 9 5068755 ENSG00000096968JAK2 cisMANUSCRIPT 0 2.76E-16 Blood 4.36E-34 rs7859390 9 5062473 ENSG00000096968JAK2 cis 0 2.99E-16 Blood 5.16E-34 rs7862042 9 5268139 ENSG00000096968JAK2 cis 0 3.05E-16 Blood 1.79E-19 rs7851556 9 5022807 ENSG00000096968JAK2 cis 0 3.37E-16 Blood 4.87E-34 rs2149555 9 5053743 ENSG00000096968JAK2 cis 0 3.75E-16 Blood 2.46E-34 rs10121491 9 5046935 ENSG00000096968JAK2 cis 0 3.76E-16 Blood 1.70E-33 rs7036761 9 5198781 ENSG00000096968JAK2 cis 0 4.95E-16 Blood 4.14E-19 rs10815149 9 5063701 ENSG00000096968JAK2 cis 0 5.23E-16 Blood 8.97E-33 rs10974944 9 5070831 ENSG00000096968JAK2 cis 0 7.01E-16 Blood 4.66E-34 rs3780366 9 5068596 ENSG00000096968JAK2 cis 0 8.17E-16 Blood 4.74E-34 rs11999802 9 5189773 ENSG00000096968JAK2 cis 0 1.22E-15 Blood 2.75E-17 rs10974914 9 5014332 ENSG00000096968ACCEPTEDJAK2 cis 0 1.54E-15 Blood 5.23E-33 rs7030260 9 5008070 ENSG00000096968JAK2 cis 0 2.96E-15 Blood 8.00E-33 rs4425810 9 5222208 ENSG00000096968JAK2 cis 0 3.12E-15 Blood 7.89E-21 rs4142064 9 5221818 ENSG00000096968JAK2 cis 0 3.48E-15 Blood 6.75E-21 rs10975003 9 5213687 ENSG00000096968JAK2 cis 0 5.21E-15 Blood 2.05E-16 ACCEPTED MANUSCRIPT

rs2224571 9 5196253 ENSG00000096968JAK2 cis 0 7.69E-15 Blood 4.20E-23 rs7034878 9 5058991 ENSG00000096968JAK2 Cis NA 2.42E-12 Whole blood 1.36E-15 rs7034878 9 5058991 ENSG00000096968JAK2 Cis NA 9.36E-12 Whole blood 1.36E-15 rs7849191 9 4988761 ENSG00000096968JAK2 cis 0 2.24E-11 Blood 5.31E-24 rs10974892 9 4964774 ENSG00000096968JAK2 cis 0 2.72E-11 Blood 1.89E-16 rs10758669 9 4981602 ENSG00000096968JAK2 cis 0 4.83E-11 Blood 7.88E-45 rs1536800 9 5055434 ENSG00000096968JAK2 cis 0 7.95E-11 Blood 3.31E-31 rs7034878 9 5058991 ENSG00000096968JAK2 Cis NA 8.67E-08 Whole blood 1.36E-15 rs10758677 9 5188078 --- ENSG00000096968JAK2 Cis 0.1 9.51E-08 Blood 2.10E-14 rs7847294 9 5097281 --- ENSG00000096968JAK2 Cis 0.1 1.42E-07 Blood 1.18E-14 rs2230724 9 5081780 --- ENSG00000096968JAK2 Cis 0.1 2.26E-07 Blood 5.57E-17 rs7032169 9 4966178 ENSG00000096968JAK2 cis 0.01 1.23E-05 Blood 5.82E-15 rs11041476 11 1875067 ENSG00000130592LSP1 Cis NA 1.11E-12 Whole blood 3.28E-10 rs11041476 11 1875067 ENSG00000130592LSP1 Cis NA 5.63E-09 Whole blood 3.28E-10 rs11041476 11 1875067 ENSG00000130592LSP1 Cis NA 3.56E-05 Whole blood 3.28E-10 rs10908812 1 160879316 ENSG00000122224LY9 cis 0 0 Blood 6.76E-07 rs11265510 1 160859559 ENSG00000122224LY9 cisMANUSCRIPT 0 0 Blood 7.76E-07 rs1333062 1 160846284 ENSG00000122224LY9 cis 0 0 Blood 2.58E-08 rs2039415 1 160854445 ENSG00000122224LY9 cis 0 0 Blood 1.02E-08 rs3820094 1 160849082 ENSG00000122224LY9 cis 0 0 Blood 2.35E-08 rs4656956 1 160847617 ENSG00000122224LY9 cis 0 0 Blood 2.21E-08 rs4656957 1 160850280 ENSG00000122224LY9 cis 0 0 Blood 2.91E-08 rs7411035 1 160856076 ENSG00000122224LY9 cis 0 0 Blood 2.94E-08 rs10763790 10 30791355 ENSG00000107968MAP3K8 cis 0 0 Blood 9.05E-07 rs2771242 10 30762973 ENSG00000107968MAP3K8 cis 0 1.58E-18 Blood 9.95E-08 rs593400 10 30762088 ENSG00000107968MAP3K8 cis 0 1.74E-18 Blood 7.18E-08 rs619864 10 30750950 ENSG00000107968MAP3K8 cis 0 3.14E-18 Blood 9.27E-08 rs603072 10 30755778 ENSG00000107968ACCEPTEDMAP3K8 cis 0 3.36E-18 Blood 4.80E-08 rs598672 10 30742388 ENSG00000107968MAP3K8 cis 0 3.85E-18 Blood 1.32E-07 rs602097 10 30747546 ENSG00000107968MAP3K8 cis 0 4.73E-18 Blood 1.56E-07 rs602966 10 30755853 ENSG00000107968MAP3K8 cis 0 6.27E-18 Blood 7.16E-08 rs10826797 10 30690376 ENSG00000107968MAP3K8 cis 0 7.43E-16 Blood 7.91E-08 ACCEPTED MANUSCRIPT

rs303428 10 30739058 ENSG00000107968MAP3K8 cis 0 5.17E-15 Blood 3.27E-08 rs10763790 10 30791355 ENSG00000107968MAP3K8 Cis NA 7.11E-13 Whole blood 9.05E-07 rs2265186 10 30691743 ENSG00000107968MAP3K8 cis 0 2.00E-12 Blood 7.04E-10 rs6481677 10 30701816 ENSG00000107968MAP3K8 cis 0 2.54E-12 Blood 7.69E-10 rs1543725 10 30698873 ENSG00000107968MAP3K8 cis 0 2.65E-12 Blood 5.02E-10 rs2480277 10 30703513 ENSG00000107968MAP3K8 cis 0 2.84E-12 Blood 9.69E-10 rs1042058 10 30728101 ENSG00000107968MAP3K8 cis 0 3.48E-12 Blood 2.10E-10 rs303436 10 30731893 ENSG00000107968MAP3K8 cis 0 7.47E-12 Blood 1.64E-08 rs694346 10 30711929 ENSG00000107968MAP3K8 cis 0 1.73E-11 Blood 5.60E-08 rs1536833 10 30699265 ENSG00000107968MAP3K8 cis 0 2.13E-11 Blood 1.53E-07 rs10763790 10 30791355 ENSG00000107968MAP3K8 Cis NA 8.58E-09 Whole blood 9.05E-07 rs306583 10 30710057 ENSG00000107968MAP3K8 cis 0 9.76E-09 Blood 6.07E-09 rs10763790 10 30791355 ENSG00000107968MAP3K8 Cis NA 7.46E-08 Whole blood 9.05E-07 rs2072711 22 37268555 LRG_159 NCF4 cis 0 0 Blood 1.00E-10 rs2072711 22 37268555 LRG_159 NCF4 cis 0 1.50E-18 Blood 1.00 E-10 rs2072711 22 37268555 LRG_159 NCF4 cis 0 1.67E-10 Blood 1.00 E-10 rs11660128 18 77227950 ENSG00000131196NFATC1 cisMANUSCRIPT 0 0 Blood 2.31E-07 rs11664153 18 77195813 ENSG00000131196NFATC1 cis 0 0 Blood 3.10E-07 rs11664725 18 77192116 ENSG00000131196NFATC1 cis 0 0 Blood 5.06E-08 rs3786185 18 77199023 ENSG00000131196NFATC1 cis 0 0 Blood 7.24E-08 rs7236492 18 77220616 ENSG00000131196NFATC1 cis 0 0 Blood 1.00E-10 rs7236492 18 77220616 ENSG00000131196NFATC1 cis 0 0 Blood 3.05E-07 rs3786185 18 77199023 ENSG00000131196NFATC1 cis 0.01 1.94E-05 Blood 7.24E-08 rs11664725 18 77192116 ENSG00000131196NFATC1 cis 0.01 2.33E-05 Blood 5.06E-08 rs10521209 16 50755709 LRG_177 NOD2 cis 0 0 Blood 7.96E-22 rs1077861 16 50759547 LRG_177 NOD2 cis 0 0 Blood 5.32E-11 rs1109863 16 50692364 LRG_177 NOD2 cis 0 0 Blood 7.05E-15 rs11640716 16 50665089 LRG_177ACCEPTED NOD2 cis 0 0 Blood 2.80E-14 rs11647841 16 50743331 LRG_177 NOD2 cis 0 0 Blood 7.76E-24 rs12913 16 50668426 LRG_177 NOD2 cis 0 0 Blood 2.52E-14 rs12925755 16 50846162 LRG_177 NOD2 cis 0 0 Blood 1.06E-17 rs1420872 16 50807779 LRG_177 NOD2 cis 0 0 Blood 5.62E-24 ACCEPTED MANUSCRIPT

rs1558663 16 50662844 LRG_177 NOD2 cis 0 0 Blood 2.63E-14 rs17221417 16 50739582 LRG_177 NOD2 cis 0 0 Blood 1.45E-51 rs17313265 16 50747704 LRG_177 NOD2 cis 0 0 Blood 1.64E-57 rs17314544 16 50820078 LRG_177 NOD2 cis 0 0 Blood 2.60E-25 rs1861758 16 50751787 LRG_177 NOD2 cis 0 0 Blood 2.57E-21 rs1861759 16 50745583 LRG_177 NOD2 cis 0 0 Blood 1.79E-20 rs2066843 16 50745199 LRG_177 NOD2 cis 0 0 Blood 2.14E-57 rs2066849 16 50687015 LRG_177 NOD2 cis 0 0 Blood 1.91E-15 rs2076756 16 50756881 LRG_177 NOD2 cis 0 0 Blood 2.83E-62 rs2111234 16 50734033 LRG_177 NOD2 cis 0 0 Blood 3.86E-08 rs2111435 16 50819910 LRG_177 NOD2 cis 0 0 Blood 4.10E-16 rs3135500 16 50766886 LRG_177 NOD2 cis 0 0 Blood 3.72E-21 rs3785142 16 50787147 LRG_177 NOD2 cis 0 0 Blood 2.51E-16 rs4486887 16 50677571 LRG_177 NOD2 cis 0 0 Blood 1.92E-15 rs4785224 16 50730446 LRG_177 NOD2 cis 0 0 Blood 6.59E-23 rs4785227 16 50842197 LRG_177 NOD2 cis 0 0 Blood 4.66E-15 rs4785450 16 50792268 LRG_177 NOD2 cisMANUSCRIPT 0 0 Blood 3.67E-20 rs4785452 16 50842077 LRG_177 NOD2 cis 0 0 Blood 4.66E-15 rs5743266 16 50731096 LRG_177 NOD2 cis 0 0 Blood 1.22E-49 rs6500331 16 50808726 LRG_177 NOD2 cis 0 0 Blood 2.66E-19 rs7186262 16 50686959 LRG_177 NOD2 cis 0 0 Blood 1.42E-15 rs7194886 16 50725193 LRG_177 NOD2 cis 0 0 Blood 8.65E-30 rs7199150 16 50676514 LRG_177 NOD2 cis 0 0 Blood 1.48E-14 rs7342715 16 50787483 LRG_177 NOD2 cis 0 0 Blood 6.85E-20 rs745230 16 50670182 LRG_177 NOD2 cis 0 0 Blood 4.06E-14 rs748855 16 50751398 LRG_177 NOD2 cis 0 0 Blood 1.37E-21 rs749985 16 50673651 LRG_177 NOD2 cis 0 0 Blood 2.52E-14 rs751271 16 50751175 LRG_177ACCEPTED NOD2 cis 0 0 Blood 1.15E-08 rs8047222 16 50660962 LRG_177 NOD2 cis 0 0 Blood 6.39E-16 rs8057341 16 50737980 LRG_177 NOD2 cis 0 0 Blood 1.79E-07 rs9635531 16 50841795 LRG_177 NOD2 cis 0 0 Blood 2.87E-19 rs9938976 16 50817219 LRG_177 NOD2 cis 0 0 Blood 2.03E-24 ACCEPTED MANUSCRIPT

rs9673419 16 50661273 LRG_177 NOD2 cis 0 3.10E-19 Blood 8.10 E-17 rs1990623 16 50565970 LRG_177 NOD2 cis 0 2.45E-18 Blood 5.97 E-11 rs2111234 16 50734033 ENSG00000167207LRG_177 NOD2 Cis 0.1 1.26E-15 Blood 3.86E-08 rs8057341 16 50737980 ENSG00000167207LRG_177 NOD2 Cis 0.1 3.23E-15 Blood 1.79E-07 rs1077861 16 50759547 ENSG00000167207LRG_177 NOD2 Cis 0.1 9.51E-15 Blood 5.32E-11 rs751271 16 50751175 ENSG00000167207LRG_177 NOD2 Cis 0.1 1.07E-13 Blood 1.15E-08 rs5743266 16 50731096 ENSG00000167207LRG_177 NOD2 Cis 0.1 5.38E-06 Blood 1.22E-49 rs17221417 16 50739582 ENSG00000167207LRG_177 NOD2 Cis 0.1 9.19E-06 Blood 1.45E-51 rs2297441 20 62327582 ENSG00000258366RTEL1 cis 0.01 2.06E-05 Blood 1.67E-20 rs2257885 20 62334220 ENSG00000258366RTEL1 cis 0.01 2.13E-05 Blood 2.62E-19 rs2427533 20 62365866 ENSG00000258366RTEL1 cis 0.01 3.07E-05 Blood 1.61E-17 rs2427530 20 62361737 ENSG00000258366RTEL1 cis 0.01 3.10E-05 Blood 1.07E-17 rs2738758 20 62349625 ENSG00000258366RTEL1 cis 0.02 4.77E-05 Blood 2.72E-18 rs2777938 20 62306208 ENSG00000258366RTEL1 cis 0.03 8.26E-05 Blood 1.07E-12 rs3016173 11 60790839 ENSG00000110446SLC15A3 cis 0 2.23E-08 Blood 4.72E-07 rs175102 11 60810757 ENSG00000110446SLC15A3 cis 0 2.91E-08 Blood 6.55E-07 rs11230563 11 60776209 ENSG00000110446SLC15A3 cisMANUSCRIPT 0 4.78E-07 Blood 9.03E-13 rs175112 11 60825022 ENSG00000110446SLC15A3 cis 0 7.30E-06 Blood 1.15E-10 rs4780355 16 11347858 ENSG00000185338SOCS1 cis 0 2.95E-11 Blood 1.20E-13 rs4451969 16 11383519 ENSG00000185338SOCS1 cis 0 6.76E-11 Blood 2.49E-12 rs243325 16 11354497 ENSG00000185338SOCS1 cis 0 1.63E-10 Blood 8.00E-14 rs243323 16 11361202 ENSG00000185338SOCS1 cis 0 4.10E-10 Blood 6.12E-15 rs7193871 16 11455606 ENSG00000185338SOCS1 cis 0 2.49E-09 Blood 1.33E-10 rs1646025 16 11379938 ENSG00000185338SOCS1 cis 0.02 3.60E-05 Blood 5.22E-12 rs423674 16 11373405 ENSG00000185338SOCS1 cis 0.02 5.65E-05 Blood 4.13E-12 rs529866 16 11373320 ENSG00000185338SOCS1 cis 0.03 5.98E-05 Blood 1.49E-12 rs730086 17 40271757 LRG_112 STAT3 cis 0 1.00E-20 Blood 4.61 E-10 rs957970 17 40519890 LRG_112ACCEPTED STAT3 cis 0 1.00E-20 Blood 2.01 E-20 rs1026916 17 40529835 LRG_112 STAT3 cis 0 2.00E-20 Blood 1.4 8E-20 rs12949918 17 40526273 LRG_112 STAT3 cis 0 2.00E-20 Blood 2. 27E-21 rs3816769 17 40498273 LRG_112 STAT3 cis 0 2.00E-20 Blood 1.5 5E-20 rs7211777 17 40534075 LRG_112 STAT3 cis 0 2.00E-20 Blood 2.5 2E-20 ACCEPTED MANUSCRIPT

rs744166 17 40514201 LRG_112 STAT3 cis 0 2.00E-20 Blood 6.15 E-22 rs1026916 17 40529835 LRG_112 STAT3 cis 0 8.00E-20 Blood 1.4 8E-20 rs7211777 17 40534075 LRG_112 STAT3 cis 0 1.10E-19 Blood 2.5 2E-20 rs957970 17 40519890 LRG_112 STAT3 cis 0 1.30E-19 Blood 2.01 E-20 rs3816769 17 40498273 LRG_112 STAT3 cis 0 2.00E-19 Blood 1.5 5E-20 rs744166 17 40514201 LRG_112 STAT3 cis 0 4.10E-19 Blood 6.15 E-22 rs12949918 17 40526273 LRG_112 STAT3 cis 0 7.80E-19 Blood 2. 27E-21 rs730086 17 40271757 LRG_112 STAT3 cis 0 7.01E-18 Blood 4.61 E-10 rs730086 17 40271757 LRG_112 STAT3 cis 0 1.08E-17 Blood 4.61 E-10 rs744166 17 40514201 LRG_112 STAT3 cis 0 9.13E-17 Blood 6.15 E-22 rs12949918 17 40526273 LRG_112 STAT3 cis 0 1.38E-16 Blood 2. 27E-21 rs7211777 17 40534075 LRG_112 STAT3 cis 0 1.50E-16 Blood 2.5 2E-20 rs1026916 17 40529835 LRG_112 STAT3 cis 0 1.68E-16 Blood 1.4 8E-20 rs957970 17 40519890 LRG_112 STAT3 cis 0 1.88E-16 Blood 2.01 E-20 rs3816769 17 40498273 LRG_112 STAT3 cis 0 2.44E-16 Blood 1.5 5E-20 rs4796793 17 40542210 LRG_112 STAT3 cis 0 8.40E-16 Blood 5.8 9E-19 rs9912773 17 40510534 LRG_112 STAT3 cisMANUSCRIPT 0 1.69E-15 Blood 1.7 2E-18 rs3785898 17 40515120 LRG_112 STAT3 cis 0 3.65E-15 Blood 1.7 2E-20 rs6503695 17 40499533 LRG_112 STAT3 cis 0 3.73E-15 Blood 1.4 1E-20 rs8069645 17 40494902 LRG_112 STAT3 cis 0 4.68E-15 Blood 2.3 9E-20 rs8078731 17 40480381 LRG_112 STAT3 cis 0 7.23E-13 Blood 1.9 7E-11 rs9912773 17 40510534 LRG_112 STAT3 cis 0 8.88E-13 Blood 1.7 2E-18 rs4796793 17 40542210 LRG_112 STAT3 cis 0 1.04E-12 Blood 5.8 9E-19 rs12601982 17 40461674 LRG_112 STAT3 cis 0 1.11E-12 Blood 8. 19E-11 rs8074524 17 40469598 LRG_112 STAT3 cis 0 1.75E-12 Blood 2.1 8E-12 rs2293155 17 40460989 LRG_112 STAT3 cis 0 1.84E-12 Blood 9.2 8E-11 rs9906989 17 40455846 LRG_112 STAT3 cis 0 2.14E-12 Blood 9.0 7E-11 rs1053005 17 40465910 LRG_112ACCEPTED STAT3 cis 0 2.34E-12 Blood 4.8 5E-12 rs8072215 17 40271970 LRG_112 STAT3 cis 0 2.57E-12 Blood 2.8 4E-07 rs3809758 17 40471980 LRG_112 STAT3 cis 0 3.04E-12 Blood 2.2 6E-12 rs739636 17 40281063 LRG_112 STAT3 cis 0 5.01E-12 Blood 3.10 E-07 rs2293154 17 40461003 LRG_112 STAT3 cis 0 7.82E-12 Blood 9.2 1E-11 ACCEPTED MANUSCRIPT

rs4796793 17 40542210 LRG_112 STAT3 cis 0 2.25E-11 Blood 5.8 9E-19 rs9912773 17 40510534 LRG_112 STAT3 cis 0 3.11E-11 Blood 1.7 2E-18 rs3785898 17 40515120 LRG_112 STAT3 cis 0 5.59E-11 Blood 1.7 2E-20 rs2293158 17 40447558 LRG_112 STAT3 cis 0 6.05E-11 Blood 4.0 6E-10 rs8072215 17 40271970 LRG_112 STAT3 cis 0 7.76E-11 Blood 2.8 4E-07 rs7217728 17 40447401 LRG_112 STAT3 cis 0 8.00E-11 Blood 4.4 3E-10 rs16967637 17 40446422 LRG_112 STAT3 cis 0 8.35E-11 Blood 3. 43E-10 rs3785898 17 40515120 LRG_112 STAT3 cis 0 8.45E-11 Blood 1.7 2E-20 rs8069645 17 40494902 LRG_112 STAT3 cis 0 9.81E-11 Blood 2.3 9E-20 rs4029774 17 40428961 LRG_112 STAT3 cis 0 1.32E-10 Blood 1.7 1E-09 rs8069645 17 40494902 LRG_112 STAT3 cis 0 1.32E-10 Blood 2.3 9E-20 rs7209222 17 40426489 LRG_112 STAT3 cis 0 1.57E-10 Blood 8.8 5E-10 rs16967611 17 40401567 LRG_112 STAT3 cis 0 1.59E-10 Blood 2. 53E-09 rs9906933 17 40410045 LRG_112 STAT3 cis 0 1.59E-10 Blood 1.8 5E-09 rs16967620 17 40422341 LRG_112 STAT3 cis 0 1.70E-10 Blood 1. 69E-09 rs6503692 17 40421513 LRG_112 STAT3 cis 0 1.70E-10 Blood 3.0 3E-10 rs7218653 17 40425320 LRG_112 STAT3 cisMANUSCRIPT 0 1.79E-10 Blood 8.1 4E-10 rs8064638 17 40424255 LRG_112 STAT3 cis 0 1.83E-10 Blood 1.3 1E-09 rs8082391 17 40398973 LRG_112 STAT3 cis 0 1.86E-10 Blood 1.9 1E-09 rs739636 17 40281063 LRG_112 STAT3 cis 0 1.92E-10 Blood 3.10 E-07 rs6503695 17 40499533 LRG_112 STAT3 cis 0 2.07E-10 Blood 1.4 1E-20 rs963987 17 40561679 LRG_112 STAT3 cis 0 2.07E-10 Blood 5.03 E-12 rs2883456 17 40552794 LRG_112 STAT3 cis 0 2.55E-10 Blood 6.6 9E-12 rs744166 17 40514201 LRG_112 STAT3 Cis NA 2.12E-09 Whole bl ood 6.15E-22 rs6503695 17 40499533 LRG_112 STAT3 cis 0 2.57E-09 Blood 1.4 1E-20 rs2883456 17 40552794 LRG_112 STAT3 cis 0 4.92E-09 Blood 6.6 9E-12 rs963987 17 40561679 LRG_112 STAT3 cis 0 5.15E-09 Blood 5.03 E-12 rs12601982 17 40461674 LRG_112ACCEPTED STAT3 cis 0 6.26E-08 Blood 8. 19E-11 rs11079045 17 40587669 LRG_112 STAT3 cis 0 7.86E-08 Blood 4. 55E-10 rs8078731 17 40480381 LRG_112 STAT3 cis 0 9.16E-08 Blood 1.9 7E-11 rs2293155 17 40460989 LRG_112 STAT3 cis 0 1.01E-07 Blood 9.2 8E-11 rs8078731 17 40480381 LRG_112 STAT3 cis 0 1.17E-07 Blood 1.9 7E-11 ACCEPTED MANUSCRIPT

rs11079045 17 40587669 LRG_112 STAT3 cis 0 1.27E-07 Blood 4. 55E-10 rs9906989 17 40455846 LRG_112 STAT3 cis 0 1.49E-07 Blood 9.0 7E-11 rs8072215 17 40271970 LRG_112 STAT3 cis 0 1.67E-07 Blood 2.8 4E-07 rs12601982 17 40461674 LRG_112 STAT3 cis 0 2.60E-07 Blood 8. 19E-11 rs739636 17 40281063 LRG_112 STAT3 cis 0 3.15E-07 Blood 3.10 E-07 rs2293155 17 40460989 LRG_112 STAT3 cis 0 3.65E-07 Blood 9.2 8E-11 rs2293154 17 40461003 LRG_112 STAT3 cis 0 3.90E-07 Blood 9.2 1E-11 rs744166 17 40514201 LRG_112 STAT3 Cis NA 4.53E-07 Whole bl ood 6.15E-22 rs9906989 17 40455846 LRG_112 STAT3 cis 0 4.94E-07 Blood 9.0 7E-11 rs2293154 17 40461003 LRG_112 STAT3 cis 0 6.94E-07 Blood 9.2 1E-11 rs8074524 17 40469598 LRG_112 STAT3 cis 0 7.72E-07 Blood 2.1 8E-12 rs3809758 17 40471980 LRG_112 STAT3 cis 0 7.87E-07 Blood 2.2 6E-12 rs1053005 17 40465910 LRG_112 STAT3 cis 0 9.04E-07 Blood 4.8 5E-12 rs744166 17 40514201 --- LRG_112 STAT3 Cis 0.1 1.00E-06 Bloo d 6.15E-22 rs963987 17 40561679 LRG_112 STAT3 cis 0 1.37E-06 Blood 5.03 E-12 rs2883456 17 40552794 LRG_112 STAT3 cis 0 1.59E-06 Blood 6.6 9E-12 rs8074524 17 40469598 LRG_112 STAT3 cisMANUSCRIPT 0 3.44E-06 Blood 2.1 8E-12 rs1053005 17 40465910 LRG_112 STAT3 cis 0 4.41E-06 Blood 4.8 5E-12 rs3809758 17 40471980 LRG_112 STAT3 cis 0 4.78E-06 Blood 2.2 6E-12 rs1905339 17 40582296 LRG_112 STAT3 cis 0 8.40E-06 Blood 4.2 0E-07 rs11079045 17 40587669 LRG_112 STAT3 cis 0.01 1.60E-05 Bloo d 4.55E-10 rs2293158 17 40447558 LRG_112 STAT3 cis 0.02 3.52E-05 Blood 4.06E-10 rs16967637 17 40446422 LRG_112 STAT3 cis 0.02 4.48E-05 Bloo d 3.43E-10 rs7217728 17 40447401 LRG_112 STAT3 cis 0.02 4.58E-05 Blood 4.43E-10 rs4029774 17 40428961 LRG_112 STAT3 cis 0.03 6.74E-05 Blood 1.71E-09 rs7209222 17 40426489 LRG_112 STAT3 cis 0.03 7.40E-05 Blood 8.85E-10 rs16967611 17 40401567 LRG_112 STAT3 cis 0.03 7.65E-05 Bloo d 2.53E-09 rs8082391 17 40398973 LRG_112ACCEPTED STAT3 cis 0.03 7.65E-05 Blood 1.91E-09 rs16967620 17 40422341 LRG_112 STAT3 cis 0.03 7.91E-05 Bloo d 1.69E-09 rs6503692 17 40421513 LRG_112 STAT3 cis 0.03 7.91E-05 Blood 3.03E-10 rs9906933 17 40410045 LRG_112 STAT3 cis 0.03 8.01E-05 Blood 1.85E-09 rs7218653 17 40425320 LRG_112 STAT3 cis 0.03 8.28E-05 Blood 8.14E-10 ACCEPTED MANUSCRIPT

rs8064638 17 40424255 LRG_112 STAT3 cis 0.03 8.28E-05 Blood 1.31E-09 rs2293154 17 40461003 ENSG00000126561STAT5A cis 0 5.62E-07 Blood 9.21E-11 rs12601982 17 40461674 ENSG00000126561STAT5A cis 0 6.57E-07 Blood 8.19E-11 rs2293155 17 40460989 ENSG00000126561STAT5A cis 0 7.65E-07 Blood 9.28E-11 rs9906989 17 40455846 ENSG00000126561STAT5A cis 0 8.49E-07 Blood 9.07E-11 rs8078731 17 40480381 ENSG00000126561STAT5A cis 0 8.51E-07 Blood 1.97E-11 rs8074524 17 40469598 ENSG00000126561STAT5A cis 0 9.86E-07 Blood 2.18E-12 rs1053005 17 40465910 ENSG00000126561STAT5A cis 0 1.17E-06 Blood 4.85E-12 rs3809758 17 40471980 ENSG00000126561STAT5A cis 0 1.30E-06 Blood 2.26E-12 rs6503695 17 40499533 ENSG00000126561STAT5A cis 0.01 1.69E-05 Blood 1.41E-20 rs8064638 17 40424255 ENSG00000126561STAT5A cis 0.01 1.97E-05 Blood 1.31E-09 rs9906933 17 40410045 ENSG00000126561STAT5A cis 0.01 1.98E-05 Blood 1.85E-09 rs16967611 17 40401567 ENSG00000126561STAT5A cis 0.01 2.08E-05 Blood 2.53E-09 rs4029774 17 40428961 ENSG00000126561STAT5A cis 0.01 2.11E-05 Blood 1.71E-09 rs7209222 17 40426489 ENSG00000126561STAT5A cis 0.01 2.11E-05 Blood 8.85E-10 rs16967620 17 40422341 ENSG00000126561STAT5A cis 0.01 2.23E-05 Blood 1.69E-09 rs6503692 17 40421513 ENSG00000126561STAT5A cisMANUSCRIPT 0.01 2.23E-05 Blood 3.03E-10 rs16967637 17 40446422 ENSG00000126561STAT5A cis 0.01 2.24E-05 Blood 3.43E-10 rs7218653 17 40425320 ENSG00000126561STAT5A cis 0.01 2.27E-05 Blood 8.14E-10 rs2293158 17 40447558 ENSG00000126561STAT5A cis 0.01 2.28E-05 Blood 4.06E-10 rs7217728 17 40447401 ENSG00000126561STAT5A cis 0.01 2.28E-05 Blood 4.43E-10 rs8082391 17 40398973 ENSG00000126561STAT5A cis 0.01 2.38E-05 Blood 1.91E-09 rs8069645 17 40494902 ENSG00000126561STAT5A cis 0.02 3.63E-05 Blood 2.39E-20 rs3785898 17 40515120 ENSG00000126561STAT5A cis 0.02 3.78E-05 Blood 1.72E-20 rs4796793 17 40542210 ENSG00000126561STAT5A cis 0.02 4.21E-05 Blood 5.89E-19 rs9912773 17 40510534 ENSG00000126561STAT5A cis 0.02 4.65E-05 Blood 1.72E-18 rs10114470 9 117547772 ENSG00000106952TNFSF8 cis 0 0 Blood 1.02E-30 rs1322055 9 117669585 ENSG00000106952ACCEPTEDTNFSF8 cis 0 0 Blood 2.87E-09 rs3810936 9 117552885 ENSG00000106952TNFSF8 cis 0 0 Blood 3.64E-31 rs4246905 9 117553249 ENSG00000106952TNFSF8 cis 0 0 Blood 2.80E-32 rs4263839 9 117566440 ENSG00000106952TNFSF8 cis 0 0 Blood 3.53E-31 rs4574921 9 117538334 ENSG00000106952TNFSF8 cis 0 0 Blood 5.05E-29 ACCEPTED MANUSCRIPT

rs6478108 9 117558703 ENSG00000106952TNFSF8 cis 0 0 Blood 4.05E-31 rs7040029 9 117619214 ENSG00000106952TNFSF8 cis 0 0 Blood 5.48E-23 rs7848647 9 117569046 ENSG00000106952TNFSF8 cis 0 0 Blood 5.02E-31 rs7866342 9 117627569 ENSG00000106952TNFSF8 cis 0 0 Blood 3.16E-21 rs911605 9 117654990 ENSG00000106952TNFSF8 cis 0 0 Blood 1.16E-19 rs7048659 9 117533289 ENSG00000106952TNFSF8 cis 0 2.76E-16 Blood 1.10E-07 rs10114224 9 117613032 ENSG00000106952TNFSF8 cis 0 1.11E-15 Blood 6.63E-09 rs10982427 9 117613696 ENSG00000106952TNFSF8 cis 0 1.11E-15 Blood 1.62E-08 rs10982441 9 117647599 ENSG00000106952TNFSF8 cis 0 1.26E-15 Blood 8.69E-07 rs7865494 9 117576479 ENSG00000106952TNFSF8 cis 0 5.91E-15 Blood 3.70E-11 rs1419134 9 117587022 ENSG00000106952TNFSF8 cis 0 1.42E-14 Blood 4.13E-11 rs2145929 9 117581940 ENSG00000106952TNFSF8 cis 0 3.14E-14 Blood 1.36E-11 rs7868351 9 117584082 ENSG00000106952TNFSF8 cis 0 8.95E-14 Blood 1.65E-11 rs10982412 9 117570856 ENSG00000106952TNFSF8 cis 0 1.42E-12 Blood 2.52E-10 rs17292046 9 117587092 ENSG00000106952TNFSF8 cis 0 2.39E-11 Blood 4.68E-10 rs10982396 9 117518929 ENSG00000106952TNFSF8 cis 0 3.64E-11 Blood 6.90E-10 rs10759736 9 117523192 ENSG00000106952TNFSF8 cisMANUSCRIPT 0 3.70E-11 Blood 4.59E-10 rs7862325 9 117567137 ENSG00000106952TNFSF8 cis 0 8.71E-11 Blood 2.90E-07 rs13300483 9 117643362 ENSG00000106952TNFSF8 cis 0 5.87E-10 Blood 4.48E-14 rs7860414 9 117541450 ENSG00000106952TNFSF8 cis 0 4.13E-09 Blood 8.50E-11 rs1407308 9 117570223 ENSG00000106952TNFSF8 cis 0 8.43E-09 Blood 1.50E-07 rs7866570 9 117577189 ENSG00000106952TNFSF8 cis 0 9.24E-09 Blood 1.26E-07 rs10982433 9 117620404 ENSG00000106952TNFSF8 cis 0 1.32E-08 Blood 1.33E-11 rs16931895 9 117609919 ENSG00000106952TNFSF8 cis 0 2.58E-08 Blood 1.80E-13 rs10982417 9 117589613 ENSG00000106952TNFSF8 cis 0 2.78E-08 Blood 4.03E-14 rs2093403 9 117611913 ENSG00000106952TNFSF8 cis 0 3.12E-08 Blood 2.03E-12 rs4262377 9 117589574 ENSG00000106952TNFSF8 cis 0 3.66E-08 Blood 7.08E-14 ACCEPTED Table S9: TRIM22 Sub network Annotation ACCEPTED MANUSCRIPT Gene Alias Category- Excerpt of reference PMID ADAR ADAR1 Inhibits HIV in macs, interferon induction. PMID: 25456137 FTSJD2 Methyltransferase responsible for cap1 formation in human cells, first nucleotide of a capped nucleotide in RNA. PMID: 20713356 CIITA MHCII transcription activation PMID: 17043423 RFX5 NLRC5 cooperates with the RFX transcription factor complex to induce MHC class I gene expression. PMID: 22490869 , PMID: 12101253 The upregulated expression of BCL6, SOCS1 and DUSP10 is integral to the signature of ILT3-Fc-induced CD8(+) T cells. These genes are known inhibitors of cytokine LILRB4 ILT3 production and TCR signaling and is a marker for CD8 T suppressor cells. PMID: 23018130 CLEC1A Natural killer c type lectin receptor H. pylori-derived peptide Hp(2-20) stimulates eosinophil migration through the engagement of FPR2 and FPR3, and also induces production of VEGF-A and TGF-beta, two FPR3 key mediators of tissue remodelling. PMID: 24067461 CXCR6 The chemokine receptor CXCR6 controls the functional topography of interleukin-22 producing intestinal innate lymphoid cells. PMID: 25456160 NAPSB Napsin B aspartic peptidase GNGT2 Phototransduction in retina Gnai2 expression affects marginal zone B-cell development, splenic architecture, lymphoid follicle size, and germinal center morphology. Gnai2 expression is also needed GNAI2 for the proper alignment of MOMA-1(+) macrophages and MAdCAM-1(+) endothelial cells along marginal zone sinuses in the spleen. PMID: 20508603 SLC2A6 GLUT6/9 Cell metabolism CSF2RA Auto antibodies correlating with aggressive disease. PMID: 24503766 ICOS Coreceptor Signaling Inactivates the Transcription Factor FOXO1 to Promote Tfh Cell Differentiation. ICOS is required for the generation of both central and effector ICOS CD4+ memory T-cell populations following acute bacterial infection. PMID: 25754933 and integrin-mediated cell adhesion. The protein encoded by this gene is a type II membrane protein which catalyzes the formation of GM3 using lactosylceramide as the ST3GAL5 GM3 substrate. PMID: 26362868 SLC39A4 A mouse model of acrodermatitis enteropathica: loss of intestine zinc transporter ZIP4 (Slc39a4) disrupts the stem cell niche and intestine integrity. PMID: 22737083 Fam65b negatively regulates chemokine-induced responses, such as adhesion, morphological polarization, and migration. Fam65b is a new transcriptional target of FOXO1 FAM65B that regulates RhoA signaling for T lymphocyte migration. PMID: 25588844 LAP3 Autophagy in yeast CXCL9 Anti microbial response: CXCR3 Chemokine Receptor Enables Local CD8+ T Cell Migration for the Destruction of Virus-Infected Cells in Skin. PMID: 25643352, PMID: 25769612 CD40LG Involved in activation and antigen presentation. Related to hyper IgM and is under selective pressure. PMID: 26644385 NFAM1 modulates B cell signaling through its ITAM, calcineurin/NFAT-activating and immunoreceptor tyrosine-based activation motif (ITAM)-containing protein (CNAIP) Y+TNF induced and activated TNF.CNAIP was preferentially expressed in neutrophils, monocytes, mast cells, and other immune-related cells. Activates TNFa and NFAM1 IL13 promoters. PMID: 15143214, PMID: 12615919 Elk-1 is a transcription factor whose activation by several mitogen-activated protein kinases (MAPKs) mediates the immediate early responses of the c-fos promoter to ELK2AP ELK growth factors and other stimuli. In heavy chain IGH gene, ELK2 is a pseudo gene. MT6-MMP regulates neutrophil and monocyte chemotaxis and by generating "eat-me" signals upon vimentin cleavage potentially increases phagocytic removal of MMP25 MT6-MMP neutrophils to resolve inflammation. PMID: 22367194 LRRC33 Identification and characterization of a unique leucine-rich repeat protein (LRRC33) that inhibits Toll-like receptor-mediated NF- κB activation. PMID: 23545260 An activator protein which stimulates the enzymatic hydrolysis of N-acetylneuraminic acid, but not N-acetylgalactosamine, from GM2. Suggested roles of Saps is their deregulating effect on various proteolytic cascades that constitute the major homeostatic systems in human hosts, including blood coagulation, fibrinolysis, and kallikrein- GM2A GM2-AP, SAP-3 kinin systems, and SAP3 generated proinflammatory bradykinin-related peptides (kinins). PMID: 8631864 , PMID: 25777036 Human adenosine deaminase 2 induces differentiation of monocytes into macrophages and stimulates proliferation of T helper cells and macrophages. Identified biallelic CECR1 ADA2 CECR1 mutations in two patients consistent with ADA2 deficiency. Both patients demonstrated an upregulation of interferon stimulated gene transcripts in peripheral blood. PMID: 20453107, PMID: 25278816 GNB4 CMTD1F GNB isoforms are required for LFA-1 activation and chemokine induced downstream signal Rac-1, PlcB2, PlcB3. PMID: 26468229 CD86 Upregulation on IECs is correlated with disease activity. PMID: 25574091 SNX10 Endosomal sorting nexin PMID: 26141367 GZMB Granzyme B-activated p53 interacts with Bcl-2 to promote cytotoxic lymphocyte-mediated apoptosis. PMID: 26156785 MMP19 and MMP23B belong to the Matrix metalloproteases (MMPs) family, which are zinc-binding endopeptidases that are capable of degrading various components of MMP19 the extracellular matrix. PMID: 18523579 PLA2G4F Pla2g4f, a phospholipase and Duox2 are colitis gene candidates- GPX metabolizes PLA2G4F and DUOX2 products. PMID: 20872835 KDM2B The H3K36 demethylase Jhdm1b/Kdm2b regulates cell proliferation and senescence through p15(Ink4b). PMID: 26237645 GPSM3 G Protein signaling modulator-3 inhibits the inflammasome activity of NLRP3, and is involved in monocyte chemotaxis. PDCD1LG2 PDL2 Regulation of T cell activation and tolerance by PDL2, which is positively regulated by IRF1. PMID:25053811 C1S Complement Regulates apoptosis, promotes targeting of mycobacteria to autophagy, the induction of the DNA damage-regulated autophagy modulator DRAM1 via the toll-like receptor DRAM1 (TLR)-MYD88-NFKB innate immunity signaling pathway/ p53 target gene that regulates autophagy and apoptosis. PMID: 25484076 SDC3 SDCN, SYND3 Syndecan-3 is a dendritic cell-specific attachment receptor for HIV-1, heparan sulfate (HS) proteoglycan . PMID: 18040049 LILRB2 LILRB2 interaction with HLA class I correlates with control of HIV-1 infection. PMID: 24603468 CCDC71L C7orf74 Gene related to athlerosclerosis. PLA2G7 Arachodinic acid program, upregulated upon neutrophil stimulation. MANUSCRIPTPMID: 26348085 SENP7 scenescence. PMID: 26527005 The plexin receptor is involved in acute inflammation and leukocyte migration. Plexin C1 (VESPR/CD232) is identified as being involved in sCD100-mediated (expressed in immature DCs) effects on human monocytes. Down-regulation of plexin C1 expression during the in vitro differentiation process of monocytes to immature DCs, while PLXNC1 concomitantly the surface expression of plexin B1 was induced. PMID: 15746246, PMID: 15746246 ING2, in the P53 pathway, can be sumoylated by small ubiquitin-like modifier 1 (SUMO1) on lysine 195 both in vitro and in vivo.ING2 sumoylation enhances its TMEM71 association with Sin3a. TMEM71 (transmembrane protein 71) expression is regulated by ING2 sumoylation. PMID: 20676127 We show that not only NOTCH but also ASB2 and SKP2 can promote the ubiquitination and degradation of JAK3. Also involved in immature DC migration, ASB2 α- ASB2 mediated effects on cell spreading are due to loss of filamins. PMID: 21969365 COL1A1 Noncanonical STAT3 Activation Regulates Excess TGF-β1 and Collagen I Expression in Muscle of Stricturing Crohn's Disease. PMID: 25740948 AKAP220/IQGAP1 networks receive and integrate calcium and cAMP second messenger signals and position signaling enzymes near their intended substrates at leading AKAP11 AKAP220 edges of migrating cells. PMID: 21890631 MX2 Interferon induced anti viral restriction factor PMID: 24760893 essential for the differentiation of intestinal microfold cells. Peyer's patch M cells derived from Lgr5(+) stem cells require SpiB and are induced by RankL in cultured SPIB "miniguts". PMID: 23439650 , PMID: 22706340, PMID: 22778137 The interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the CD1E presentation of lipid antigens. PMID: 22880058 MTX3 Metaxin deficiency alters mitochondrial membrane permeability and leads to resistance of TNF induced killing. PMID: 21088703 UbcH8, an ubiquitin E2 conjugating enzyme, was shown to be involved in RIG-I ISGylation. UbcH8 suppressed RIG-I ubiquitination by RNF125, and this suppression was UBE2E2 UBCH8 relieved by ectopic expression of ISG15. PMID:25960396 CXCL5 Neutrophil recruitment. De-regulation in the intestinal epithelium in perdiatric IBD reported in this pathway. PMID: 25738378 AT-rich-interactive domain-containing protein 5A functions as a negative regulator of retinoic acid receptor-related orphan nuclear receptor γt-induced Th17 cell ARID5A differentiation. Controls IL6 levels. PMID: 23676272 TIMP1 MMP-3 and TIMP-1: inhibitor of MMPs activity by myofibroblast involved in ulcers in CD. PMID: 24583398 Binding of the sialic acid-binding lectin, Siglec-9, to the membrane mucin, MUC1, induces recruitment of β-catenin and subsequent cell growth. Colonic epithelial cells express specific ligands for mucosal macrophage immunosuppressive receptors siglec-7 and -9. 9 turned out to be resident macrophages characterized by low expression of CD14/CD89 and high expression of CD68/CD163. A minor subpopulation of CD8(+) T lymphocytes also expressed siglec-7/-9. Siglec-7/-9 ligation suppressed LPS-induced SIGLEC9 cyclooxygenase-2 expression and PGE(2) production by macrophages. PMID: 24045940 , PMID: 22467657 Intermolecular binding between TIFA-FHA and TIFA-pT mediates tumor necrosis factor alpha stimulation and NF- κB activation. Identification of TIFA as an adapter TIFA protein that links tumor necrosis factor receptor-associated factor 6 (TRAF6) to interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK-1) in IL-1 receptor signaling. PMID: 22566686, PMID: 12566447 Targeted resequencing identifies defective variants of decoy receptor 3 in pediatric-onset inflammatory bowel disease, Differential expression of the TL1A/DcR3 system of TNF/TNFR-likeACCEPTED proteins in large vs. small intestinal Crohn's disease, High intestinal and systemic levels of decoy receptor 3 (DcR3) and its ligand TL1A in active ulcerative TNFRSF6B DCR3 colitis. An additive gene-gene interaction involving TLR4, PSMG1, TNFRSF6B and IRGM was identified with CD. PMID: 23965943 , PMID: 21978578, PMID: 21079743 CEBPB Inflammatory cytokine production by human neutrophils involves C/EBP transcription factors. PMID: 19109189 PARP9 DTX3L and ARTD9 inhibit IRF1 expression and mediate in cooperation with ARTD8 survival and proliferation of metastatic prostate cancer cells. PMID: 23487038 CENPV Kinetochore/ mitotic spindles CD40 The interaction between CD40 and its ligand, CD40L/CD154, is crucial for the efficient initiation and regulation of immune responses against viruses. PMID: 25765994 DOK3 DOK3 is required for IFN- β production by enabling TRAF3/TBK1 complex formation and IRF3 activation. Also involved in LPS response in macrophages. PMID: 24929003 S1PR4 S1PR4 is required for plasmacytoid dendritic cell differentiation. PMID: 25720060 Glia maturation factor- γ negatively modulates TLR4 signaling by facilitating TLR4 endocytic trafficking in macrophages. GMFG is a component of human T cell GMFG pseudopodia required for migration. Glia maturation factor- γ mediates neutrophil chemotaxis. PMID: 23677465, PMID: 22510515, PMID: 21653232 A tartrate-resistant acid phosphatase. Disruption of the gene results in elevated serum interferon alpha activity, and gene expression profiling in whole blood defined a type I ACP5 interferon signature. PMID: 21217755 TNFAIP2 Inhibits NFKB and is an autoinhibitor of TNF during sepsis. PMID: 22078882, PMID: 26347487 LYST Lysosomal trafficking regulator. LYST controls the biogenesis of the endosomal compartment required for secretory lysosome function. PMID: 25425525 VPS13C Glucose insulin metabolism (potentially autophagy) PMID: 22956255 Involved in eosinophil effector functions, HV1 is among genes which correlate significantly with Crohn's clinical activity. Hv1 proton channels differentially regulate the HV1 HVCN1 pH of neutrophil and macrophage phagosomes by sustaining the production of phagosomal ROS that inhibit the delivery of vacuolar ATPases. PMID: 23437289, PMID: 24415791, PMID: 23231444 KCNAB2 Voltage pottassium channel CD11b/CD18 (Mac-1) is a novel surface receptor for extracellular double-stranded RNA to mediate cellular inflammatory responses. DNA methylation-associated colonic ITGB2 CD18 mucosal immune and defense responses in treatment-naïve pediatric ulcerative colitis. PMID: 23209319 CLEC7A Downregulated by Th17, polymorphism in the gene for Dectin-1 (CLEC7A) is strongly linked to a severe form of ulcerative colitis. PMID: 22674328 CXCL6 Neutrophil cytokine in response to IEC cell lineA cuCCEPTEDltured with TNF and IL17. MANUSCRIPT PMID: 26617980 LIMS3L LIM and senescent cell antigen-like domains 3-like PLEKHO2 Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities in signalling. FBXO6 FBS2 Fbs2 is a new member of the E3 ubiquitin ligase family that recognizes sugar chains.. Inhibits ER stress by functioning as ubiquitin ligase in ERAD system. PMID: 12939278, PMID: 25374377 SLAMF8 Slamf8 is a negative regulator of Nox2 activity in macrophages. PMID: 22593622 PSTPIP2 Associated with autoinflammatory disease and anti HCV replication. PMID: 22130530, PMID: 24395802 NCF2 NADPH / NCF2/p67phox: A novel player in the anti-apoptotic functions of p53 and can be induced by TNF α. PMID: 21900546 Activation of IL-27 signalling promotes development of postinfluenza pneumococcal pneumonia.In response to il10, supresses Th17 1l17 and ifng related processes, copy IL27RA induces TH1, also can be anti viral. PMID: 24408967, PMID: 25088998, PMID: 24726498, PMID: 25073675 ARHGAP30 ArhGAP30 promotes p53 acetylation and function in colorectal cancer. PMID: 25156493 CSF3R G-CSF preferentially supports the generation of gut-homing Gr-1high macrophages in M-CSF-treated bone marrow cells. PMID: 24503766, PMID: 24981628 MNDA The human PYHIN proteins, AIM2, IFI16, IFIX, and MNDA, are critical regulators of immune response, transcription, apoptosis, and cell cycle. FAH Fumarylacetoacetate hydrolase- an enzyme that catalyzes the last step of tyrosine metabolism. PMID: 24879068 DCIR-mediated enhancement of HIV-1 infection requires the ITIM-associated signal transduction pathway. DCIR is endocytosed into human dendritic cells and inhibits CLEC4A DCIR TLR8-mediated cytokine production. PMID: 19028959 Associated with pneumoccocal disease, PAF signaling in peritoneal macrophages requires TLR4, MyD88, and TRIF. In peritoneal macrophages, the PTAFR agonist carbamyl PAF induces production of inflammatory markers previously demonstrated to be upregulated during bacterially induced labor, including: inducible nitric oxide synthase (Nos2), the chemokine Ccl5 (RANTES), tumor necrosis factor (Tnf), and level of their end-products (NO, CCL5, TNF) in a process dependent upon both IkappaB PTAFR PAF kinase and calcium/calmodulin-dependent protein kinase II. PMID: 25253732, PMID: 25253732 CD300A PPAR β/δ activation of CD300a controls intestinal immunity in M1 macs and binds phophatidyserine on dying cells. PMID: 24958459 CDC42SE1 SPEC1 SPECs may play important roles in Cdc42-mediated F-actin accumulation at the immunological synapse. PMID: 15840583 GBP4 GBP4 is a negative regulator of virus-triggered IFN-I production, and it is identified as a novel protein targeting IRF7 and inhibiting its function. PMID: 23029268, PMID: 22095711 KYNU Kynureninase involved in the L-tryptophan aerobic degradation pathway. PMID: 26347385, PMID: 26347385 TLR8 Toll-like receptor 8 (TLR8) was required for inducing pro-IL-1 β expression, whereas the NLRP3 inflammasome was required for IL-1 β maturation and release. PMID: 24939850 Tumor necrosis factor alpha triggers Bid-dependent lysosomal permeabilization, followed by release of cathepsin B into the cytosol and activation of caspase 2. Caspase 2 BID then facilitates efficient mitochondrial cytochrome c release and apoptosis. PMID: 16012953, PMID: 16012953 Src family kinases were required for the generation of the inflammatory environment in vivo and for the release of proinflammatory mediators from neutrophils and macrophages in vitro, likely due to their role in Fc γ receptor signal transduction, but not involved in recruitment. ROS-triggered phosphorylation of complex II by Fgr kinase FGR regulates cellular adaptation to fuel use. PMID: 25225462, PMID: 24856931 SLAMF7 CS1 monocytes. PMID: 23695528 Downregulated by CD40L on DCs. This structure and expression protein belonging to the CD8 family suggests a function as a co-receptor, perhaps in an antigen uptake IGSF6 DORA complex, or in homing or recirculation of DC. PMID: 9809579 DYSF Inflammasome up-regulation and activation in dysferlin-deficient skeletal muscle. PMID: 20413686 Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that is classically activated through cleavage of the N-terminal exodomain by the serine protease F2R PAR1 thrombin. Most recently, 2 MMPs have been discovered to have agonist activity for PAR1:MMP-1 and MMP-13 PMID: 23086754 EMR2 receptor ligation modulates cytokine secretion profiles and cell survival of lipopolysaccharide-treated neutrophils. CD312, the human adhesion-GPCR EMR2, is EMR2 differentially expressed during differentiation, maturation, and activation of myeloid cells. PMID: 22035891, PMID: 17174274 LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis, selectively induces pro-inflammatory cytokines and IL-10 and is regulated by TNF-alpha, IL- LILRA5 10 and IFN-gamma. PMID:19009525 EMILIN2 Regulation of the extrinsic apoptotic pathway by the extracellular matrix glycoprotein EMILIN2. PMID: 17698584 TBXAS1 Thromboxane A synthase (TBXAS1) converts prostaglandin H to thromboxane A. A distal locus element mediates IFN- γ priming of lipopolysaccharide-stimulated TNF gene expression. Data suggests that during intestinal inflammation, the TNF- α- mediated activation of IRF1 is related to the subsequent suppression of OPN expression, further reducing p-AKT, p-P38, and p-ERK activities and resulting in aggravation of IRF1 the injury to intestinal epithelial cells. PMID: 25482561, PMID: 25208103 ZYX ESP-2, HED-2 ZYX-1 protein is expressed in the striated body-wall muscles and is involved in dystrophin-dependent muscle. GBP1P1 Gamma interferon-induced guanylate binding protein 1 is a novel actin cytoskeleton remodeling factor. PMID: 24190970 APOL4 Lipid exchange and transport FCGR3A CD16 Associated with susceptibility to autoimmunity. Influences response to Infliximab through ADCC. PMID: 26407570, PMID: 23358932 UBE2D1 UBCH5 UbcH5 is an E2 ubiquitin-conjugating enzyme catalyzing ubiquitination during TNF- α-triggered NF- κB activation. Inhibition leads to p53 apoptosis. PMID: 25200604 GPNMB Lysosomal stress in obese adipose tissue macrophages contributes to MITF-dependent Gpnmb induction. PMID: 24789918 MRAS RRAS3 Ras oncogene, MRAS, through its ability to recruit a complex with paradoxical components, coordinates ERK pathway. Involved in intestinal fibrosis in CD rats. PMID: 23970928, PMID: 23863483, PMID: 17538012 DNASE2 Seven nonsynonymous SNPs in the gene encoding human deoxyribonuclease II may serve as a functional SNP potentially implicated in autoimmune dysfunction. PMID: 24242851 MSANTD3 Anti viral response, DNA binding. PMID: 25547032 LIMS3 PINCH3 Pinch-3 protein is an important constituent of cell membranes, which directly affects the cell morphology and mechanical properties. PMID: 23865313 IL2RA Il2 receptor chain expressed on FoxP3 regulatory T cells. PMID: 24483245 PLEK p47 p47 negatively regulates IKK activation by inducing the lysosomal degradation of polyubiquitinated NEMO. PMID: 22990857 STAT1 Mendelian myco bacteria response, type I and type II interferon signalling. PML Induction of IFNB MANUSCRIPTPMID: 25733689 TGFB TGFB1 Controls proliferation and cellular differentiation. Involved in wound healing and mucosal immunity. PMID: 26559094 Expression of suppressor of cytokine signaling 1 (SOCS1) impairs viral clearance and exacerbates lung injury during influenza infection. Has been reported to play an important role in Treg cell integrity and function by protecting the cells from excessive inflammatory cytokines. Co-immunoprecipitation studies revealed that SOCS1 SOCS1 physically interacted with RhTRIM5 α. PMID: 25500584, PMID: 25133199, PMID: 25310711 DNAJC5B CSP-beta Cysteine string proteins (CSPs) are associated with regulated secretory organelles. CTLA4 P695. A mutation in CTLA4 associated with early-onset Crohn's disease and autoimmunity. PMID: 25718204 SLC43A3 A candidate gene linking systemic inflammation to atherosclerosis; results of a human in vivo LPS infusion study. PMID: 21827714 CHST2 CHST1 and CHST2 contribute to the generation of optimal L-selectin ligands in vascular endothelial cells at sites of inflammation. PMID: 11310842 Pharmacological inhibition of TPL2/MAP3K8 blocks human cytotoxic T lymphocyte effector functions.TPL2 mediates autoimmune inflammation through activation of the MAP3K8 TAK1 axis of IL-17 signaling. PMID: 24642963, PMID: 24980047 BATF2 IFN induced, interact with IRF, DCs PMID: 23787991 CXCL10 NOD2 synergized with IFN- γ to induce CXCL9 and CXCL10 secretion in dendritic cells, macrophages, and intestinal stromal cells in vitro. PMID: 24591373 APOL1 APOL1 expression is induced by proinflammatory cytokines gamma interferon (IFN- γ) and tumor necrosis factor alpha (TNF- α). PMID: 24173214 APOL2 A novel anti-apoptotic role for apolipoprotein L2 in IFN- γ-induced cytotoxicity in human bronchial epithelial cells. PMID: 20665705 PDE4B Inhibition of PDE4B suppresses inflammation by increasing expression of the deubiquitinase CYLD. PMID: 23575688, PMID: 25748781 Type I interferon related TB response. IL-15 modulates the balance between Bcl-2 and Bim via a Jak3/1-PI3K-Akt-ERK pathway to promote CD8 αα+ intestinal IL15RA intraepithelial lymphocyte survival. PMID: 23754237 CD274 Biochemical signaling of PD-1 on T cells and its functional implications. PMID: 25098287 Hypoxia/IL-10 showed the enhanced induction of a set of genes including PLOD2 and SLC2A3. In cancer, mTORC1 enhances Glut3 expression through the activation of SLC2A3 GLUT3 the IKK/NF κB pathway. PMID: 25450522 NNMT Related to pathogenesis of metabolic disease. IL10RA Clinical features of interleukin 10 receptor gene mutations in children with very early-onset inflammatory bowel disease. PMID: 25373860 γ-Interferon-inducible lysosomal thiol reductase (GILT) maintains phagosomal proteolysis in alternatively activated macrophages.GILT expression in B cells diminishes IFI30 cathepsin S steady-state protein expression and activity. PMID: 25253686 DANGER, a novel regulatory protein of inositol 1,4,5-trisphosphate-receptor activity. Individual subtypes of enteroendocrine cells in the mouse small intestine exhibit ITPRIP DANGER unique patterns of inositol 1,4,5-trisphosphate receptor expression. PMID: 14688217 SCIMP SCIMP, a transmembrane adaptor protein involved in major histocompatibility complex class II signaling. PMID: 21930792 The transcription factor GFI1 negatively regulates NLRP3 inflammasome activation in macrophages. There are many other transcription factors, such as RUNX family GFI1 proteins, IRF4, Dec2, Gfi1, Hlx, and JunB that can impair TH1/TH2 cells differentiation. PMID: 25447538, PMID: 25261203 E3 Ubiquitin ligase, Deltex1 antagonizes HIF-1 α and sustains the stability of regulatory T cells in vivo, Deltex1 is a target of the transcription factor NFAT that promotes T DTX1 cell anergy. ACCEPTED PMID: 25695215, PMID: 19592273 CP CP-2 Ceruloplasmin (ferroxidase) / iron. CFH Complement Factor H. Intestinal glycoprotein activates the alternative complement pathway by reacting with factor H. PMID: 6213029 GBP5 GBP5 promotes NLRP3 inflammasome assembly and immunity in mammals. PMID: 22461501 BASP1 Repression of transcription by WT1-BASP1 requires the myristoylation of BASP1 and the PIP2-dependent recruitment of histone deacetylase./ apoptosis. PMID: 22939983 TRIM22 TRIM proteins regulate autophagy: TRIM5 is a selective autophagy receptor mediating HIV-1 restriction and is a ubiquitin ligase that stimulates type I interferon & NFKB TRIM5 pathways with TRAF6. PMID: 25587751, PMID: 26503954 TRIM38 inhibits TNF α- and IL-1 β-triggered NF- κB activation by mediating lysosome-dependent degradation of TAB2/3. TRIM38 negatively regulates TLR3-mediated TRIM38 IFN- β signaling by targeting TRIF for degradation. PMID: 24434549, PMID: 23056470 Interferon alpha induced. STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1. Upon binding to dsRNA or dsDNA, OAS proteins and cGAS produce nucleotide second messengers to activate RNase L and STING (stimulator of interferon genes, gene symbol: TMEM173), OAS2 respectively; this leads to the initiation of antiviral responses and interferon beta. PMID: 25564224, PMID: 25752600 SERPING1 C1 inhibitor PMID: 22576004 IFI44L c1orf29 Induced by IFNalpha, downregulated by HIV, HIV-1 Vpr induces interferon-stimulated genes in human monocyte-derived macrophages. PMID: 25170834 IRF8 directs stress-induced autophagy in macrophages and promotes clearance of Listeria monocytogenes. IFN Regulatory Factor 8 Represses GM-CSF Expression in T IRF8 Cells To Affect Myeloid Cell Lineage Differentiation. Negatively regulates plasma cells. Mendelian myco bacteria susceptibility PMID: 25398936, PMID: 25775030, PMID: 25646302 Syntaxin11 serves as a t-SNARE for the fusion of lytic granules in human cytotoxic T lymphocytes, Syntaxin-11, but not syntaxin-2 or syntaxin-4, is required for platelet STX11 secretion and NK cell de granulation. PMID: 24227526 , PMID: 22767500 The nuclear receptor nr4a1 controls CD8 T cell development through transcriptional suppression of Runx3. Runt-related transcription factor 3 is involved in the altered RUNX3 phenotype and function in ThPok-deficient invariant natural killer T cells. PMID: 25762306, PMID: 24561456 SH2D1A SLAM-SAP signaling promotes differentiationA of CCEPTEDIL-17-producing T cells and progressionMANUSCRIPT of experimental autoimmune encephalomyelitis. PMID: 25362182 RASSF5 RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF- α and TNF-related apoptosis-inducing ligand. PMID: 20810663 ARL4C ARL7 membrane. PMID: 21187453 ZNF222 PMID: 25236797 Phostensin was abundant in helper T-lymphocytes, cytotoxic T-lymphocytes, mature monocytes, macrophages, B-lymphocytes, natural killer cells, and granulocytes. PPP1R18 Phostensin binds to the pointed ends of actin filaments and modulates actin dynamics. PMID: 21804078 PIK3R5 Involved in IL23R/IL17 production. PMID: 23024273 SELL CD62L Marker in t cell colitis transfer model and adoptive therapy for Crohn's. PMID: 25715355, PMID: 24132160 Combined use of biomarkers for distinguishing between bacterial and viral etiologies in pediatric lower respiratory tract infections. CD53 Characterization of tetraspanins CD9, CD53, CD63, and CD81 in monocytes and macrophages in HIV-1 . PMID: 25712729 , PMID: 23570947 BIN2 Cell motility, adhesion and phagocytosis are controlled by actin and membrane remodelling processes. PMID: 23285027 p53, nutlin-3-activated ERK1/2 may stimulate the transcription of BCL2A1 via the activation of ELK1, and BCL2A1 expression may contribute to the inhibitory effect of BCL2A1 ERK1/2 on nutlin-3-induced apoptosis, thereby constituting a negative feedback loop of p53-induced apoptosis. PMID: 24867259 TNFSF13B BAFF Methylation of TNFRSF13B and TNFRSF13C in duodenal mucosa in canine inflammatory bowel disease and its association with decreased mucosal IgA expression. PMID: 24814046 SRGN PPG, PRG, PRG1 Pro apoptosis in DCs. PMID: 24475103 CMKLR1 Levels are associated with DSS colitis. PMID: 26265756 SAMSN1 Hacs1 SH3 and SAM domain-containing adaptor protein, is up-regulated by IL-4 in activated B cells and strongly expressed in dendritic cells. PMID: 21859567, PMID: 19923443 GBP1 Downstream of interferon gamma. Activates T cells. PMID: 24337748 LINC00847 RTN1 ER localization and retention. Interacts with HDAC enzymes. PMID: 17303085, PMID: 20000484 LAIR1 Inhibitory immuno receptor (esp in b cells). PMID: 24694245, PMID: 14559247 DTNB Dystrobrevin, scaffold in nucleolus. PMID: 25959029 PPT1 Identification of palmitoyl protein thioesterase 1 in human THP1 monocytes and macrophages. PMID: 24083319 Chronic granulomatous disease (CGD) is a primary immunodeficiency disease that is characterized by susceptibility to bacterial and fungal infections. Various mutations in CYBB encoding the gp91(phox) subunit of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase impair the respiratory burst of all types of CYBB CGD/NOX2 phagocytic cells disrupting the response to IFN- γ. PMID: 25752509, PMID: 25301784 , PMID: 25188296, PMID: 25453225 NOD2 Crohns gene, MDP sensor PMID:11385577 TIFAB Negative regulator of TRAF6. PMID: 19470519 MMP12 Macrophage metalloelastase (MMP12) regulates adipose tissue expansion, insulin sensitivity, and expression of inducible nitric oxide synthase in M2 macrophages. PMID: 24914938 ADCY7 Macrophages in ADCY7 knock downs produce more TNFa in response to LPS. ADCY7 also affects memory T cells and antibody responses. PMID: 23229509 , PMID: 15550390, PMID: 20505140 41518

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ACCEPTED Table S10: Functional Categories of Genes in TRIM22 Sub network ACCEPTED MANUSCRIPT Functional Categories from NOD2 from TRIM22 vortex: Correlation within each category (induced by, repressed by pathway signalling or directly modulates) ADAR1, CSF2RA,CXCL9, CECR1, PPT1, MMP12, GMFG, ITGB2, SPI1, SLAMF8, Il27RA, PTGES, CSF3R, PTAFR, CD300A, FGR, SLAMF7, Monocyte, Macrophage EMR2, LILRA5, FCGR3A, GPNMB, HCK, CXCL10, IL10RA, IFI44L, PPP1R18, CD53, TGFB, ADCY7, PLXNC1 Neutrophil CXCL5, CEBPB, GMFG, CXCL6, FGR, EMR2, TGFB DCs CXCL9, SPIB, S1PR4, ITGB2, CLEC4A, KYNU (tolerance), IGSF6, BATF2, CXCL10, IL10RA, SDC3 T effector CD4 CSF2RA, ICOS, FAM65B, CD40LG, SPIB, GMFG, RUNX3 (suppression to induce IELs), CDC42SE1, MAP3K8, SELL CD8/NK CLEC1A, GZMB, CLEC1A, STX11, RUNX3 T17 PIK3R5, ARID5A, MAP3K8, SH2D1A, CXCL6, CLEC7A, IL41I, SH2D1A T regulatory SOCS1, IL4I1, CTLA4, CD274 CD4 PDCD1LG2, CLEC7A, IL27RA, IL2RA, Il10R, DTX1, PPP1R18, TGFB CD8 LILRB4, IL27RA B LAIR1, NFAM1, SPIB, DOK3, IL27RA, IFI30, PPP1R18, ELK2AP Cytoskeleton (migration & phagocytosis/ immunological synapse/ mechanics CDC42SE1, GBP1P1,PPP1R18, BIN2 migration FPR3, CXCR6 (ILC), FAM65B, CXCL9, MMP25, GPSM3, PLXNC1, AKAP11, GMFG, LIMS3 Endosomal SLC15A3, DNAJC5B, SNX10 Complement C1S, CFP, CFH, SERPING1 Tissue re-modelling/ thrombins FPR3, MMP19, COL1A1, TIMP1,F2R E2/E3 ligase, ubiquitin pathway SENP7, ASB2, UBE2E2, FBXO6, UBE2D1, PDE4B, DTX1 Inflammasome GPSM3, MNDA, inflammassome (apoptosis), DYSF, GFI1, GBP5 Autophagy LAP3, DRAM1, TRIM5 Lysosomal LYST, GPNMB Platelets STX11 CECR1, MX2, PML , PARP9,CD40 , DOK3, BST2, ACP5, PSTPIP2, PTGES, CLEC4A, GBP4, IRF1, GBP1P1, MSANTD3, STAT1, PML, BATF2, Viral infection and response (interferon inducible) APOL1, IL15RA, IFI30, OAS2, IFI44L, SDC3 LRRC33, NFAM1, TIFAB, GZMB, CYBB, DRAM1, TIFA, TNFRSF6B, BST2, TNFAIP2, NCF2, PTGES, PTAFR, BID (TNFa only, SLAMF7), NFKB/TNF α/ apoptosis EMILIN2 (apoptosis), IRF1 (TNFa induced), PLEK,APOL1, SLC2A3, TRIM38, RASSF5, DNASE, APOL2/ BASP1, ADCY7, UBE2D1 ROS/NADPH/ phagosome CYBB, CYBA, HV1, SLAMF8, NCF2, FGR, NCF4, IFI30 Mycobacteria response (NOD2/IFN synergy (part viral)) SLC15A3, STAT1, CXCL10, IL15RA, NCF4 P53 (same as Trim22) NCF2, ARHGAP30 Prostaglandin/arachadonic acid PLA2G7, TBXAS1 Antigen Presentation/ MHC CIITA, RFX5, LILRB2, CD1E, SCIMP Miscellaneous Inflammatory (adhesion)/signalling/muscle GNAI2, CD18, PLA2G4F, CHST2, DTNB, ST3GAL5, GM2A, GNB4, SDC3, PLEKHO2, LIMS3L/ ZYX/ DYSF Activation CD86, CD40 Cell cycle/ prolif CECR1, KDM2B, CENPV, MRAS Metabolic/lipids APOC1, APOC1P1, APOE, CCDC71L, VPS13C, KCNAB2, FAH, SLC43A3, NNMT, CP, ARL4C, SLC2A6, APOL4 Misc (biochem) FTSJD2, NAPSB, GNGT2, ZNF222 IEC/ enteroendocrine SLC39A4, SPIB, ITPRIP ER RTN1

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ACCEPTED Table S11: Adult and VEO IBD NetworkACCEPTED Predicted MANUSCRIPT Key Drivers Adult Key Drivers Overlap of Adult IBD Key Drivers in 179 gene TRIM22 Subnetwork CYYR1 TRIM22 ZNF521 EMR2 INSR CLEC7A GLCCI1 CYBB CREB5 CEBPB ASAP1 IL10RA LOC100132891 ITGB2 HIP1 TNFSF13B ST3GAL1 UBE2E2 DOCK8 DNASE2 TRIM22 GBP4 EMR2 LAP3 C1R PLXNC1 MMP14 CLEC4A PMAIP1 GZMB KIF26B CXCL9 IL8 RUNX3 BTN2A2 MMP19 FPR2 CD86 LOC100505687 FGR PDE4A TIFA LDHB KYNU KLHDC7B NFAM1 MANUSCRIPT OAS2 PPP1R18 COL1A1 CXCL6 NCF1C STX11 DARC MRAS TMTC1 SERPING1 HLA-DPA1 CD53 GJD3 SAMSN1 SLAMF8 CTLA4 CCR1 TLR8 C9orf91 DYSF CRLF3 DRAM1 TNS1 STAT1 CDK14ACCEPTED IGSF6 CYBB GBP1 IL13RA2 CCDC71L CXCL10 MX2 RAMP2 RASSF5 FSCN1 BID FCN3 GMFG SELE OAS2 ITGAM COL1A1 ME1 SLAMF8 ACCEPTED MANUSCRIPT ABLIM3 CXCL10 CD37 TNFAIP2 FNDC3B BCL2A1 CSGALNACT2 PLA2G7 INHBA NCF2 PHACTR1 SOCS1 EMR1 SLC2A6 PPM1F SELL ASPHD2 LAIR1 S1PR3 CXCL5 WNT5A ST3GAL5 SFRP2 LILRB4 VWF ICOS TNFAIP2 GM2A SMARCB1 MNDA FBN1 CSF2RA CNN2 BASP1 MAFB BCL2A1 MAPK1 BST2 IL10RA IFI44 ARHGAP15 MANUSCRIPT GRHL1 C17orf96 CDC42BPG GPX8 STEAP4 TPP1 TRPM6 GIMAP7 PIP4K2A FLJ43663 PIK3R3 HERC5 ENTPD1 ACCEPTED ITGB2 ADAMTS2 DUSP10 GPRIN2 ADAM19 TNFSF13B MLKL GAS7 COL5A1 ACCEPTED MANUSCRIPT HCK WHAMMP2 IL23A OSMR PLA2G7 FCGR2A ITGA4 SLC7A11 FLI1 C4orf32 TREM1 HTRA1 NFKBIA CDX2 NPEPL1 NCF2 CCL8 FPR1 UBE2E2 CD80 CD300LF RNF166 GAD1 GCA MANUSCRIPT THBS2 IL6 PKHD1L1 SOCS1 ADAMTS5 ERG LOC100506941 IL1R1 GIMAP2 LAP3 SNX20 TBL1X ST8SIA4 ACCEPTED PLXNC1 DOCK2 GABBR1 EBI3 SLC22A15 CLYBL ADAMTS1 TBC1D9 CCL11 ACCEPTED MANUSCRIPT PTGS1 CLEC4A PFKFB3 GPR4 HSD11B1 PEA15 HSH2D RBPMS TNFSF4 ANKRD44 LCP2 LAMP3 AGTRAP ARHGAP20 CH25H SLC6A14 IL12RB1 MCAM GLUL IER3 TLR2 TGIF2 RGS1 EGFL6 MANUSCRIPT SLC2A6 CXCL9 UCP2 RUNX3 GAS1 IL1B SLC9B2 ZNF618 CXCL11 IL1RAP BICC1 PDZK1IP1 SEMA4A ACCEPTED FCGR1A CTGF KMO CLIC2 RHOQ CAV1 TICAM2 TNFAIP6 SELP ACCEPTED MANUSCRIPT SOCS3 SLC16A6 RGS5 IFITM2 SELL IFITM1 IL11 LAIR1 CD86 CYP27B1 C1orf162 LSP1 SLC25A23 EVI2B FKBP11 TSPAN2 DENND3 AKT3 TIFA GPR68 FCGR2B FYB ARMCX1 PIK3CD MANUSCRIPT KYNU RAPGEF6 IL33 CACNA2D1 SLC1A3 TNF ITPR1 CCL18 CXCL5 CXCL1 ST3GAL5 GPR183 CHI3L1 ACCEPTED TMEM154 NINJ2 HLA-DOB GJA5 FCHSD2 ORAI2 F5 NPL PRDM1 ACCEPTED MANUSCRIPT KCNJ15 CCDC3 CHRDL2 TPST1 LAMC1 TYROBP ZNF331 SLC9A1 MXRA5 CXCL6 GZMK COL5A2 CLIP4 DOCK10 LIX1L MIR155 IGDCC4 LILRB4 GJA1 ROBO4 DENND5A CKAP2 LHFP IL18R1 MANUSCRIPT PEAR1 MRAS SERPING1 ARHGAP39 IFITM3 CFI TDO2 EDNRA CD53 EHD3 CXCR2 ABCA1 KIAA1199 ACCEPTED CDH11 EOMES S100B IL1RN RASA3 PAPLN SLFN11 ST6GAL1 SAMSN1 ACCEPTED MANUSCRIPT CYP26B1 STC1 CD180 ICOS MLK7-AS1 CTLA4 C13orf44-AS1 CD79A LAMP5 WNK3 GM2A IDO1 FAM20A LOC648987 DISC1 FAM110B ANKRD55 TSHZ2 TWIST2 DDR2 LRRK2 STAT1 IGSF6 PPM1M MANUSCRIPT SLC2A14 RILPL2 SASH3 IRS1 GBP1 CCDC71L MMP3 PDE10A COL15A1 LINC00152 TTC7B MX2 SCRN1 ACCEPTED ITGAX SCUBE2 LAIR2 IL4I1 CD300C TSPAN4 FCER1G RASSF5 GBP2 ACCEPTED MANUSCRIPT MNDA CD109 BID CSF2RA B9D1 CHST15 GMFG ACSL4 RGS10 CLIC4 VNN2 LITAF PECAM1 GPR176 COL1A2 LPAR6 LY96 VEGFC NOS2 CD84 EMID1 CLIC6 TRIB2 CTSK MANUSCRIPT PDPN TMEM45A HAVCR2 CLEC7A MCL1 PTPN11 CTHRC1 RNF24 AKAP1 DUOXA2 MAT2A LST1 NR4A3 ACCEPTED CXCR1 CEBPB CCL4 APCDD1 IRAK3 CD48 DUOX2 ALOX5AP MMP1 ACCEPTED MANUSCRIPT QPCT HAPLN3 ZEB1 TBC1D4 TNFSF11 RHOH MS4A1 SNN FGF7 SULF1 GPR116 PRNP SERPINE1 KRT23 OLFML2B TRDV3 DAZAP2 SERPINI1 PXDN DNASE2 IL7R C12orf5 ESAM WISP1 MANUSCRIPT ACSL1 WBP5 GBP4 TESC CCL3 RASSF2 SLC15A3 DUSP6 COL4A1 C4BPB FCRL1 CCL2 LOC100129518 ACCEPTED GZMB CLEC4D SERPINB5 SEMA4D KLRF1 JAZF1 ZNF277 CYSLTR1 LIN7A ACCEPTED MANUSCRIPT CD83 LOC100652805 MMP19 EVI2A TRERF1 GNA12 FGR IPCEF1 SPP1 C5AR1 CEP170 SAMD3 LMBR1 SALL1 MMP10 ATP6AP2 KIRREL NFAM1 JAK3 GZMH CFP CMTM2 COG1 PPP1R18 MANUSCRIPT ADRBK2 STX11 SELT FAM26F RHOA RAB8B TANK SLC28A2 GIMAP1-GIMAP5 STEAP1B MYEOV ZG16 AIDA ACCEPTED VNN3 C13orf44 IGHA1 TLR8 DYSF IFIT1 BASP1 KCNJ10 TLR1 ACCEPTED MANUSCRIPT C12orf29 RGS19 PLSCR1 DRAM1 CSF2RB DSE PRR15 FCRLA S100A12 CHSY1 FCGR2C ARL6IP5 HS3ST3B1 RAC2 SP110 NAMPT ARF4 IL24 COL3A1 THBD SFMBT2

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SUPPLEMENTAL MATERIALS FOR:

Variants in TRIM22 that Affect NOD2 Signaling Are Associated With Very Early Onset Inflammatory Bowel Disease

Qi Li 1,2,§,* , Cheng Hiang Lee 3,4,§,* , Lauren A Peters 5,6,* , Lucas A Mastropaolo 2,§,# , Cornelia Thoeni 1,2,§,# , Abdul Elkadri 1,2,7,#,§ , Tobias Schwerd 8,# , Jun Zhu 6, Bin Zhang 6, Yongzhong Zhao 6, Ke Hao 6, Antonio Dinarzo 6, Gabriel Hoffman 6, Brian A Kidd 6, Ryan Murchie 1,2,§ , Ziad Al Adham 1,2,7,§ , Conghui Guo 2,§* , Daniel Kotlarz 9, Ernest Cutz 10 , Thomas D Walters 1,2,§ , Dror S Shouval 11,§ , Mark Curran 12 , Radu Dobrin 12 , Carrie Brodmerkel 12 , Scott B Snapper 11,13 , Christoph Klein 9, John H Brumell 1,7,14§ , Mingjing Hu 3,4 , Ralph Nanan 3,4 , Brigitte Snanter-Nanan 3,4 , Melanie Wong 15 , Francoise Le Deist 16 , Elie Haddad 17 , Chaim M Roifman 18 , Colette Deslandres 19 , Anne M Griffiths 1,2,§ , Kevin J Gaskin 3,4 , Holm H Uhlig 8, Eric E Schadt 6,% , Aleixo M Muise 1,2,7,§, % ^

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T Table of Contents

Methods

Detailed Case Reports: Family 1 Family 2 Family 3

Supplemental Tables

Table S1: Clinical (A) and Genetic Features (B) of Patients with TRIM22

Table S2: Autosomal Recessive Variants Identified by WES in Patient 1.

Table S3: VEOIBD Genes See excel sheet

Table S4. VEOIBD Key Drivers See excel sheet

Table S5. IIBDGC GWAS Enrichment in TRIM22 Locus See excel sheet. MANUSCRIPT Table S6. IIBDGC GWAS Enrichment in TRIM22 eQTL Enrichment See excel sheet

Table S7. eQTL Pathway Enrichment See excel sheet

Table S8. Crohn’s GWAS Enriched Blood eQTL Overlap with the VEO Network

Table S9. Annotation of 179 VEO Network See excel sheet

Table S10. Functional Categories of Genes in the TRIM22 Sub network

Table S11. Inflamed Sigmoid and Rectum Key Drivers

TableACCEPTED S12. Correlations of the Expression of TRIM22 to the Clinical Response Characteristics Stratified by Distinct Intestine Anatomy Sites

Table S13. Correlation of the Expression of TRIM22 in blood to CDAI

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Supplemental Figures

Figure S1: Variants Identified in WES of Patient 1.

Figure S2: Heterozygous TRIM22 Variants R150T and S244L Occur in the Coiled- coil Domain and May Affect Domain Oligomerization.

Figure S3: Blood and Intestine Derived eQTLs: Gene Set Enrichment Analysis of Genes Controlled by IBD SNPs (Metacore)

Figure S4: Computational Design

Figure S5: First Neighbors of TRIM22 and NOD2 in VEOIBD Intestine Network

Figure S6: TRIM22 and NOD2 Co-localize in Healthy Controls and Disrupted in Patient Tissue Sections.

Figure S7: TRIM22 Variants and shRNA Abrogate NOD2 signaling.

Figure S8: TRIM22 shRNA Knockdown.

Figure S9: TRIMM22 Abrogates NOD2 Signaling in Patient 2 with Coiled-Coil Variants.

Figure S10: TRIM22 Directly MediatesMANUSCRIPT the Polyubiquitination of NOD2 but not MAVS or RIPK2.

Figure S11: TRIM22 does not Mediate K48-linked Polyubiquitination of NOD2.

References

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Methods

Subjects: All experiments were carried out with the approval of the Research Ethics Board (REB) at the Hospital for Sick Children. Informed consent to participate in research was obtained. A copy of the consent is available on the interNational Early Onset Pediatric IBD Cohort Study (NEOPICS) website at http://www.neopics.org/NEOPICS_Documents.html.

Genetics

Whole exome sequencing: For patient 1 and her parents (trio), whole exome sequencing (WES) was performed using the Agilent SureSelect Human All Exon 50Mb kit with high-throughput sequencing conducted using the SOLiD 4 System at The Center for Applied Genomics (TCAG) through the Hospital for Sick Children (Toronto, ON). Sanger sequencing was used to verify variant genotypes in the Family 1 and infantile patients from the collaborating institutions were screened for TRIM22 variants.

Analysis: Whole exome sequencing of Patient 1 and her parents resulted in greater than 100 times mean exome coverage and identified 152,020 variants. Using SNP and Variation Suite Version 8.1 (Golden Helix), variant call files (VCF) were organized by the family pedigree. Using the 1000 genomes Variant Frequencies (Phase 1) and the NHLBI Exome Sequencing Project V2 Exome Variant Frequencies, rare variants with a minor allele frequency below 1% were filtered. This resulted in 16,097 rare variants. Variants were then selected based upon whether they were deemed to be coding. From this list, we then identified 12 non-synonymous homozygous variants (Table S2) that were inherited in an autosomal recessive manner. Non- synonymous and unclassified variants were thenMANUSCRIPT scored using the database for non-synonymous functional predictions (dbNSFP 2.9), filtering out variants found to have no damaging score (Polyphen2, SIFT, MutationTaster, MutationAssessor, FATHMM). As well, variants were scored using PhyloP and GERP++ to determine conservation (Figure S1). Based on known protein function, expression profiles, animal models, mutation conservation, and network analysis, this list was narrowed down to a single candidate variant in TRIM22 .

Validation: In order to validate these findings, we examined WES results from 150 infantile international VEOIBD patients without a genetic diagnosis who were previously sequenced in Toronto (NEOPICS), Oxford, and Munich (Care-For-Rare) and did not identify predicted damaging variants in the TRIM22 gene. Therefore, we performed targeted exome sequencing of the TRIM22 gene in ten IL10RA/B, and IL10 negative patients without previous WES, and identified two additional patients with TRIM22 variants (Patients 2 and 3) with a very similar severe phenotype of granulomatous colitis and severe perianal disease.

Computational Analysis ACCEPTED Dataset: Blood and biopsy data was collected at baseline from anti-TNF resistant CD patients enrolled in the Ustekinumab trial previously described 1. Two 2.5 mL blood samples were collected at the trial baseline using PAXgene tubes from all subjects (1528 blood samples in total). All blood samples were stored at -80°C until RNA isolation was performed. A subset of 150 blood samples was hybridized by microarray for genome-wide mRNA expression profiling. Ileal, rectal, and colonic biopsies were collected at the screening visits from a sub-group of

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subjects who consented to this procedure. Biopsies were obtained from selected colonic sections, including the ascending, transverse, and descending colonic regions, and from both inflamed and non-involved areas. The inflamed regions were defined as the colonic segments with the most severe disease activity. Additional biopsies were collected from the terminal ileum and from the rectum (i.e., the distal 10 cm of the colon).

Total RNA including micro ribonucleic acids (miRNA) was isolated with PAXgene Blood RNA MDx Kit plus customized reagent BM3 (Qiagen Inc., Valencia, CA). RNA quantity and quality were determined with a LabChip GX (Caliper Life Sciences, Hopkinton, MA). Gene expression were carried out using Affymetrix GeneChip HTHGU133+ (Affymetrix, Santa Clara, CA, Cat# 901262) according to the manufacturer’s protocol with the exception that DMSO replaced the Tetramethylammonium Chloride Solution (TMAC) in the hybridization buffer. Arrays were washed and stained on the Affymetrix GeneChip Array Station then scanned on an HTAPS scanner.

Reconstruction of the Bayesian Networks: Bayesian networks are directed acyclic graphs in which the edges of the graph are defined by conditional probabilities that characterize the distribution of states of each node given the state of its parents. The network topology defines a partitioned joint probability distribution over all nodes in a network, such that the probability distribution of states of a node depends only on the states of its parent nodes: formally, a joint probability distribution p( X ) on a set of nodes X can be decomposed as pX( )= ∏ pX (i | Pa( X i )) , where Pa(X i ) represents the parent set of X i . In our networks, i each node represents transcription expression of a gene. These conditional probabilities reflect not only relationships between genes, but also theMANUSCRIPT stochastic nature of these relationships, as well as noise in the data used to reconstruct the networ k. Bayes formula allows us to determine the likelihood of a network model M given observed data D as a function of our prior belief that the model is correct and the probability of the observed data given the model: PMD( |)
PDM (| )*() PM . The number of possible network structures grows super-exponentially with the number of nodes, so an exhaustive search of all possible structures to find the one best supported by the data is not feasible, even for a relatively small number of nodes. We employed Monte Carlo Markov Chain (MCMC) 2 simulation to identify potentially thousands of different plausible networks, which are then combined to obtain a consensus network (see below). Each reconstruction begins with a null network. Small random changes are then made to the network by flipping, adding, or deleting individual edges, ultimately accepting those changes that lead to an overall improvement in the fit of the network to the data. We assess whether a change improves the network model using the Bayesian Information Criterion (BIC) 3, which avoids over fitting by imposing a cost on the addition of new parameters. This is equivalent to imposing a lower prior probability P( M ) on models with ACCEPTEDlarger numbers of parameters. Even through edges in Bayesian networks are directed, we can’t infer causal relationships from the structure directly in general. For example, in a network with two nodes, X 1 and X 2 , the two models X1→ X 2 and X2→ X 1 have equal probability distributions as pXX(,)12= pXXpX (|)() 21 1 = pXX (|)() 12 pX 2 . Thus, by data itself, we can’t infer whether X 1

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is causal to X 2 , or vise versa. In a more general case, a network with three nodes, X 1 , X 2 , and X 3 , there are multiple groups of structures that are mathematically equivalent. For example, the following three different models, M1:X1→ XX 22 , → X 3 , M2:X2→ XX 12 , → X 3 , and M3:X2→ XX 13 , → X 2 , are Markov equivalent (which means that they all encode for the same conditional independent relationships). In the above case, all three structures encode the same conditional independent relationship, X1⊥ X 3| X 2 , X 1 and X 3 are independent conditioning on X 2 , and they are mathematically equal. pX()= p (M1|) D = pX (21 | XpXpXX )( 1 )( 32 | ) =p(M2|) D = pXXpXpXX (12 | )( 2 )( 32 | ) . =p(M3|) D = pX (23 | XpXpXX )( 3 )( 12 | ) Thus, we can’t infer whether X 1 is causal to X 2 or vise versa from these types of structures. However, there is a class of structures, V-shape structure (eg. Mv :X1→ XX 23 , → X 2 ), which has no Markov equivalent structure. In this case, we can infer causal relationships. There are more parameters to estimate in the Mv model than M1, M2, or M3, which means a large penalty in BIC score for the Mv model. In practice, a large sample size is needed to differentiate the Mv model from the M1, M2, or M3 models.

Incorporating genetic data as a structure prior in the Bayesian network reconstruction process: In general, Bayesian networks can only be solved to Markov equivalent structures, so that it is often not possible to determine the causal direction of a link between two nodes even through Bayesian networks are directed graphs. MANUSCRIPT However, the Bayesian network reconstruction algorithm can take advantage of the experimental design by incorporating genetic data to break the symmetry among nodes in the network that lead to Markov equivalent structures, thereby providing a way to infer causal directions in the network in an unambiguous fashion 4. We modified the reconstruction algorithm to incorporate eSNP data as priors as follows: genes with cis-eSNP 5 are allowed to be parent nodes of genes without cis-eSNPs, but genes without cis- eSNPs are not allowed to be parents of genes with cis-eSNPs, p(trans >− cis ) = 0 . We have shown that integrating genetic data such as cis-acting eSNP or eQTLs (excluding edges into certain nodes) improves the quality of the network reconstruction by simulations 6 and by experimental validations 4, 7. We note that in applying this particular version of the Bayesian network reconstruction algorithm (incorporating genetic information as a prior), if genetic information is not available or is ignored, the population is simply treated as a population with random genetic perturbations.

Averaging network models: Searching optimal BN structures given a dataset is an NP-hard problem. WeACCEPTED employed an MCMC method to do local search of optimal structures as described above. As the method is stochastic, the resulting structure will be different for each run. In our process, 1,000 BNs were reconstructed using different random seeds to start the stochastic reconstruction process. From the resulting set of 1,000 networks generated by this process, edges that appeared in greater than 30% of the networks were used to define a consensus network. A 30% cutoff threshold for edge inclusion was based on our simulation study 6, where a 30% cutoff yields the best tradeoff between recall rate and precision. The consensus network resulting from

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the averaging process may not be a BN (a directed acyclic graph). To ensure the consensus network structure is a directed acyclic graph, edges in this consensus network were removed if and only if (1) the edge was involved in a loop, and (2) the edge was the most weakly supported of all edges making up the loop.

Bayesian network for individual co-expression module: Searching optimal BN structures given a dataset is an NP-hard problem. The computational complexity of our MCMC method for described above is O(N 4 ) , where N is the number of nodes included in the network reconstruction process. It is practically impossible to construct a global Bayesian network including all 39,000 genes from the intestinal tissue. Following the procedure described above, 1,000 BNs were reconstructed using different random seeds to start the reconstruction process. From the resulting set of 1,000 networks generated by this process, edges that appeared in greater than 30% of the networks were used to define a consensus network. Our previous simulation study shows that the 30% inclusion threshold results in a stable structure and achieves the best tradeoff between precision and recall 6. The histogram of percentage of occurrences of all potential edges shows that 30% is a reasonable cutoff threshold for inclusion (a typical histogram is shown in Figure here).

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Identification of Key Causal Regulators: For each Bayesian network, we further identified the regulators by examining the number of N-hob downstream nodes (NHDN) for each gene in the 8 directed network . For a given network, let be the numbers of N-hop downstream nodes and d be the out degrees for all the genes. The genes with the number of N-hop downstream nodes

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(NHDN) greater than µ + σ µ )( are nominated as causal regulators. The regulators with degree above d + 2σ d )( , where d denote the number of downstream genes, become key causal regulators of a corresponding sub-network associated with disease status. These criteria identified genes with number of downstream nodes and number of out links significantly above the corresponding average value. This method was performed on the IBD RISK network to determine VEOIBD Key Causal Regulators.

Weighted Gene Coexpression Network Analysis: The weighted network analysis begins with a matrix of the Pearson correlations between all gene pairs, then converts the correlation matrix into an adjacency matrix using a power function f(x)=x^ β. The parameter β of the power function is determined in such a way that the resulting adjacency matrix (i.e., the weighted co-expression network) is approximately scale-free. To measure how well a network satisfies a scale-free topology, we use the fitting index 9 (i.e., the model fitting index R2 of the linear model that regresses log(p(k)) on log(k) where k is connectivity and p(k) is the frequency distribution of connectivity). The fitting index of a perfect scale-free network is 1. For this dataset, we select the smallest β which leads to an approximately scale-free network. The distribution p(k) of the −γ resulting network approximates a power law: kp ~)( k .

To explore the modular structures of the co-expression network, the adjacency matrix is further transformed into a topological overlap matrix 9. As the topological overlap between two genes reflects not only their direct interaction, but also their indirect interactions through all the other genes in the network, previous studies 9, 10 have shown that topological overlap leads to more cohesive and biologically meaningful modules. To identify modules of highly co-regulated genes, we used average linkage hierarchical clusteringMANUSCRIPT to group genes based on the topological overlap of their connectivity, followed by a dynamic cut-tree algorithm to dynamically cut clustering dendrogram branches into gene modules 11 . To distinguish between modules, each module was assigned a unique color identifier, with the remaining, poorly connected genes colored grey. To examine how each gene module was related to clinical traits we first performed principal component analysis for each module, and then computed module-trait correlation between the first principal component (module eigengene) and each trait 12 . The significance (p value) of each correlation was also calculated.

Key Driver Analysis: Key driver analysis (KDA) takes as input a set of genes (G) and a directed gene network N (eg Bayesian network) 12-15 . The objective is to identify the key regulators for the gene sets with respect to the given network. KDA first generates a sub-network N G, defined as the set of nodes in N that are no more than h-layers away from the nodes in G, and then searches the h-layer neighborhood (h=1,..,H) for each gene in N G (HLN g,h ) for the optimal h*, such that ES h* = max( ES h, g ) ∀ g ∈ N g, h ∈ {1 .. H } where ES h,g is the computed enrichment statistic for HLN g,h . A node becomesACCEPTED a candidate driver if its HLN is significantly enriched for the nodes in G. Candidate drivers without any parent node (i.e., root nodes in directed networks) are designated as global drivers and the remaining are local drivers.

Key Driver Analysis was performed by projecting Inflamed Sigmoid and Inflamed Rectum signatures on the Bayesian Ileum network constructed from the ustekinumab trial data. Differential Expression: Whole-genome transcriptional expression data were collected from

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biopsy tissue samples and whole blood of individuals in the Janssen ustekinumab trial (T26). We wanted to identify differential expression signatures of disease status, degree of tissue inflammation (inflamed versus non-involved).

Sample Selection and Data Curation Biopsy data: A total of 321 biopsy tissue samples were collected from 86 individuals (mean of 4 samples from differential anatomical regions of the terminal ileum, colon, and rectum per individual) at the screening time point of the study. Whole-genome expression data were collected on the Affymetrix ht_hg-u133_pm_ap platform, which contains 54,715 probes (_21,000 genes). Pre-processing of expression data included background correction, log2 transformation, quantile normalization, and Robust Multi-array Averaging to stabilize the variance and rescale the levels across arrays1. Further quality control measures of clustering and principal component analysis (PCA) were employed to identify potential outliers in the data. Age and gender adjustments to expression levels were performed on each tissue location using robust linear regression. Only samples with valid age and gender data were used for analysis.

Blood data: A total of 836 blood samples were collected from 275 individuals during the course of the trial. Whole-genome expression data were collected on the Affymetrix ht_hg-u133_pm_ap platform, which contains 54,715 probes (21,000 genes). Pre-processing and quality control steps were similar to biopsy procedures listed above. Expression levels were adjusted by age, gender, and batch using robust linear regression. Only samples with valid age and gender data were used for analysis.

To identify differential expression signatures, we used an unbiased univariate filter to select top- varying genes and then applied significance analysiMANUSCRIPTs of microarrays (SAM) 16 to whole-genome expression data collected from biopsy tissue sample s and whole blood of individuals in T26 trial. To control for multiple hypothesis testing, we used the Benjamini & Hochberg adjustment on the raw p-values to control the family-wise error rate, and set a false discovery rate threshold of 1% or 5%. All analyses performed using the R statistical package, version 2.15.24.1.

Comparisons: Biopsy – anatomical region and inflammatory signatures. To ascertain tissue-specific signatures, we examined inflamed versus non-involved tissue from differential anatomical regions of the small intestine, colon, and rectum.

Clinical variable correlation: We used Spearman correlation to examine the relationship between the mRNA expression of TRIM22 in intestine and blood and several key IBD traits. The correlation test was performed with the R function “corr.test”. We applied a simple Bonferroni correction to a 0.01 significance level across all gene-trait correlation tests performed.

Functional StudiesACCEPTED

Constructs : HA-epitope tagged mammalian expression plasmids for NOD1 and NOD2 were obtained from Dr. Dana Philpott (University of Toronto), GFP-tagged Ubiquitin was obtained for Dr. John Brumell (University of Toronto), HA-tagged K48 Ubiquitin and HA-tagged K63 Ubiquitin were obtained for Dr. Jane McGlade (University of Toronto), FLAG-tagged MAVS and FLAG-tagged RIP2 were obtained for Dr. Hong-bing Shu (Wuhan University) and

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mammalian expression plasmids for FLAG-tagged TRIM22 and its mutants were constructed by site-directed mutagenesis.

Transfection and Luciferase Reporter Assays: NF-κB luciferase reporter plasmids (Promega) and ISRE promoter and IFN β promoter luciferase reporter plasmids were obtained from Dr. Hong-bing Shu (Wuhan University). A total of HEK 293 cells (1 × 10 5) were seeded on 24-well dishes and transfected the following day using Lipofectamine 2000 method. Empty control plasmid was added to ensure that each transfection received the same amount of total DNA. To normalize for transfection efficiency, 0.01 g of pRL-TK (Renilla luciferase) reporter plasmid was added to each transfection. Approximately 24h after transfection, luciferase assays were performed using a dual-specific luciferase assay kit (Promega). Firefly luciferase activities were normalized on the basis of Renilla luciferase activities. Cells were treated with recombinant TNF α, RSV, or L18-MDP (InvivoGen). Statistical analysis of luciferase assay data consisted of two-way ANOVA followed by Bonferroni post-hoc testing to compare across conditions. Statistical significance was assigned to tests with p<0.05 after Bonferroni correction.

Quantitative real-time PCR: Total RNA was isolated from cells using Trizol reagent (Ambion), reverse-transcribed in cDNA using Superscript VILO (Invitrogen), and subjected to qPCR analysis to measure mRNA expression levels of tested genes. Gene-specific primer sequences were as follow: ISG15 : 5 ′-AATGGGCTGGGACCTGACGG-3′ (forward), 5 ′- TTAGCTCCGCCCGCCAGGCT-3′ (reverse); IL-6: 5 ′- CCT GAACCTTCCAAAGATGGC-3′ (forward), 5 ′-TTCACCAGGCAA GTCTCCTCA-3′ (reverse); TNF : 5 ′- GAGGCCAAGCCCTGGTATG-3′ (forward), 5 ′-CGGGCCGATTGATCTCAG C-3′ (reverse); GAPDH : 5 ′-GAGTCAACGGATTTGGTCGT-3′ (forward), 5 ′- GACAAGCTTCCCGTTCTCAG-3′ (reverse). StatisticalMANUSCRIPT analysis of qPCR data consisted of two- way ANOVA followed by Bonferroni post-hoc testing to compare across conditions. Statistical significance was assigned to tests with p<0.05 after Bonferroni correction.

Coimmunoprecipitation and Immunoblot Analysis: For transient transfection and coimmunoprecipitation experiments, HEK 293 cells (1 × 10 6) were seeded for 24 hr then transiently transfected for 18–24 hr using Lipofectamine 2000 according to the manufacturer’s protocols. Transfected cells were lysed in 1 mL of lysis buffer (150mM NaCl, 50mM HEPES, 1% Triton X-100, 10% glycerol, 1.5mM MgCl 2, 1.0mM EGTA) supplemented with protease and phosphatase inhibitors (aprotinin (1:1000), leupeptin (1:1000), pepsin (1:1000) and, PMSF (1:100). For each immunoprecipitation, a 0.9mL aliquot of the lysate was incubated with 0.5 g of the indicated antibody and 40 L of a 1:1 slurry of Protein G Sepharose (Bioshop) for 1 h. Sepharose beads were washed 3X with 1 mL of high salt lysis buffer containing 0.5 M NaCl. The precipitates were analyzed by standard immunoblot procedures using the following antibodies: mouse monoclonal anti-FLAG (Sigma), anti-HA (Origene), anti-NOD2 (Novus), anti-GFP (Invitrogen), rabbit polyclonal anti-TRIM22 (Abnova) and goat polyclonal anti-NOD2 (Ingenix). For endogenousACCEPTED coimmunoprecipitation experiments, HT29 cells (5 × 10 7) were stimulated with MDP (10 µg/mL) for the indicated times or left untreated. The coimmunoprecipitation and immunoblot experiments were performed as described above. shRNA Transduced Stable HT29 cells: shRNA targeted to TRIM22 was obtained from Dr. Barr’s lab (University of Western Ontario). The HT29 cells were transfected with two packaging

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plasmids, pMD2G and pSPA, and the GFP control or TRIM22 shRNA retroviral plasmid by calcium phosphate precipitation. Cells were washed 12 hr after transfection and new medium without antibiotics was added for 24 hr. The recombinant virus-containing medium was filtered and used to infect HT29 cells in the presence of polybrene (4 g/mL). The infected HT29 cells were selected using puromycin (2 g/mL) for 2 weeks before additional experiments were performed.

Immunohistochemistry: Colonic biopsies and tissues from colon resection specimen were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections cut at 5 µm were placed on Superfrost Plus (Fisher Scientific, Waltham, MA) microscope slides. Following incubation at 60°C for 30 min, the tissues sections were deparaffinized and rehydrated using sequential washes (100% xylenes, 5 min x 2; 50% xylenes: 50% ethanol, 5 min; 100% ethanol, 5 min x 2; 95% ethanol, 3 min; 75% ethanol, 3 min; 50% ethanol, 3 min; cold H 2O, 5 min). Sections were then stained with haematoxylin and eosin (H&E) for 10 min, followed by sequential washes in cold H2O. Stained tissue was mounted using xylene based mounting media. Stained sections were photographed with a Nikon Light Microscope and adjusted for brightness and contrast using Adobe Photoshop.

Fluorescent Confocal Microscopy Immunofluorescence on biopsy samples: Paraffin sections were deparaffinized using xylene and rehydrated in ethanol at different concentrations. Antigen retrieval was performed using high pressure-cooking in EDTA buffer, pH 9. Afterwards tissue sections were blocked for 1 hr at RT with 5% BSA and 15% goat serum in 1X PBS without Ca2+ and Mg2+ to avoid nonspecific antibody binding. First antibody incubation was performed overnight at 4°C. Next day, sections were washed 3X for 10 min with 1X Ca2+, Mg2+MANUSCRIPT free PBS. Tissue slides were incubated with secondary antibody for 1 hr in darkness at RT and afterwards washed 3X for 10 min with 1X PBS without Ca2+ and Mg2+. Finally mounting of stained sections were performed overnight using Vectoshield Mounting Medium. Polyclonal rabbit anti-TRIM22 (Sigma Aldrich), monoclonal mouse anti-NOD2 (Novus Biologicals), polyclonal rabbit anti-Flag (Sigma Aldrich) and mouse monoclonal HA (Thermo Scientific) were used as first antibodies in a 1:100 dilution. ALEXA 488 goat anti-mouse and ALEXA 568 goat anti-rabbit were used as secondary antibodies at a dilution of 1:500. Nuclei were stained using Dapi (Thermo Scientific) in a dilution 1:10 000. Images of stained slides were taken on an Olympus Spinning disk confocal microscope and adjusted for brightness and contrast in the Velocity Software 6.1.1.

Peptide inhibition of NOD2 in human biopsy samples: NOD2 peptide (ab39813, Abcam) was incubated overnight at 4°C with the NOD2 antibody (ab36836, Abcam) in the following proportion: 10X peptide concentration to 1X antibody. Next day immunofluorescence analysis on biopsy samples was performed as described above.

ImmunofluorescenceACCEPTED on HEK 293 cells: Cells were fixed in 4% paraformaldehyde and permeablized with 0.01% Triton-X-100 for 1 minute. Blocking was performed at RT for 1 hour and first antibody incubation overnight at 4°C. Afterwards cells were washed 3X for 10 minutes with 1X PBS without Ca2+ and Mg2+. Next cells were incubated with secondary antibodies for 1 hr at RT in darkness, washed 3X for 10 minutes with 1X PBS without Ca2+ and Mg2+ and

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finally mounted with Dako Fluorescent Mounting Medium (Dako). Nuclei counter-staining as well as first and secondary antibodies were the same as used for biopsy staining.

Intracellular TNF staining and flow cytometry: Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from whole blood from patient, parent or healthy donor. NOD2-dependent TNF response in monocytes was evaluated as previously described (please cite PMID 24611904). In brief, PBMCs were cultured overnight in RPMI1640 supplemented with 10% FCS. The following day, cells were gently washed with PBS and activated for 2.5 hours with 200 ng/ml L18-MDP (Invivogen) or 200 ng/mL LPS (Enzo) in the presence of Golgiplug (BD Bioscience). After stimulation, cells were harvested by scraping, put on ice and labelled with fixable viability dye (eBioscience) and surface monocyte markers. Following fixation and cell permeabilization (Cytofix/Cytoperm Kit, BD Bioscience) cells were stained for intracellular production of TNF. The following antibodies were used: anti-TNF (clone MAb11, eBioscience), anti-CD14 (clone M5E2) and anti-HLA-DR (clone L243, both BioLegend). Results were acquired by flow cytometry (LSRFortessa, BD Biosciences) and data analyzed with FlowJo software (Version 10.0.6, Treestar).

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Detailed Case Reports – Patients 1 to 3

Patient 1: Patient 1 was born at term with a birth weight of 3.8 kg to consanguineous parents of Lebanese descent. At 8 days old, she presented with fever, poor feeding, mouth ulcers, diarrhea, and perianal excoriation. Chest X-ray showed increase opacity over right upper zone, consistent with right upper lope collapse. She was treated with empiric antibiotics and started on amino acid based formula for presume cow’s milk protein allergy. During the next few months, she had recurrent fever, mouth ulcers and poor feeding.

At 7 months old, Patient 1 experienced severe pain associated with defecation and bloody diarrhea. She was later referred for investigation of possible VEOIBD. Clinical examination revealed multiple perianal skin tags and fissures. Colonoscopy showed active chronic proctitis with granuloma formation. She was treated with prednisolone leading to some symptomatic relief, however she later proved to be steroid dependent. Gastrostomy was inserted at 9 months to facilitate continuous feed in view of her significant growth failure.

Infliximab was started at 16 months old but she developed anaphylaxis after 2 doses. As her perianal disease deteriorated, she experienced very severe pain while defecating. In order to reduce fecal output and hence reduce the perianal symptoms, she was commenced on 6 months of total parenteral nutrition (TPN) with complete gut rest. She remained symptomatic despite these interventions.

Simultaneously, Patient 1 also had partial upper airway obstruction with non-granulomatous inflammatory polyps in the pharynx; these were removed surgically. After the upper airway obstruction was resolved, Adalimumab wasMANUSCRIPT introduced with no clinical improvement. Subsequently, her course was punctuated by multiple infections, even though screening tests for immunodeficiency were normal (discussed below).

At 25 months old, a diversion colostomy was performed to control her perianal disease. Initially there were marked improvements after fecal stream diversion and topical tacrolimus treatment. However she continued to have colitis and poor growth.

At 3 years old, she developed fistulae around her colostomy site. Due to persistent disease, she had sub-total colectomy and formation of end ileostomy at 4 years old.

At nearly 5 years old, Patient 1 had type-2 respiratory failure secondary to Coronavirus respiratory tract infection. Further investigation revealed that she had severe bronchiectasis, probably secondary to recurrent respiratory tract infection.

Presently, the combination of supplementary parental nutrition, fecal stream diversion and no immunosuppressiveACCEPTED medication, Patient 1 has been relatively symptom free with appropriate growth.

She had numerous central venous access associated sepsis; Candida parapsilosis , Enterobacter cloacae , Coagulase negative staphylococcus , pseudomonas aeruginosa, Escherichia coli, Klebsiella oxytoca were cultured in her blood. She also had multiple respiratory infections, microorganism isolated included Respiratory syncytia virus , Influenza A , Coronavirus , Human

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metapneumovirus , and Pseudomonas aeruginosa . The majority of her infections of viral origin occurred within 48 hours post-Adalimumab injection.

Her immunological workup showed on multiple occasions with ongoing inflammation normal to mildly elevated IgG, IgM, IgE, and IgA. Immunophenotyping showed normal populations of CD3, CD4, CD8, CD4:CD8 ratio, CD19, and NK cells. There was normal expression of CD11b and CD18 compared to normal controls indicating no evidence of leukocyte Adhesion Deficiency Type 1. C3 and C4 levels were noted to be normal. The following antibodies were noted to be negative: Anti-dsDNA, Anti-Myeloperoxidase, Anti-PR3, and Anti-Nuclear Antibody. She was found to have C-Anti-Neutrophil Cytoplasmic Antibody positivity 1/80. NBT testing was normal on several occasions, and Neutrophil Oxidative Burst Index was found to be normal as well. Screening for Familial Mediterranean Fever was found to be negative. Functional studies demonstrated a normal TNF α response to L18-MDP stimulation (data not shown). Whole exome sequencing performed showed no variants within NOD2, IKBKG, XIAP or other genes known to cause primary immunodeficiency or other genes know to cause colitis 17 .

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Patient 2: Patient 2 was born premature at 35 weeks of gestation to non-consanguineous parents of Jamaican descent. There is a family history of Crohn’s disease in his uncle. At 3 ½ months old, he presented with a fever and a rash and was found to have hypoalbuminemia and severe microcytic anemia (hemoglobin of 67 g/l, MVC 60). His septic workup was negative and he was treated with a change in his diet to a semi-hydrolyzed formula.

At 6 months of age, he presented with an acute lower GI bleed and was found to have a major coagulopathy, hypoalbuminemia and severe anemia (69 g/L). During this admission, he developed a perianal abscess on his left gluteal region that was surgically drained. He had multiple infections during this admission ( E. coli urinary tract infection, Rotavirus and a positive urine culture for CMV). Colonoscopy found large ulcerations in the sigmoid and descending colon with multiple pseudopolypoid lesions and marked background inflammation with loss of vasculature. Biopsy pathology noted inflamed granulation tissue lined by histiocytes and fibrino- purulent exudate. His upper endoscopy was visually normal, but granulomas were noted on patholgoy of the stomach biopsies. He was started on an amino acid formula.

At 9 months old, he presented with polyarthritis and dactylitis. He continued to have severe anemia with elevated acute phase reactants. He was started on steroids and subsequently azathioprine for maintenance therapy.

At 13 months of age, he was admitted with a severe flare of his colitis and a severe drop of his hemoglobin to (39 g/L). Colonoscopy reconfirmed pancolitis. Biopsies showed noncaseating granulomata with changes of chronic colitis. Azathioprine was changed to methotrexate. He also was found to have a perianal abscess on his right buttocks that was drained surgically. MANUSCRIPT At 18 months, he presented with severe diarrhea resulting in metabolic acidosis requiring admission and rehydration. Prednisone was restarted.

At 20 months, his perianal disease worsened with the development of multiple fistulae. Infliximab was attempted at 4.2 mg/kg/dose.

At 23 months, his perianal disease worsened. Colonoscopy showed again pancolitis with serpigineous ulcers from rectum to proximal descending colon.

At 24 months, he continued to flare and his perianal disease worsened. He was noted to have a loss of response to Infliximab, and he underwent a left-sided colectomy with placement of a colostomy.

He presented again at 28 months with a further ischio-rectal abscess of the right buttock that was drained surgically. ACCEPTED At 38 months, he developed a large peri-colostomy abscess with a generalized worsening of disease. Infliximab was restarted, and he developed an allergic reaction after the second dose. He continued to worsen and subsequently required a right hemicolectomy with ileostomy formation.

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Abscess continued to form for the next three years, requiring admission and drainage. Adalimumab was attempted with improvement of weight and symptoms.

As for extra-intestinal manifestations, he has had dactylitis and polyarthropathy since 9 months of age. These have improved on steroid therapy. He also developed orchitis and epididymitis requiring treatment with clavulin. He developed pyoderma gangrenosum near the site of ileostomy.

His initial immune workup noted a normal NBT test and Neutrophil Oxidative Burst Index (NOBI) and normal IgG, IgM and IgA level. Lymphocyte proliferation was noted to be normal. Immunophenotyping showed normal percentages of CD3, CD4, CD8, CD19, CD4:CD8 ratio and NK cells. He was negative for HLA-B27. The following autoantibodies were noted to be negative: Anti-Nuclear Antibody (ANA), Anti-Smooth Muscle Antibody (Anti-SMA), Anti-Parietal Cell Antibody, Anti-Cardiolipins IgM and IgG, and Rheumatoid Factor. IL10, IL10RA and IL10RB genes were sequenced and no mutations were identified, and STAT3 phosphorylation was noted to be normal after stimulation of PBMCs with IL-10. His most recent immunological work up showed a normal Neutrophil Oxidative Burst Index (NOBI) and a normal immunophenotyping analysis of lymphocytes. Functional studies demonstrated abnormal TNF α response to L18-MDP stimulation (Figure S8). Because of this abnormal MDP response, the patient had directed sequencing of XIAP on 4 separate occasions at 2 institutions and no disease causing mutations were identified.

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Patient 3: Patient 3 presented at age 7 years with severe hypoalbuminemia, bloody diarrhea and weight loss of a few weeks duration. Previously she had an unremarkable medical history and was born to consanguineous parents. She had been unwell for approximately 1 year prior to presentation and found to have severe anemia requiring a transfusion and elevated acute phase reactants. She presented with pericarditis and was admitted to the ICU. After initiating steroids, she responded initially. Her prior medical history was unremarkable and included only reactive airway disease. There was a strong family history of Crohn’s disease in both her mother and maternal aunt. Her initial colonoscopy was limited due to severity but showed continuous colitis with cobble stoning and friability. Pathology showed chronic inflammation of the colon with partial villous blunting of the duodenal villi.

She continued on steroids but continued to be steroid dependent despite azathioprine. She was noted to develop a rectovaginal fistula, along with perianal disease. Due to continued disease activity, perianal disease and steroid dependence, infliximab was initiated with no significant improvement. She had repeated endoscopic evaluation showed pseudopolyps and discontinuous disease of the colon. Her terminal ileum showed edema and her perianal disease required multiple interventions using Seton placement and skin graft surgery.

Due to continued disease and lack of response, she subsequently underwent diversion ileostomy, followed by total proctocolectomy and abdominal perineal resection. Pathology of the resected specimen noted granulomas and inflammation involving the terminal ileum. Subsequent biopsies from colonoscopies done after surgery have all shown granulomas and mild patchy chronic colitis. She was started on Adalimumab therapy.

Despite diversion and second line anti-TNF therapy,MANUSCRIPT she continued to have perianal disease with partial response. She also developed iritis and erythema nodosum. At last follow-up, she had transitioned to adult care while on azathioprine and steroid and antibiotic therapy. For her immunological workup, she was noted to have a normal level of IgG, IgA, and IgM. NOBI was repeatedly found to be normal. She did not have mutations in IL10RA/B, IL10 , or NEMO. The patients had a c:109 del C, p: P37QfsX32 mutation in XIAP but is female and a heterozygote carrier. As the variants in TRIM22 are also found in databases it is possible that the XIAP variant may contribute to disease in this patient. However, Patient 3 has transferred to adult care and no longer consented to any further immunological testing. Therefore no MDP functional studies were carried out.

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Supplemental Tables

S1A) Clinical Features

Age of Patient Ethnicity/ Onset of Gender Consanguinity Clinical Features # History of IBD Symptoms Diarrhea, rectal bleeding, perianal Lebanese 1 8 days Female Yes disease, growth failure, weight loss, No family history epithelial granuloma of IBD

Severe growth failure, perianal disease, Jamaican fever, petechiae, hepatomegally, Strong maternal 2 < 3 months Male No diarrhea, polyarthritis involving mainly family history of large joints. Crohn's Disease ~ 6 years Caucasian Diarrhea, abdominal pains, weight loss, and Strong maternal 3 Female No diffuse edema of lower extremities, presented at family history of significant anemia. 7 years Crohn's Disease

S1B) Genetic Features

Variant Position Prediction Patient MANUSCRIPTGERP++ PhyloP SNP Exon Genomic MAF Polyphen2 SIFT # cDNA Protein Rank Rank (GrCh37.p13) Rank Score Rank Score Score Score Probably Damaging 1 rs187416296 7 g.5730705C>T c.1324C>T p.Arg442Cys 0.0041 Damaging 1.68 0.36288 (0.90636) (0.87578 ) Probably Damaging rs61735273 2 g.5719756C>T c.731C>T p.Ser244Leu 0.0035 Damaging 0.40419 0.77407 (0.90636) (0.8513) 2 Possibly Tolerated rs200924168 3 g.5718503G>C c.449G>C p. Arg150Thr 0.0044 Damaging 0.01094 0.00845 (0.3317) (0.47786)

Probably Damaging 3 rs12364019 7 g.5730343G>A c.962G>A p.Arg321Lys 0.015 Damaging 0.32553 0.34637 (0.70945) (0.83275)

Table S1. Clinical (A) and Genetic Features (B) of Patients with TRIM22. MAF minor allelic frequency based on 1000 Genomes and 6503 healthy individuals genotyped in the NHLBI Exome Sequencing Project (http://evs.gs.washington.edu). The variant prediction are based ACCEPTED Polyphen2 18 , SIFT (http://sift.jcvi.org), GERP++ (http://mendel.stanford.edu/SidowLab/downloads/gerp/), and PhyloP (http://ccg.vital- it.ch/mga/hg19/phylop/phylop.html).

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Position Chromosome Position Identifier Reference Gene Name Parent Parent Patient

1:76226952-SNV 1 76226952 ? T ACADM, C_T C_T C_C DLSTP1 3:111793206- 3 111793206 ? C TMPRSS7 C_T C_T T_T SNV 3:112648127-Del 3 112648127 rs4596117; ? T CD200R1 -_T G_T G_G

6:132206095- 6 132206095 ? C ENPP1 A_C A_C A_A SNV 7:21698453-SNV 7 21698453 ? A DNAH11 A_G A_G G_G

7:21781702-SNV 7 21781702 ? A DNAH11 A_G A_G G_G

7:91623968-SNV 7 91623968 ? G AKAP9 A_G A_G A_A

10:75442543- 10 75442543 ? C AGAP5 C_T C_T T_T SNV 11:5730705-SNV 11 5730705 ? C TRIM22 C_T C_T T_T

11:73796902- 11 73796902 rs78878933 T C2CD3 C_T C_T C_C SNV 11:78419504- 11 78419504 ? G ODZ4 A_G A_G A_A SNV 19:15730499-Ins 19 15730499 rs55848814 - CYP4F8 -_C -_C C_C

Table S2. Autosomal Recessive Variants Identified by WES in Patient 1.

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Table S3: VEO Genes See excel sheet

Table S4. VEOIBD Key Drivers See excel sheet

Table S5. IIBDGC GWAS Enrichment in TRIM22 Locus See excel sheet.

Table S6. IIBDGC GWAS Enrichment in TRIM22 eQTL Enrichment See excel sheet

Table S7. eQTL Pathway Enrichment See excel sheet

Table S8. Crohn’s GWAS Enriched Blood eQTL Overlap with the VEO Network

Table S9. Annotation of 179 VEO Network See excel sheet

Table S10. Functional Categories of Genes in the TRIM22 Sub network

Table S11. Inflamed Sigmoid and Rectum Key Drivers

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TRIM22 CDAI CDAI Baseline_CRP Baseline_CRP Baseline_Fecal Baseline_Fecal # of samples Levels at Correlation P Value Correlation P Value Calprotectin Calprotectin Intestine Site Correlation P Value Ileum -1.23E-01 9.34E-02 4.41E-02 5.53E-01 4.15E-02 5.76E-01 193 Ascending -5.39E-02 5.57E-01 1.64E-01 7.52E-02 2.45E-01 6.93E-03* 128 Transverse 1.70E-01 5.53E-02 1.74E-01 5.30E-02 3.66E-01 2.26E-05* 134 Descending 4.84E-02 6.49E-01 1.89E-01 7.67E-02 5.50E-01 1.40E-08* 96 Sigmoid -1.52E-01 2.05E-01 8.79E-02 4.63E-01 4.96E-01 9.43E-06* 75 Rectum -5.02E-02 5.09E-01 1.85E-01 1.53E-02 3.85E-01 1.46E-07* 184 Total -2.65E-03 9.41E-01 1.26E-01 5.21E-04* 2.86E-01 6.54E-16* 810

Table S12. Correlations of the Expression of TRIM22 to the Clinical Response Characteristics Stratified by Distinct Intestine Anatomy Sites (Includes Both Inflamed and Non Inflamed Samples).

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CDAI Rho P value TRIM22 0.0829 0.0203

Table S13. Correlation of the Expression of TRIM22 in blood to CDAI

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Supplemental Figures

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Figure S1: Variant Identification Pipeline for WES of Patient 1.

Whole exome sequencing identified 152,020 exonic variants in Patient 1. Eliminating low-quality and synonymous variants further refined the WES variant set. To isolated potential causative variants, only rare and predicated damaging (by dbNSFP) variants were included in inheritance analysis. Variants identified according to each inheritance model are shown. ACCEPTED

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Figure S2: Heterozygous TRIM22 Variants R150T and S244L Occur in the Coiled-coil Domain and May Affect Domain Oligomerization.

A) Protein sequence for TRIM22 indicating the position of the two coiled-coil regions termed region 1 (green) and region 2 (purple) including the position of R150 and S244.

B, C) PrOCoil analysis of coiled coil region 1 (B) and region 2 (C) for TRIM22 with the correspondingACCEPTED variants reveals potential alterations in domain oligomerization patterns. PrOCoil evaluates the relative weighted contribution of each amino acid to the oligomeric character of the coiled-coil domain. The introduction of the R150T and S244L variants alter the weighted effect of positions near the site of variant. These alterations have potential to impact the ability of the coiled-coil domain to fold correctly.

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Figure S3: Blood and Intestine Derived eQTLs: Gene Set Enrichment Analysis of Genes Controlled by IBD SNPs (Metacore) See Table S7 for genes in pathway.

Top enriched gene sets among genes controlled by IBD SNPs: NHGRI catalog entries for IBD, UC and CD, plus all WTCCC IBD GWAS SNPs with p-value ≤ 0.001. 362 genes showed IBD SNP regulation in the blood, 98 in the intestine (56 genes were shared between the two tissues). Log10 p-value of enrichment on the horizontal axis: intestine (blue bar) and blood (orange bar).

Different Gene Set database categories are reported in each panel: TRIM22 Pathways: See below 1. Transcription P53 signaling (TRIM22 is a P53 target gene) 4. Immune response: Antiviral actions of interferons 9. Ubiquitin PathwayACCEPTED

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COMPUTATIONAL DESIGN FOR ADULT IBD NETWORK

B.

Inflamed and non inflamed tissue biopsies collected from anti TNFα refractory Crohn’s patients enrolled in clinical trial for Ustekinumab (anti P40- IL12/IL23)

A. Pan intestine Bayesian Network C. Differential expression of inflamed versus non inflamed per region D. Key Driver Analysis

mRNA Ascending Transverse expression colon colon profiling

Descending colon

Sigmoid Ileum PROJECTION OF GENES UPREGULATED IN Rectum INFLAMED MANUSCRIPTIleum Bayesian Network

Figure S4: Computational Design.

A) Inflamed and non inflamed tissue biopsies were collected from anti TNF α refractory Crohn’s patients enrolled in clinical trial for ustekinumab (anti P40- IL12/IL23).

B) Bayesian pan intestine network was constructed from microarray expression profiling on biopsies collected from various regions of the intestine.

C) Differential expression was performed on inflamed vs. non-inflamed for each region of the intestine collected.

D) Differential expression signatures of genes up regulated in inflamed sigmoid and inflamed rectum were projected on Bayesian ileum network for key driver analysis. ACCEPTED

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Figure S5: First Neighbors of TRIM22 and NOD2 in VEOIBD Intestine Network. MANUSCRIPT Genes within one path of TRIM22 in the VEOIBD RISK network have putative functional roles in autophagy.

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Figure S6: TRIM22 and NOD2 Co-localize in Healthy Controls and Disrupted in Patient Tissue Sections.

A, B) Immunofluorescence analysis of TRIM22 and NOD2 in colonic biopsy samples from Healthy Controls and Patients . TRIM22 (red) and NOD2 (green) localization was analyzed in the villus (A) and crypts (B) of both healthy controls and Patients 1-3. TRIM22 and NOD2 colocalization was observed in the both the villus and crypts of the healthy control biopsy samples. Both TRIM22 and NOD2 expression and colocalization were reduced in the patient biopsy samples Scale bars represent10 µm.

C) Exogenous TRIM22 co-localize with Exogenous NOD2. HEK293T cells seeded on coverslips were co-transfected with FLAG-TRIM22 and HA-NOD2. TRIM22 (red) and NOD2 (green) immunostaining show strong cytoplasmic colocalization. Scale bars represent 10 µm.

D) TRIM22 does not co-localize with NOD1 . To evaluate the specificity of the observed TRIM22 and NOD2 co-localization, immunofluorescence microscopy was performed on HEK293T cells transiently expressing FLAG-TRIM22 and HA-NOD1, a NOD family member that does not interact with TRIM22. TRIM22 (red) and NOD1 (green) staining did not show significant overlap. Scale bars represent 10 µm.

E, F) Peptide inhibition of NOD2 in colonic biopsy samples of Healthy Controls and Patients . To demonstrate NOD2 antibody specificity, endogenouMANUSCRIPTs NOD2 immunostaining was performed on health and disease control biopsy samples as well as Patients 1-3 in the presence or absence of a NOD2 antibody peptide inhibitor. Without peptide incubation, NOD2 showed a cytoplasmic expression in healthy controls and disease controls, and is reduced in the patients. The specific NOD2 peptide is blocking the antigen-antibody binding and therefore no staining is detected when the peptide is added concurrently.

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T K C 0 2 T 5 44L T 2 FLAG-TRIM22: W R1 S R321R44 WT W A HA-NOD2: + + + + + -- B HA-NOD1: ----- + - Untreated HA-empty vec: ------+ TNF α + TNF α kDa 20 IP: HA

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Figure S7: TRIM22 Variants and shRNA Abrogate NOD2 Signaling. ACCEPTED

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A) NOD2 co-immunoprecipitates TRIM22 from in vitro cell lysates after transient co-expression . HEK293T cells were transfected with HA-epitope tagged NOD2 and either wild type (WT) or mutant (R150T, S244L, R321K, R442C) FLAG-epitope tagged TRIM22 constructs. Twenty-four hours post-transfection, cells were lysed and NOD2 was immunoprecipitated with anti-HA antibody. Western blotting identified bound proteins. WT-TRIM22 interacted with and pulled- down NOD2; however, mutant TRIM22 showed reduced or absent NOD2 binding. NOD1, which does not interact with TRIM22, and an empty vector construct were included as negative controls. All lucerifase assay and Western blotting experiments were carried out in triplicate independent experiments and blots are representative.

B) TRIM22 overexpression has no effect on TNF α mRNA levels following stimulation by TNF α. HCT116 cells were transiently transfected with control (empty vector), TRIM22-WT, or TRIM22-R442C plasmids. Twenty-four hours post-transfection, cells were stimulated for 18 hr with TNF α (20 ng/mL). mRNA of TNF α were assessed by Q-PCR. No significant increase in TNF α mRNA was observed following TNF α stimulation of cells overexpressing TRIM22.

C, D, E) TRIM22 mutants reduce mRNA level of genes downstream of the NOD2 signalling. HCT116 cells were transiently co-transfected with NOD2 and TRIM22-WT or TRIM22 mutants (R150T, S244L, R321K, R442C). Twenty-four hours post-transfection, cells were stimulated with MDP (10 µg/mL) (C) or RSV (0.1 g) (D, E) for 18 hr and then qPCR were performed. Cells transfected with TRIM22 mutants showed significantly reduced expression of TNF α (C), IL-6 (D), and ISG15 (E) (*p < 0.001 after Bonferroni post-hoc testing).

F) Overexpression of TRIM22 increases ISRE activation. HEK293T cells were transiently transfected with control (empty vector), TRIM22,MANUSCRIPT or NOD2. ISRE reporter activation was evaluated by luciferase assay following stimulation with respiratory syncytial virus (RSV) (0.1 g). TRIM22 overexpression significantly increased ISRE reporter activation compared to empty vector control (*p<0.001 after Bonferroni post-hoc testing). All experiments were carried out in triplicate, independent experiments.

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A 1 A # #4 N A A #3 A R N N N h s hR hR e s 2 s bl 22 shRNA 22#2 shR ram IM22 IM IM IM2 c R kDa S TR TR T TR 75 TRIM22 63

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o l MANUSCRIPT 2 20 20 0 0 0 Scramble TRIM22 Scramble TRIM22 Scramble TRIM22 shRNA shRNA shRNA shRNA shRNA shRNA

Figure S8: TRIM22 shRNA Knockdown.

A) To demonstrate the degree of knockdown present in cells expressing TRIM22 shRNA, HEK293T cells were transiently transfected with control (scramble) or various TRIM22 shRNA clones for 48 hours. Western blotting analysis was performed using anti-TRIM22 antibodies with β-actin staining as a loading control.

B) Knockdown of TRIM22 reduces mRNA level of genes downstream of the NOD2. mRNA of TNF α, IL-6, andACCEPTED ISG15 were assessed by qPCR in HT29 cells stably transduced with control (scramble) or TRIM22 shRNA following stimulation by MDP (10 µg/mL) or respiratory syncytial virus (RSV) (0.1 g). (*p<0.001 after Bonferroni post-hoc testing).

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Figure S9: TRIM22 Variants Abrogates NOD2 Signaling in Patient 2 with Coiled-coil Variants.

Representative FACS blots and quantificationMANUSCRIPT of TNFα production in monocytes following stimulation with NOD2 and TLR4 ligand. Monocytes obtained from Patient 2 did not generate TNF α production in monocytes following stimulation with NOD2 and TLR4 ligand. Monocytes obtained from patient in response to L18-MDP stimulation but demonstrate normal reactivity to LPS. The patient’s father and healthy donors are shown as controls. Monocytes are gated for single, live, CD14+, HLA-DR+ cells. ∆TNF (%) is determined by subtracting frequency of TNF- positive monocytes in stimulated from unstimulated conditions. Overnight travel of blood sample from Patient 2 and father of Patient 2 caused high baseline TNF without affecting results calculated as ∆TNF (%). Grey background indicates normal range. Similar results with patient 2 were obtained in two independent experiments performed ten months apart. Open symbols L18- MDP stimulation, closed symbols LPS stimulation. Similar results were not obtained from Patient 1 and Patient 3 was not tested as she did not consent to further testing.

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Figure S10: TRIM22 Directly Mediates the Polyubiquitination of NOD2 but not MAVS or RIPK2.

A) Wildtype TRIM22 enhances polyubiquitination of NOD2 in HEK293 cells. HEK293 cells were transiently co-transfected with HA-NOD2, GFP-ubiquitin, and either wildtype (WT) or variant (R150T, S244L, R321L or R442C) FLAG-TRIM22. Following cell lysis, NOD2 was immunoprecipitated using anti-HAMANUSCRIPT antibody bound to Protein G-Sepharose and NOD2 ubiquitination was observed by GFP immunoblott ing. Co-transfection with WT-TRIM22 increased NOD2 ubiquitination that was lost in cells transfected with the TRIM22 variants.

B) TRIM22 does not mediate the ubiquitination of MAVS or RIPK2 . To determine if TRIM22 also regulates downstream NOD2 signaling modulators, HEK23T cells were co-transfected with FLAG-TRIM22, GFP-ubiquitin, and either HA-MAVS or HA-RIPK2. Twenty-four hours post-transfection, cells were lysed followed by immunoprecipitation of MAVS or RIPK2 using anti-HA-agarose. Immunoblotting for GFP-ubiquitin demonstrated no significant increase compared to empty TRIM22 vector controls. All experiments were carried out in triplicate independent experiments and blots are representative.

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A tor B Control TRIM22 KD vec T K C 0 4L 1 2 15 4 32 44 FLAG-NOD2: + ++ + + + V5-TRIM22: Empty WT R S2 R R HA-Ub(K48): + ++ + + + FLAG-NOD2: ++ + + + + HA-Ub(K48): + ++ + + + MDP (10µg/mL): 0 1 3 0 1 3 hours kDa 245 kDa 180 245 IP: FLAG (NOD2) 135 IB: HA (Ub) 180 IP: FLAG (NOD2) IB: HA (Ub) 100 135

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180 IP: FLAG (NOD2) 135 IB: HA (Ub) MANUSCRIPT 100 75 TRIM22 63 lysate FLAG-NOD2 100

Figure S11: TRIM22 Does Not Mediate K48-linked Polyubiquitination of NOD2.

A) TRIM22 does not mediate K48-specific polyubiquitination of NOD2. HEK293T cells were transiently co-transfected with FLAG-NOD2, HA-(K48-specific) ubiquitin, and either wildtype (WT) or variant (R150T, S244L, R321K, or R442C) V5-TRIM22. Twenty- four hours post-transfection, cells were lysed and NOD2 was immunoprecipitation with anti- FLAG-agarose. Immunoblotting for HA-tagged ubiquitin demonstrated no significant increase in cells transfected with WT-TRIM22 compared to empty vectors controls. Likewise, TRIM22 variants did not show significant K48-ubiquitin staining. ACCEPTED B, C) TRIM22-shRNA does not reduce MDP and RSV induced K48-linked polyubiquitination of NOD2. HEK293T cells stably transduced with control (scramble) or TRIM22 shRNA were transfected with a HA-tagged K48-specific ubiquitin construct and FLAG-NOD2. Forty-eight hours post transfection, cell lysates were stimulated with MDP (B) or RSV (C) for the indicated time points followed by cell lysis and immunoprecipitation of NOD2 by anti-FLAG-agarose. In both control

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and TRIM22 knockdown cells, stimulation did not result in increased NOD2 K48-specific poly- ubiquitination. Western blotting experiments were carried out in triplicate, independent experiments.

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