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Exome Sequencing Analysis Reveals Variants in Primary Immunodeficiency Genes in Patients With Very Early Onset Inflammatory Bowel Disease

      Background & Aims

      Very early onset inflammatory bowel disease (VEO-IBD), IBD diagnosed at 5 years of age or younger, frequently presents with a different and more severe phenotype than older-onset IBD. We investigated whether patients with VEO-IBD carry rare or novel variants in genes associated with immunodeficiencies that might contribute to disease development.

      Methods

      Patients with VEO-IBD and parents (when available) were recruited from the Children’s Hospital of Philadelphia from March 2013 through July 2014. We analyzed DNA from 125 patients with VEO-IBD (age, 3 wk to 4 y) and 19 parents, 4 of whom also had IBD. Exome capture was performed by Agilent SureSelect V4, and sequencing was performed using the Illumina HiSeq platform. Alignment to human genome GRCh37 was achieved followed by postprocessing and variant calling. After functional annotation, candidate variants were analyzed for change in protein function, minor allele frequency less than 0.1%, and scaled combined annotation-dependent depletion scores of 10 or less. We focused on genes associated with primary immunodeficiencies and related pathways. An additional 210 exome samples from patients with pediatric IBD (n = 45) or adult-onset Crohn’s disease (n = 20) and healthy individuals (controls, n = 145) were obtained from the University of Kiel, Germany, and used as control groups.

      Results

      Four hundred genes and regions associated with primary immunodeficiency, covering approximately 6500 coding exons totaling more than 1 Mbp of coding sequence, were selected from the whole-exome data. Our analysis showed novel and rare variants within these genes that could contribute to the development of VEO-IBD, including rare heterozygous missense variants in IL10RA and previously unidentified variants in MSH5 and CD19.

      Conclusions

      In an exome sequence analysis of patients with VEO-IBD and their parents, we identified variants in genes that regulate B- and T-cell functions and could contribute to pathogenesis. Our analysis could lead to the identification of previously unidentified IBD-associated variants.

      Keywords

      Abbreviations used in this paper:

      CADD (combined annotation dependent depletion), CVID (common variable immunodeficiency), EVS (exome variant server), ExAC (Exome Aggregation Consortium), GWAS (genome-wide association studies), IBD (inflammatory bowel disease), IL (interleukin), PBMC (peripheral blood mononuclear cell), SNP (single-nucleotide polymorphism), Th (T helper), TNF (tumor necrosis factor), VEO-IBD (very early onset inflammatory bowel disease), WES (whole-exome sequencing)
      Inflammatory bowel disease (IBD) is a multigenic and environmentally triggered disease resulting in a dysregulated immune response. The genomic contribution of IBD has been evaluated extensively through genome-wide association studies (GWAS), and more than 163 IBD-associated risk loci
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      Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.
      have been identified. However, these studies were performed primarily in adult-onset IBD and in children 10 years of age and older and thus did not include children with very early-onset IBD (VEO-IBD), diagnosed at younger than 5 years of age. Furthermore, GWAS often do not capture rare variants, specifically those with a minor allele frequency less than 5%. VEO-IBD is a heterogeneous disease with different degrees of disease severity, including some children who have a relatively mild disease course.
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      Incidence, outcomes, and health services burden of very early onset inflammatory bowel disease.
      However, a subset of patients with VEO-IBD present with a distinct phenotype, including extensive colonic involvement and more severe disease than older children and adults. In addition, because of poor response to conventional therapies, severity of inflammation, and greater duration of disease, there are higher rates of morbidity in this population.
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      IBD and IBD mimicking enterocolitis in children younger than 2 years of age.
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      There is currently no standard of care in the evaluation and treatment for this population. Although IBD is a complex disease involving an environmental trigger, it is thought that the host genetics play a more prominent role in this young population. In both mice
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      and human beings, single-gene defects are known to be associated with severe IBD phenotypes.
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      Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.
      For example, several interleukin 10 (IL10)
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      and IL10-receptor
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      Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.
      gene mutations have been associated with a phenotype of severe perianal disease and colitis in patients with VEO-IBD, particularly in infants.
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      Defective IL10 signaling defining a subgroup of patients with inflammatory bowel disease.
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      IL-10R polymorphisms are associated with very-early-onset ulcerative colitis.
      In general, single-gene defects are hypothesized to be enriched in the VEO-IBD population.
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      Defective IL10 signaling defining a subgroup of patients with inflammatory bowel disease.
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      In addition to IL10 defects, additional underlying immunodeficiency or genetic disorders have been associated with VEO-IBD.
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      These include common variable immunodeficiency (CVID); Wiskott–Aldrich syndrome; immunodysregulation, polyendocrinopathy and enteropathy, X-linked; and chronic granulomatous disease, as well as many others.
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      Inflammatory bowel disease: is it a primary immunodeficiency?.
      Identifying the driving forces in patients with particularly severe early onset disease may lead to group-specific therapeutic approaches.
      We hypothesized that rare or novel variants, including mutations in genes associated with primary immunodeficiencies, are enriched in patients with severe VEO-IBD and may contribute to the development of disease. Because of the lack of ability to detect rare variants using the GWAS approach, we used next-generation sequencing technology to study specific genes or pathways involved in this disease process. Whole-exome sequencing (WES) can sequence the protein encoding component of the genome and has revolutionized our ability to study rare variants and determine the genetic basis of disease. Here, we report our experience using WES to identify candidate causal variants in 125 patients with VEO-IBD. The focus of our analysis was on the genes associated with primary immunodeficiencies and related pathways.

      Materials and Methods

      The Institutional Review Board at The Children’s Hospital of Philadelphia approved the protocol (2002-07-2805), and all parents of patients provided written informed consent. Patients with onset of IBD at 5 years of age and younger, and, when available, their parents, were recruited. This was an unselected cohort, with a heterogeneous disease presentation and severity. Patients with a previously identified immunodeficiency were excluded from the study. All probands had a confirmed diagnosis of IBD by standard methods, including endoscopy, radiologic, laboratory, and clinical evaluation. Phenotypic classification was based on the Paris Classification (Table 1).
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      Disease activity was quantified by the Pediatric Crohn’s Disease Activity Index. Because of early age of onset of disease presentation, the majority of patients underwent an immunology evaluation for primary immunodeficiencies with a gastrointestinal presentation. The extent of this work-up varied among subjects, and is described later (Table 1). Clinical information was obtained from the electronic medical records and the following information was extracted: date and age at diagnosis, disease classification and phenotype based on the Paris Classification, disease severity, surgical history, medication history, and family history. After consent was obtained, blood samples were drawn from study subjects.
      Table 1Patient Demographics and Baseline Immunophenotyping
      n%
      Total sample size = 125.
      Demographics
       Age, y (range)(0.06–5)-
       Male6955.2
      Disease location
       Perianal2116.8
       Ileal75.6
       Ileocolonic3729.6
       Colonic8568
       Upper129.6
      Surgery and therapy
       Surgery1713.6
       Diverted1713.6
       TNF5947.2
       Immunomodulators7862.4
      Immunophenotyping
       Immunoglobulins5342.4
       Vaccine titers4636.8
       T lymphocytes4536
       B lymphocytes4132.8
       DHR5241.6
       NK cell counts4132.8
       TLR129.6
       FOXP364.8
       IL1064.8
       None5644.8
      DHR, dihydrorhodamine; NK, natural killer; TLR, Toll-like receptor; TNF, tumor necrosis factor.
      a Total sample size = 125.

       Whole-Exome Sequencing

      Exome capture was accomplished using the Agilent SureSelect V4 (Agilent Technologies, Santa Clara, CA), and sequencing was performed using the Illumina HiSeq platform (Illumina Cambridge Ltd, Cambridge, UK) at an average coverage depth of 100×. Whole-exome library preparation was based on modification of the protocol using the Agilent SureSelect Whole Exome version 4 kit (51 MB target size). Sequence read alignments were completed using Novoalign (V2.07.18) against the human reference genome GRCh37.p10 (http://www.novocraft.com). The Broad Institute’s Genome Analysis Toolkit
      • McKenna A.
      • Hanna M.
      • Banks E.
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      The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.
      best practices for variant detection were used for single-nucleotide polymorphism (SNP) and insertions/deletions calls. Annotation of the variants was performed with the use of SNPEff,
      • Cingolani P.
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      A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3.
      using publicly available information from RefSeq (hg19) and Ensembl (GRCh37.66). Variants with sequence coverage of 5 or greater and a variant quality score of 10 or greater then were loaded into the Center for Biomedical Informatic's Varify database (unpublished data). Additional annotations at the variant, exon, and gene level were obtained using information from the 1000 Genomes Project (www.1000genomes.org/), National Heart, Lung, and Blood Institute GO Exome Sequencing Project Exome Variant Server (EVS) (http://evs.gs.washington.edu/EVS/), Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org; release 0.3), Sorting Intolerant from Tolerant,
      • Ng P.C.
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      Predicting deleterious amino acid substitutions.
      PolyPhen2,
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      • Jordan D.M.
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      Predicting functional effect of human missense mutations using PolyPhen-2.
      dbSNP,
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      dbSNP: the NCBI database of genetic variation.
      HGNC,
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      Genenames.org: the HGNC resources in 2011.
      HGMD,
      • Stenson P.D.
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      Human Gene Mutation Database (HGMD): 2003 update.
      combined annotation dependent depletion (CADD),
      • Kircher M.
      • Witten D.M.
      • Jain P.
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      A general framework for estimating the relative pathogenicity of human genetic variants.
      and HPO.
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      The human phenotype ontology.
      After functional annotation, only variants likely to alter protein function, such as missense and loss-of-function mutations, were kept for subsequent analysis. Variants were filtered further to include only those with a minor allele frequency less than 0.1% in data from the 1000 Genomes Project, EVS, and ExAC. Variant frequencies also were checked against those reported in the Children’s Hospital of Philadelphia’s internal whole-exome cohort (containing >400 exomes). We prioritized our focus on a panel of known genes associated with primary immunodeficiencies and related pathways. The list of primary immunodeficiency-associated genes and regions was generated from the NCBI gene database (http://www.mcbi.nlm.nih.gov/gene/; accessed on February 23, 2014) using the terms “homo sapiens” and “primary immunodeficiency,” resulting in 400 genes and genomic regions (Supplementary Table 1). For each of these, we evaluated the number of probands that carry one or more variants and examined the parents, when available, for presence of the variant.
      To identify candidate causative alleles, we used as a further filter criterion the CADD score,
      • Kircher M.
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      • Jain P.
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      A general framework for estimating the relative pathogenicity of human genetic variants.
      and only included variants with a scaled CADD score of at least 10. The CADD system, a recently described annotation score, is unique in that it scores the deleteriousness of single-nucleotide variants as well as insertion/deletion variants through integration of multiple networks and functions.
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      A general framework for estimating the relative pathogenicity of human genetic variants.

       Control Whole-Exome Sequencing Data

      An additional 210 exome samples from pediatric IBD (n = 45), adult-onset Crohn’s disease (n = 20), and healthy controls (n = 145) were obtained from the Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Germany, and were used as control groups. Age at onset for pediatric IBD ranged from 6 to 18 years. The Illumina TruSeq exome kit was used for library preparation and sequencing was performed using the Illumina HiSeq2000. The same filtering criteria for identifying variants in primary immunodeficiency genes in our VEO-IBD data were applied to the different control groups. Variants were limited to those with the following: (1) a minor allele frequency less than 0.1% in the 1000 Genomes Project, EVS, and ExAC; (2) a minimum CADD score of 10; and (3) a high or moderate effect determined using SNPEff. The total number of variants in the primary immunodeficiency genes satisfying these conditions was calculated for each of the 3 groups. The average sample variant rate was computed by dividing the total number of variants in a group by the number of samples in that group.

       Permutation Bioinformatics Analysis

      To assess the enrichment of primary immunodeficiency genes in our VEO-IBD data, we simulated the variant data in the healthy control group, for which the sample size was larger than 125. One hundred variant matrices were simulated by randomly assigning the total number of variants observed across the samples in that group. We then randomly selected 125 samples from each of the generated matrixes, equal to the number of VEO-IBD patients in our study, repeating the sampling process 100 times for a total of 10,000 permutations for each data set. The total number of variants observed in the 125 selected samples was calculated for every iteration and a normal distribution was fit into each data set to calculate the significance of the number of variants in our cohort compared with the healthy control group.

       Sanger Sequencing

      Sanger sequencing of purified polymerase chain reaction products was performed in the Applied Biosystems 3730 DNA (Applied Biosystems, Waltham, MA) Analyzer and analyzed using Sequencher (GeneCodes, Ann Arbor, MI). Potential novel variants and rare variants identified in this cohort were validated by resequencing in the forward and reverse directions.

       IL10RA analysis

      Biopsy samples were obtained from the terminal ileum, transverse colon, and rectum during colonoscopy. Two punch biopsy specimens were obtained and the lamina propria cells were isolated after a digestion protocol.
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      Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4+ T cells.
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      Peripheral blood also was obtained and peripheral blood mononuclear cells (PBMCs) were isolated on a Ficoll gradient (Ficoll-Paque Plus; GE Healthcare, Piscataway, NJ). Single-cell suspensions then were viably cryopreserved until the day of the experiment. On the day of the experiment, cells were thawed and restimulated with 50 ng/mL phorbol myristate acetate and 750 ng/mL ionomycin in the presence of 10 ug/mL of Brefeldin A (all obtained from Sigma-Aldrich, St Louis, MO) for 4 hours. Cells then were stained intracellularly for cytokines and transcription factors and analyzed on a flow cytometer. Cells were stained with antibodies to the following markers: anti-CD3 PE-Cy7 (clone UCHT1; eBioscience, San Diego, CA), and anti-CD4 PE-Texas red (clone S3.5; Life Technologies, Carlsbad, CA). For intracellular staining, cells were fixed and permeabilized using a commercially available kit (eBioscience) and stained with anti-FoxP3 eFluor 450 (clone 236A/E7; eBioscience), anti–tumor necrosis factor (TNF)α Alexa 700 (clone Mab11; eBioscience), and anti-interferon γ fluorescein isothiocyanate (clone GZ-4; eBioscience).
      For pSTAT3 analyses, T cells were expanded from PBMCs using anti-CD3 and anti-CD28–coated Dynabeads (Thermofisher Waltham, MA) and recombinant IL2, according to the manufacturer’s protocol for 7–10 days (Invitrogen). T cells then were rested for approximately 16 hours and stimulated with 200 ng/mL recombinant IL10 (eBioscience) for 15 minutes. Cells then were stained for pSTAT3 Y705 Alexa Fluor 647 (clone 4/P-STAT3) using BD Phosphoflow buffers (eBioscience).
      For macrophage experiments, macrophages were isolated from PBMCs and stimulated with lipopolysaccharide as previously described
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      A new functional assay for the diagnosis of X-linked inhibitor of apoptosis (XIAP) deficiency.
      in the presence or absence of recombinant IL10 200 ng/mL. Cells were analyzed by intracellular cytokine staining as described earlier.

       Immunologic Assessments

      The extent of the immunology evaluation at baseline was variable among the subjects as shown in Table 1. Upon evaluation of identified variants, it was evident that many patients had variants related to lymphocyte function and we developed a screen to evaluate for evidence of lymphocyte dysfunction. We focused on B cells because low switched memory B cells are seen in many T-cell defects, B-cell defects, and myeloid defects. A research protocol was developed to analyze these subsets. In addition, many patients had clinical testing of T-cell subsets. B cells were identified by physical characteristics and CD19. B-cell subsets were identified by additional antibodies IgM, IgD, CD27, CD38, and CD21. Cells were analyzed on a LSR Fortessa (BD Biosciences) in the Children’s Hospital of Philadelphia Flow Cytometry Core. We defined transitional B cells as IgM+CD27-CD38++, naive mature B cells as CD27-CD38+, nonswitched memory B cells as CD27+IgM+, switched memory B cells as CD27+IgM-, and naive B cells as CD27-IgM+. Plasmablasts were defined as IgM- CD38++ or CD27++.

      Results

      A total of 125 patients with VEO-IBD were enrolled, including 2 sets of siblings. We also had access to DNA from 19 parents (both parents for 8 children) for this analysis. The range of age of onset of the affected children was from 3 weeks to 4 years, with the majority (21; 84%) younger than 2 years of age. Briefly, 37 (29.6%) patients had ileocolonic disease, and the remainder had primarily colonic distribution of disease. Disease activity was severe as measured by the Pediatric Crohn’s Disease Activity Index or the PUCAI in 76 patients (61%). Forty-six (37%) patients were treated with anti-TNF therapy and 76 patients were treated with an immunomodulator (61%). Fourteen patients underwent a colectomy, 15 patients underwent diverting ileostomies, and 2 patients had perianal disease requiring surgical intervention (Table 1). There was a positive family history in first-degree relatives in 13 patients (10%), and in second-degree relatives in 23 patients (18%).
      Approximately 40% of patients had testing for chronic granulomatous disease with dihydrorhodamine. A subset of patients had quantitative immunoglobulins (42%) and/or vaccine titers (37%) to identify CVID and immunoglobulin defects. In addition, B- and T-cell lymphocyte analysis was performed in 33% and 36% of the cohort, respectively, for the evaluation of severe combined immunodeficiency disease, Wiskott–Aldrich syndrome, or specific B- and T-cell defects. A specific genetic defect analysis was performed on 6% of patients (Table 1). There were no identified genetic defects in any subject before study enrollment.

       Analysis of Variants in Primary Immunodeficiency Pathways

      Four hundred genes (Supplementary Table 2) and genomic regions are associated with primary immunodeficiency, covering approximately 6500 coding exons, totaling more than 1 Mbp of coding sequence. In these regions, 86.9% of coding exons were fully covered at more than 20 times. The percentages of partially covered (between 40% and 99%) and not covered exons (<40%) were 7.4% and 5.7%, respectively. The analysis showed novel and rare putative causative variants within these genes in multiple patients as described later. Variants remained in this analysis if they were rare (minor allele frequency, <0.1%) or novel in EVS, 1000 Genomes, and ExAC. To meet criteria for potential pathogenicity, we only included variants that were predicted to be deleterious, by altering amino acids or splicing patterns, and had CADD scaled scores of at least 10. In addition, variants in HLA genes were removed because of the inherent variation in their sequence. After applying these filters to the initial 400 genes and regions associated with primary immunodeficiencies, 473 missense and 12 nonsense variants in 267 different genes fulfilled our filter criteria (Supplementary Table 1). There were 204 novel variants identified. All variants occurred in heterozygosity except for 4 homozygous variants found in 3 different patients. Of the 485 variants, 464 were detected only once each. Four variants, TINF2 Ala436Val, HIVEP3 Glu813Gln, ACOT8 Glu182Lys, and RNASEH2A Phe139Leu, were each present in 2 affected siblings. Of the remaining variants, 13 were found in 2 unrelated patients and 2 were found in 3 unrelated patients. In addition, the MUC21 Gly386Arg and the ANKS1A Gly49Ser variants were seen in 5 and 8 unrelated patients, respectively. The number of variants per patient ranged from 1 to 17. In the 8 cases in which both parental samples were available, the variants also were detected in 1 of the parents for 7 of the 8 children (all were heterozygous except 2). In the remaining case, all except for 1 variant were detected in 1 of the parents. However, reads containing the variant represented only 9% of all reads at that position and therefore the variant was likely to be a sequencing error. Of the 43 variants collectively found in these 8 patients, 5 variants in 3 children came from affected parents.
      We also considered gene combinations, in which the same 2 or more genes contained variants in multiple unrelated probands. We found 11 gene combinations (Supplementary Table 3), each harboring different variants found in 2 unrelated probands. Of the 11 combinations, 10 were gene pairs comprising variants in 2 genes and 1 was a 3-gene combination, with variants found in HIVEP3, TNXB, and GNAI1. However, the significance of these gene combinations cannot be determined without further insight into the inheritance mode of the different variants.

       Enrichment of Variants in Primary Immunodeficiency Genes in VEO-IBD

      The same filtering criteria were applied to 3 different control groups: pediatric IBD (n = 45), adult IBD (n = 20), and healthy controls (n = 145), to obtain a total count of variants in primary immunodeficiency genes in each of these cohorts (Table 2). The variant rate in our VEO-IBD cohort was 4.14 variants per child, compared with 3.69, 3.45, and 3.44 variants per sample in pediatric IBD, adult-onset IBD, and healthy controls, respectively. These results support the enrichment of variants in primary immunodeficiency genes in VEO-IBD because there were 1.12–1.20 times more variants on average per child in our cohort, compared with the older IBD onset and healthy controls.
      Table 2Summary of the Primary Immunodeficiency Variants Found in VEO-IBD and the Three Control Groups
      CohortnTotal variants, nVariant rateVEO-IBD/control
      Ratio of variant rate in VEO-IBD to controls.
      P value
      VEO-IBD1255184.14N/AN/A
      Pediatric IBD451663.691.12N/A
      Adult-onset IBD20693.451.20N/A
      Healthy controls1454993.441.20<1 × 10-4
      a Ratio of variant rate in VEO-IBD to controls.
      To further test our hypothesis, we randomly generated 100 data sets from the observed data in the healthy controls. For each iteration, we performed 100 random selections of 125 samples to calculate the expected total number of variants observed in a sample size equivalent to that of our VEO-IBD cohort. The resulting data followed a normal distribution with a mean of 430.22 (SD, ±7.74) in the healthy control data. Comparing the 518 observed variants in our cohort with the expected number of variants in the healthy control groups resulted in a P value less than 1 × 10-4, thus confirming the over-representation of variants in primary immunodeficiency genes in our cohort.

       Functional Analysis of the IL10RA Variant in Terminal Ileum Biopsy

      A known rare heterozygous IL10RA missense variant (rs143538561; Arg412Trp) was detected in a male patient born to nonconsanguineous parents, who presented during the first few months of life with diarrhea. At 15 months of life he was diagnosed with CD after undergoing endoscopy and colonoscopy. His disease was classified as ileocolonic and was characterized as inflammatory with multiple granulomas present throughout the gastrointestinal tract. By 2 years of age he developed atopic dermatitis and keratosis that was difficult to control. This patient had a normal immunology evaluation, including a dihydrorhodamine study, T-cell function analysis, immunoglobulin analysis, and appropriate vaccination titers. His intestinal disease course progressed and he proved to be refractory to multiple medical therapies, including mesalamine, antibiotics, immunomodulators, and infliximab, to which he developed an anaphylactic reaction at the second dose. This patient had a second variant in a gene of interest: IL21R. This was a novel nonsynonymous coding variant (Val341Met).
      After Sanger sequencing was used to verify variant genotypes in the proband, we sought to determine the functional effect of the IL10RA variant. Analysis of immune cells in the PBMCs and terminal ileum biopsy specimens through gating live CD4+ CD3+ T cells showed a trend toward a reduction in FoxP3+ regulatory T-cell frequency in both compartments of the patient as compared with a non-IBD healthy control and a patient with IBD (who lacked the variant) (Figure 1A).
      Figure thumbnail gr1
      Figure 1Preliminary functional analysis of immune cells in the IL10RA variant patient via punch biopsy specimens from terminal ileum and peripheral blood. Cells were analyzed intracellularly for cytokines and transcription factors and analyzed on a flow cytometer. (A) Suggests a trend toward decreased expression of FoxP3 in CD4+ T cells, (B) suggests a trend toward an increase in proinflammatory cytokine expression in gated (live, CD3+, CD4+ T cells) in both compartments of the IL10RA variant relative to a non-IBD control and a patient with IBD, but without the variant. (C) CD4 T cells were expanded and rested overnight. Cells then were stimulated with recombinant IL10, and pSTAT3 (Y705) was analyzed by flow cytometry. (D) Macrophages derived from blood were rested overnight, stimulated with lipopolysaccharide (LPS) in the presence of absence of recombinant IL (rIL)10, and analyzed for tumor necrosis factor (TNF) production by flow cytometry. MHC, major histocompatibility complex; PBS, phosphate-buffered saline.
      Consistent with a potential reduction in FoxP3+ regulatory T cells, these analyses also showed that there was an increase in proinflammatory cytokine production from CD4+ T cells as measured by an increased frequency of TNFα+ and interferon γ+ CD4+ T cells in the patient with the IL10RA variant as compared with a non-IBD healthy control and a patient with IBD, but without the variant (Figure 1B). Further analysis showed that expanded T cells from all patients can phosphorylate STAT3 in response to IL10 stimulation in vitro (Figure 1C), suggesting that it is not a complete loss of function of the IL10R. However, IL10 was less efficient at reducing TNF production from lipopolysaccharide-stimulated, blood-derived macrophages in the patient with the IL10R variant relative to control patients (Figure 1D). These findings suggest that in the heterozygous state, there may not be a complete loss of function, however, there are still potential consequences on IL10–IL10R function, which is associated with reduced regulatory T cells, increased proinflammatory cytokine response, and impaired regulation of macrophages. These studies are consistent with the recently identified role for macrophage-intrinsic IL10R in regulating intestinal homeostasis.
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      Additional analyses will be required to determine the exact effects of the variant on immune cell function.

       Analysis of Variants in CVID-Associated Genes MSH5 and CD19

      The genes CD19 and MSH5 were identified previously to be involved in the CVID pathway, a disease whose main phenotype is loss of B-cell function and humoral immunity.
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      • Cunningham-Rundles C.
      Perspectives on common variable immune deficiency.
      A rare nonsynonymous variant in the MSH5 gene (rs370037482; Ser537Thr) was detected in heterozygosity in a 3-year-old boy with severe ileocolonic IBD and fistulizing perianal disease. Laboratory evaluation showed low IgG and IgM levels, and levels of IgA and IgE at the low end of normal.
      A nonsynonymous novel variant (Gln345His) in CD19 was detected in 1 female patient with VEO-IBD diagnosed at the age of 2. This variant was inherited from her unaffected father. This subject had low IgG, IgM, and IgE levels.
      After Sanger sequencing confirmation of the variants, we sought to investigate the B-cell function in the patients with these variants using the B-cell assay described in Figure 2. Figure 3 shows stages of B-cell development of the patients with variants in MSH5 and CD19, also associated with CVID. There was a decrease in memory B cells (CD27+/IgM+), switch memory cells (CD27+/IgM-), and plasmablasts (CD38hi/IgM-). In addition, there was an increase in CD21lo/CD38lo, indicative of naive transition cells, often seen in a stressed state. Together, these findings suggest that in a subset of patients with VEO-IBD, there may be potential effects on the B-cell pathway, associated with B-cell differentiation functional deficits. Additional analyses will be required to determine the exact effects of these variants in the CVID pathway on immune cell function.
      Figure thumbnail gr2
      Figure 2B cells were identified by physical characteristics and CD19+. Subsets were identified by the following additional antibodies: IgM, IgD, CD27, CD38, and CD21.
      Figure thumbnail gr3
      Figure 3Analysis of (A) B-cell function (CD19%), (B) memory B cells (CD27+/IgM+), (C) switch memory cells (CD27+/IgM-), (D) naive transition cells (CD27-/IgM+), (E) CD21lo/CD38lo, and (F) plasmablasts (CD38hi/IgM) in subjects with variants in MSH5, CD19, as well as variants in LRBA, and also in the CVID pathway. Error bars represent the standard error.

      Discussion

      VEO-IBD in young children is challenging to both evaluate and treat. This likely is owing to the unique genomic and immune triggers of the disease that do not respond to conventional approaches used in older-onset IBD. WES provides a method to identify some of the underlying genetic defects and may allow for targeted therapy in these children. We performed a candidate analysis using WES on 125 patients with VEO-IBD to detect causative variants, and subsequent functional analysis on 2 primary immunodeficiency pathways in 3 subjects. This was a heterogeneous unselected cohort included in this analysis based on age of diagnosis alone. The variants we identified highlight the spectrum of immunologic defects potentially relevant in this population, including T-cell and regulatory pathways, as well as pathways involving B-cell differentiation. Of interest, the relationship between IL10R and CVID has been explored in several studies, specifically regarding the link between T-cell deficiency and subsequent B-cell function via impaired IL10.
      • Holm A.M.
      • Aukrust P.
      • Aandahl E.M.
      • et al.
      Impaired secretion of IL-10 by T cells from patients with common variable immunodeficiency–involvement of protein kinase A type I.
      Prior studies have shown monogenic defects in patients with VEO-IBD, including mutations identified in IL10,
      • Glocker E.O.
      • Kotlarz D.
      • Boztug K.
      • et al.
      Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.
      XIAP,
      • Worthey E.A.
      • Mayer A.N.
      • Syverson G.D.
      • et al.
      Making a definitive diagnosis: successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease.
      NCF2,
      • Muise A.M.
      • Xu W.
      • Guo C.H.
      • et al.
      NADPH oxidase complex and IBD candidate gene studies: identification of a rare variant in NCF2 that results in reduced binding to RAC2.
      MEFV,
      • Kuloglu Z.
      • Kansu A.
      • Ustundag G.
      • et al.
      An infant with severe refractory Crohn's disease and homozygous MEFV mutation who dramatically responded to colchicine.
      and LRBA.
      • Alangari A.
      • Alsultan A.
      • Adly N.
      • et al.
      LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency.
      IL10RA (encoding IL10R1) homozygous mutations have been associated with VEO-IBD, specifically in neonatal-onset disease with a phenotype of severe enterocolitis and perianal disease.
      • Glocker E.O.
      • Kotlarz D.
      • Boztug K.
      • et al.
      Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.
      In addition, compound heterozygote mutations of IL10RA have been reported with neonatal CD and enterocolitis.
      • Shim J.O.
      • Hwang S.
      • Yang H.R.
      • et al.
      Interleukin-10 receptor mutations in children with neonatal-onset Crohn's disease and intractable ulcerating enterocolitis.
      IL10 is an anti-inflammatory cytokine secreted by a variety of cells, including dendritic cells, natural killer cells, eosinophils, mast cells, macrophages, B cells, and CD4+ T-cell subsets (including T-helper [Th]2, Th1, Th17, and T-regulatory cells).
      • Moore K.W.
      • de Waal Malefyt R.
      • Coffman R.L.
      • et al.
      Interleukin-10 and the interleukin-10 receptor.
      IL10 maintains homeostasis through suppression of an excessive proinflammatory response.
      • Hutchins A.P.
      • Diez D.
      • Miranda-Saavedra D.
      The IL-10/STAT3-mediated anti-inflammatory response: recent developments and future challenges.
      IL10 exerts its effect through binding to the tetrametric complex IL10 receptor. It is composed of 2 distinct chains, 2 molecules of IL10R1 (α chain) and 2 molecules of IL10R2 (β chain). IL10 binding to IL10R activates the IL10/JAK1/STAT3 cascade.
      • Murray P.J.
      The primary mechanism of the IL-10-regulated antiinflammatory response is to selectively inhibit transcription.
      STAT3 activates effector genes, which subsequently suppresses the proinflammatory genes. Our patient presented here was found to have a rare variant of IL10RA. Although he had similar phenotypic findings to IL10R defects depicted in the literature, such as disease onset in infancy, severe disease, and skin rashes, notable distinctions were found, particularly the absence of fistulae and perianal disease. This novel patient illustrates that varying phenotypic expression may occur in children with VEO-IBD who have heterozygous IL10R mutations.
      CVID, a primary immunodeficiency, is a complex and heterogeneous disease, with the responsible mutations known for only a minority of cases. The association between IBD and CVID has been well recognized. In a large cohort study of 473 patients with CVID, 20 developed IBD.
      • Conley M.E.
      • Notarangelo L.D.
      • Etzioni A.
      Diagnostic criteria for primary immunodeficiencies. Representing PAGID (Pan-American Group for Immunodeficiency) and ESID (European Society for Immunodeficiencies).
      A recent study showed the importance of the CVID pathway in VEO-IBD, with the identification of an IL21 defect in 3 children in a consanguineous family. These patients had defects in B-cell development.
      • Salzer E.
      • Kansu A.
      • Sic H.
      • et al.
      Early-onset inflammatory bowel disease and common variable immunodeficiency-like disease caused by IL-21 deficiency.
      The variants detected in our subjects in genes associated with CVID included variants in CD19 and MSH5. The B-cell analysis performed in our study provides further evidence that these CVID-associated genes may be functionally relevant in VEO-IBD.
      MSH5 belongs to the MutS family of proteins, which are part of DNA mismatch repair and meiotic homologous recombination. There are 7 known eukaryote MutS homologues, and MSH5 and MSH4 form an exclusive heterocomplex. This complex has been found to play a role in DNA damage response and double-strand base repair, immunoglobulin diversity, and has been linked to neoplasia (including colorectal cancer), immune disease, and reproductive disorders.
      • Clark N.
      • Wu X.
      • Her C.
      MutS Homologues hMSH4 and hMSH5: genetic variations, functions, and implications in human diseases.
      MSH5 also has been associated with systemic lupus erythematosus,
      • Fernando M.M.
      • Freudenberg J.
      • Lee A.
      • et al.
      Transancestral mapping of the MHC region in systemic lupus erythematosus identifies new independent and interacting loci at MSH5, HLA-DPB1 and HLA-G.
      Kawasaki disease, type 1 diabetes, IgA deficiency, and CVID.
      • Clark N.
      • Wu X.
      • Her C.
      MutS Homologues hMSH4 and hMSH5: genetic variations, functions, and implications in human diseases.
      • Lougaris V.
      • Gallizzi R.
      • Vitali M.
      • et al.
      A novel compound heterozygous TACI mutation in an autosomal recessive common variable immunodeficiency (CVID) family.
      • Sekine H.
      • Ferreira R.C.
      • Pan-Hammarstrom Q.
      • et al.
      Role for Msh5 in the regulation of Ig class switch recombination.
      Its role in immunoglobulin diversity may be associated with the phenotype of VEO-IBD seen in our subject with the S554T variant, as it relates to B-cell function.
      The CD19 gene is located on the short arm of chromosome 16 (16p11.2).
      • van Zelm M.C.
      • Reisli I.
      • van der Burg M.
      • et al.
      An antibody-deficiency syndrome due to mutations in the CD19 gene.
      Similar to CD21, CD19 is a B-cell–specific antigen. It is a member of the B-cell–receptor complex together with CD21 and CD81.
      • Carter R.H.
      • Fearon D.T.
      CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes.
      • Fearon D.T.
      • Carroll M.C.
      Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex.
      B-cell development is dependent on signal transduction through this complex. The complex also acts as a link between the innate and adaptive immune systems.
      • Carter R.H.
      • Fearon D.T.
      CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes.
      • Fearon D.T.
      • Carroll M.C.
      Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex.
      Defects in CD19 show a decrease in serum Ig secretion and defective response to T-cell–dependent antigens.
      • Bacchelli C.
      • Buckridge S.
      • Thrasher A.J.
      • et al.
      Translational mini-review series on immunodeficiency: molecular defects in common variable immunodeficiency.
      • Yazdani R.
      • Hakemi M.G.
      • Sherkat R.
      • et al.
      Genetic defects and the role of helper T-cells in the pathogenesis of common variable immunodeficiency.
      Patients with CD19 mutations have been shown to have normal B-cell development; however, with the loss of CD19 signal transduction, they have a poor response to antigenic stimuli and thus are unable to have effective humoral response.
      • Bacchelli C.
      • Buckridge S.
      • Thrasher A.J.
      • et al.
      Translational mini-review series on immunodeficiency: molecular defects in common variable immunodeficiency.
      The CD19 variant in our patient may be important in the VEO-IBD phenotype owing to B-cell functional deficits.
      These studies and our findings outlined earlier show the importance of genetic overlap between disease processes. In fact, GWAS that included multiple disease processes have identified risk variants that show significant overlap among different immune-related diseases.
      • Virgin H.W.
      • Todd J.A.
      Metagenomics and personalized medicine.
      In addition, most complex disorders show a high degree of genetic heterogeneity, and this also seems to be the case in VEO-IBD. It therefore is likely that there are more pathways involved in VEO-IBD, and the outcome of these children can be improved through further study and identification of the associated variants. We identified 518 rare or novel nonsynonymous variants in primary immunodeficiency pathways in our cohort of VEO-IBD. Patient presentation varied, as did severity of disease. The phenotypic variation in this population likely is secondary not only to the great genetic heterogeneity of disease-causing variants but also to genetic modifiers of disease as well.
      We recognize several limitations of this study. Because of the limited sample size, we focused on the known primary immunodeficiency pathways because they represent strong candidate genes for VEO-IBD. However, because IBD is a complex genetic disease, it is very likely that there are variants that affect genes and pathways that are equally important in this cohort, but have not yet been evaluated. Conversely, although we attempted to use strict filter criteria, we may determine as we proceed with further functional analyses that some of the variants we identified may indeed have no effect on gene function.
      Despite these limitations, this mode of analysis allows us to begin to understand the complex genomics of VEO-IBD, and may help to identify the pathways that contribute to the disease process in this population. Although it now is understood that an immunologic evaluation in children who present with IBD at younger than 5 years of age is critical, many of the patients in this study had limited or no immune work-up before WES. As more is understood about the etiology of disease in this population a systematic approach to evaluating the disease pathogenesis has been suggested.
      • Uhlig H.H.
      • Schwerd T.
      • Koletzko S.
      • et al.
      The diagnostic approach to monogenic very early onset inflammatory bowel disease.
      As we, and others, have shown, defects in both B- and T-cell development, migration, and proliferation can drive disease in this population. Therefore, a framework to identify potential immune defects is an important part of the analysis and clinical care in these children. We therefore recommend a more complete immunologic evaluation be performed in patients with VEO-IBD, including studies depicted in Supplementary Table 3. As we begin to understand the different components of the immune system that are involved, including B- and T-cell pathways, we can begin to individualize our therapy to the specific patient.

      Supplementary Material

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