Exome sequencing analysis reveals variants in primary immunodeficiency genes in patients with very early onset inflammatory bowel disease

Abstract

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: CVID; Common Variable Immune Deficiency; IBD; Inherited Defects; Innate and Adaptive Immunity.

Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Preliminary functional analysis of immune cells in the IL-10RA variant patient via punch biopsies from terminal ileum and perhiperal blood. Cells were analyzed intracellularly for cytokines and transcription factors and analyzed on a flow cytometer. (A) Suggests a trend towards decreased expression of FoxP3 in CD4+ T cells and (B) suggest a trend toward an increase in pro-inflammatory cytokine expression in gated (live, CD3+, CD4+ T cells) in both compartments of the IL-10RA 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 were then stimulated with recombinant IL-10, and pSTAT3 (Y705) was analyzed by flow cytometry. (D) Macrophages-derived from blood were rested overnight, stimulated with LPS in the presence of absence of rIL-10, and analyzed for TNF production by flow cytometry.

Figure 2

B Cells were identified by physical characteristics and CD19+. Subsets were identified by additional antibodies: IgM, IgD, CD27, CD38, CD21

Figure 3

Analysis of (A) B cell function (CD19%), (B) memory B cells (CD27+ / IgM+), (C) switch memory cells (CD27+/IgM−), (D) naïve ransition cells (CD27−IgM+), (E) CD21lo/CD38lo, and (F) Plasmablasts (CD38hi/IgM) in subjects with variants in MSH5, CD19 as well as variant in LRBA, also in CVID pathway. Error bars represent the standard error.