IL-10R polymorphisms are associated with very-early-onset ulcerative colitis

Abstract

Background: Interleukin-10 (IL-10) signaling genes are attractive inflammatory bowel disease (IBD) candidate genes as IL-10 restricts intestinal inflammation, IL-10 polymorphisms have been associated with IBD in genome-wide association studies, and mutations in IL-10 and IL-10 receptor (IL-10R) genes have been reported in immunodeficient children with severe infantile-onset IBD. Our objective was to determine if IL-10R polymorphisms were associated with early-onset IBD (EO-IBD) and very-early-onset IBD (VEO-IBD).

Methods: Candidate-gene analysis of IL10RA and IL10RB was performed after initial sequencing of an infantile onset-IBD patient identified a novel homozygous mutation. The discovery cohort included 188 EO-IBD subjects and 188 healthy subjects. Polymorphisms associated with IBD in the discovery cohort were genotyped in an independent validation cohort of 422 EO-IBD subjects and 480 healthy subjects.

Results: We identified a homozygous, splice-site point mutation in IL10RA in an infantile-onset IBD patient causing a premature stop codon (P206X) and IL-10 insensitivity. IL10RA and IL10RB sequencing in the discovery cohort identified five IL10RA polymorphisms associated with ulcerative colitis (UC) and two IL10RB polymorphisms associated with Crohn's disease (CD). Of these polymorphisms, two IL10RA single nucleotide polymorphisms, rs2228054 and rs2228055, were associated with VEO-UC in the discovery cohort and replicated in an independent validation cohort (odds ratio [OR] 3.08, combined P = 2 x 10(-4); and OR 2.93, P = 6 x 10(-4), respectively).

Conclusions: We identified IL10RA polymorphisms that confer risk for developing VEO-UC. Additionally, we identified the first splice site mutation in IL10RA resulting in infantile-onset IBD. This study expands the phenotype of IL10RA polymorphisms to include both severe arthritis and VEO-UC.

Figure 1. Clinical Features of Patient with Severe, Infantile-Onset IBD

The patient had skin folliculitis (A), perianal disease with fistulae (B), joint effusions (C), and colonic erosions (D). Histology showed colitis with gland branching and mixed lamina propria cell infiltrate with fibropurulent exudate (arrow) (E).

Figure 2. Identification of IL10RA g.IVS5+2T>C Mutation in Genomic DNA

(A) Sequencing of Exon 5 of IL10RA revealed a homozygous g.IVS5+2T>C mutation (Bottom), compared to wild-type (Top). The splice donor site, putative cryptic splice donor site, and T>C mutation are underlined. (B) RT-PCR of IL10RA (Exon 4-7) from RNA isolated from blood and ileum. Lane 1 and Lane 1-ileum is from the patient. Lanes 2-6 are controls. Right: RT-PCR of Exon 4-7 of IL10RA from RNA from the patient and her parents. (C) Sequence of abnormal PCR product shows 76bp deletion (c.690_765del) leading to a premature stop codon (underlined).

Figure 3. Altered IL-10 Signaling in Patient with IL-10RA Mutation

(A) Truncated IL10RA protein product found in the patient compared to full-length IL10RA in parents/controls. Extracellular (ECD), intracellular (ICD), signaling peptide (SP), and transmembrane (TM) domains are shown. (B) Western blot analysis of IL-10-induced STAT3 phosphorylation in PBMCs from patient and control. (C) Flow cytometric analysis of peripheral blood FoxP3⁺ Tregs of patient and mother. Cells depicted are CD4+ in small lymphocyte gate and are plotted CD25 vs. intracellular FoxP3.