Genome-wide association study identifies variants associated with autoimmune hepatitis type 1.

BACKGROUND & AIMS: Autoimmune hepatitis (AIH) is an uncommon autoimmune liver disease of unknown etiology. We used a genome-wide approach to identify genetic variants that predispose individuals to AIH. METHODS: We performed a genome-wide association study of 649 adults in The Netherlands with AIH type 1 and 13,436 controls. Initial associations were further analyzed in an independent replication panel comprising 451 patients with AIH type 1 in Germany and 4103 controls. We also performed an association analysis in the discovery cohort using imputed genotypes of the major histocompatibility complex region. RESULTS: We associated AIH with a variant in the major histocompatibility complex region at rs2187668 (P = 1.5 × 10(-78)). Analysis of this variant in the discovery cohort identified HLA-DRB1*0301 (P = 5.3 × 10(-49)) as a primary susceptibility genotype and HLA-DRB1*0401 (P = 2.8 × 10(-18)) as a secondary susceptibility genotype. We also associated AIH with variants of SH2B3 (rs3184504, 12q24; P = 7.7 × 10(-8)) and CARD10 (rs6000782, 22q13.1; P = 3.0 × 10(-6)). In addition, strong inflation of association signal was found with single-nucleotide polymorphisms associated with other immune-mediated diseases, including primary sclerosing cholangitis and primary biliary cirrhosis, but not with single-nucleotide polymorphisms associated with other genetic traits. CONCLUSIONS: In a genome-wide association study, we associated AIH type 1 with variants in the major histocompatibility complex region, and identified variants of SH2B3and CARD10 as likely risk factors. These findings support a complex genetic basis for AIH pathogenesis and indicate that part of the genetic susceptibility overlaps with that for other immune-mediated liver diseases.

Cutting edge: a variant of the IL-23R gene associated with inflammatory bowel disease induces loss of microRNA regulation and enhanced protein production.

IL-23R gene variants have been identified as risk factors for two major inflammatory bowel diseases (IBDs), Crohn's disease and ulcerative colitis, but how they contribute to disease is poorly understood. In this study, we show that the rs10889677 variant in the 3'-untranslated region of the IL-23R gene displays enhanced levels of both mRNA and protein production of IL-23R. This can be attributed to a loss of binding capacity for the microRNAs (miRNAs) Let-7e and Let-7f by the variant allele. Indeed, inhibition and overexpression of these miRNAs influenced the expression of the wild type but not the variant allele. Our data clearly demonstrate a role for miRNA-mediated dysregulation of IL-23R signaling, correlated with a single nucleotide polymorphism in the IL-23R strongly associated with IBD susceptibility. This implies that this mutation, in combination with other genetic risk factors, can lead to disease through sustained IL-23R signaling, contributing to the chronicity of IBD.

Cytotoxic T lymphocyte antigen-4 +49A/G polymorphism does not affect susceptibility to autoimmune hepatitis.

BACKGROUND & AIMS: Single nucleotide polymorphisms (SNP) in the Cytotoxic T lymphocyte antigen-4 gene (CTLA-4) have been associated with several autoimmune diseases including autoimmune Hepatitis (AIH). In this chronic idiopathic inflammatory liver disease, conflicting results have been reported on the association with a SNP at position +49 in the CTLA-4 gene in small patient cohorts. Here, we established the role of this SNP in a sufficiently large cohort of AIH patients. METHODS: The study population consisted of 672 AIH patients derived from academic and regional hospitals in the Netherlands and was compared with 500 controls selected from the 'Genome of the Netherlands' project cohort. Genotype frequencies were assessed by PCR for patients and by whole genome sequencing for controls. RESULTS: No significant differences in allele frequencies were found between patients and controls (G Allele: 40% vs 39%, P = 0.7). Similarly, no significant differences in genotype frequencies between patients and controls were found. Finally, there was no relation between disease activity and the G allele or AG and GG genotypes. CONCLUSION: The Cytotoxic T Lymphocyte Antigen-4 +49 A/G polymorphism does not represent a major susceptibility risk allele for AIH in Caucasians and is not associated with disease severity at presentation.

A polymorphism in the coding region of Il12b promotes IL-12p70 and IL-23 heterodimer formation.

IL-12 and IL-23 are heterodimeric cytokines involved in the induction of Th1 and Th17 immune responses. Previous work indicated that a region on chromosome 11 encoding the IL-12p40 subunit regulates strain differences in susceptibility to murine trinitrobenzene sulfonic acid-induced colitis. In addition, this region determines strain differences in LPS-induced IL-12 responses. In this study, we investigated how polymorphisms in the coding region of murine Il12b influence IL-12 and IL-23 heterodimer formation. Transfection studies using constructs containing IL-12p35 linked to IL-12p40 from the colitis-resistant C57BL/6 strain or to the polymorphic p40 variant from the colitis-susceptible SJL/J strain demonstrated that SJL/J-derived p40 constructs synthesized significantly more IL-12p70 than did constructs harboring the C57BL/6-p40 variant. This could not be attributed to differences in synthesis rate or secretion, implicating a greater affinity of SJL/J-derived IL-12p40 for its IL-12p35 subunit. This greater affinity is also associated with increased IL-23 synthesis. In addition, C57BL/6 mice transgenic for the SJL/J 40 variant synthesized significantly more IL-12p70 upon LPS challenge and were more prone to develop colonic inflammation than did C57BL/6 mice transgenic for the C57BL/6-p40 variant. The more efficient binding of the polymorphic Il12b variant to p35 and p19 is most likely due to conformational changes following differential glycosylation as a consequence of the polymorphism. The high synthesis rate of the mature cytokines resulting from this efficient binding can lead to rapid proinflammatory skewing of immune responses and distortion of the homeostatic balance underlying the greater susceptibility for colitis.

Celiac disease associated SNP rs17810546 is located in a gene silencing region.

GWAS studies have identified variant rs 17810546 in a non-coding region on chromosome 3 as a risk factor for several auto-immune diseases, including Celiac Disease. In silico analysis reveals that this variant is located in a transcription regulatory site. By means of reporter constructs we show that this region can override the expression rate of a gene as determined by its native promoter and that this modulation is influenced by the genetic composition of the haplotype which rs17810546 forms with a nearby other variant, rs761008. Secondly, we present data that this genetically imprinted modulation could be involved in Celiac Disease through the IL12A gene which is located 40 Kb downstream of this regulatory region. Based on our findings it is most likely that the IL12A gene does so as part of the cytokine IL-35.

Dendritic Cell Migration to Skin-Draining Lymph Nodes Is Controlled by Dermatan Sulfate and Determines Adaptive Immunity Magnitude.

For full activation of naïve adaptive lymphocytes in skin-draining lymph nodes (LNs), presentation of peptide:MHC complexes by LN-resident and skin-derived dendritic cells (DCs) that encountered antigens (Ags) is an absolute prerequisite. To get to the nearest draining LN upon intradermal immunization, DCs need to migrate from the infection site to the afferent lymphatics, which can only be reached by traversing a collagen-dense network located in the dermis of the skin through the activity of proteolytic enzymes. Here, we show that mice with altered collagen fibrillogenesis resulting in thicker collagen fibers in the skin display a reduced DC migration to the draining LN upon immune challenge. Consequently, the initiation of the cellular and humoral immune response was diminished. Ag-specific CD8+ and CD4+ T cells as well as Ag-specific germinal center B cells and serum immunoglobulin levels were significantly decreased. Hence, we postulate that alterations to the production of extracellular matrix, as seen in various connective tissue disorders, may in the end affect the qualitative outcome of adaptive immunity.

Acute experimental colitis and human chronic inflammatory diseases share expression of inflammation-related genes with conserved Ets2 binding sites.

BACKGROUND: Ulcerative colitis (UC) and Crohn's disease (CD) are characterized by chronic inflammation of the gastrointestinal tract, with overlapping clinical characteristics and unknown etiology. We reasoned that in intestinal inflammation the initial activation of the innate immune response fails to resolve, finally resulting in uncontrolled chronic inflammatory bowel disease. METHODS: To identify the early inflammatory events in colitis that remain active in human chronic colitis, we analyzed the changes of the colonic transcriptome during acute experimental colitis and compared the outcome with previously published profiles of affected tissues from patients with UC and CD, and as a control for intestinal inflammation in general, tissues from celiac disease patients. Rheumatoid arthritis synovial tissues were included as a nonintestinal inflammatory disease. The expression profiles of each disease were analyzed separately, in which diseased tissues were compared to unaffected tissues from the same anatomical location. RESULTS: Gene ontology analysis of significantly regulated genes revealed a marked activation of immunity and defense processes in all diseases, except celiac disease, where immune activation is less prominent. The control region of upregulated genes contained an increase in Ets2 binding sites in experimental colitis, UC, and rheumatoid arthritis, and were associated with upregulated immune activity. In contrast, upregulated genes in celiac disease harbored the transcription factor binding site GLI, which binds to the Gli family of transcription factors involved in hedgehog signaling, affecting development and morphogenesis. CONCLUSION: Ets2 may be an important transcription factor driving inflammation in acute as well as chronic inflammatory disease

Increased expression of the tight junction molecule claudin-18 A1 in both experimental colitis and ulcerative colitis.

BACKGROUND: We previously identified a major quantitative trait locus (qtl) on mouse chromosome 9 (Tnbs1) that confers resistance/susceptibility to trinitrobenzene sulfonic acid (TNBS) induced colitis. Here we wanted to identify possible candidate genes in this locus. METHODS: We applied micro-array technology and identified claudin-18 as a plausible candidate gene in the Tnbs1 region. Subsequently we studied the expression profile of this gene by means of RT-PCR in resistant and susceptible mice as well as in human inflammatory bowel disease. RESULTS: Expression of this gene was markedly upregulated during colitis in mice. Also in humans relative expression of claudin-18 in patients with ulcerative colitis was significantly upregulated as compared to healthy individuals undergoing surveillance endoscopy (n = 13, P < 0.0005). Expression was not related to the histological severity of the disease. CONCLUSIONS: Claudins belong to the integral membrane proteins of the tight junction, a structure that seals off the intercellular space between adjacent epithelial cells and regulates passive diffusion of solutes and macromolecules. This study demonstrates for the first time that claudin-18 is expressed in human and mouse colon. Expression is upregulated during experimental colitis and in patients with ulcerative colitis. The observation that this is unrelated to the severity of inflammation might point to a primary defect in regulation in patients with ulcerative colitis and warrants further genetic examination.

Positive coeliac serology in irritable bowel syndrome patients with normal duodenal biopsies: Video capsule endoscopy findings and HLA-DQ typing may affect clinical management.

OBJECTIVES: To investigate a group of IBS patients (Rome criteria) with positive coeliac serology (EMA, TTG, IgG or IgA AGA) and normal small bowel biopsies. Video capsule endoscopy (VCE) findings of the small bowell were compared with DQ-typing. METHODS: Twenty-two patients with chronic abdominal pain (with or without diarrhea) and at least one positive result of any of the coeliac serological markers (AGA, TTG, EMA) and normal duodenal biopsy were enrolled and underwent VCE. Twelve healthy volunteers with VCE served as control group. Coeliac related HLA DQ2 or DQ8 markers were determined. RESULTS: 12/ 22 (55%) patients had small bowel abnormalities with VCE. No mucosal abnormalities were recognized in the control group (p = 0.002). Inflammatory changes were classified as moderate or pronounced. Eight patients (36%) had moderate changes and four patients (18%) demonstrated pronounced changes. Only 6 of the 21 IBS patients were positive for DQ2 and/or DQ8. CONCLUSIONS: The patients in this study fulfilled the diagnostic Rome criteria for Irritable Bowel Syndrome. We suggest that patients with positive coeliac serology and normal duodenal biopsies should undergo HLA typing. In patients positive for DQ2 and/or DQ8, a VCE should be performed. Patients with mucosal abnormalities compatible with CD should be considered as a group distinct from IBS patients and could be tested with gluten challenge or treated with a gluten free diet.