Antonie Zwiers

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.

Genetic variations in interleukin-12 related genes in immune-mediated diseases

The interleukin-12 (IL-12) family comprises a group of heterodimeric cytokines and their respective receptors that play key roles in immune responses. A growing number of autoimmune diseases has been found to be associated with genetic variation in these genes. Based on their respective associations with the IL-12 genes, autoimmune diseases appear to cluster in two groups that either show strong associations with the Th1/Th17 pathway (as indicated by genetic association with IL12B and IL23R) or the Th1/IL-35 pathway as the consequence of their association with polymorphisms in the IL12A gene region. The genetic associations are described in relation to what is known of the functionality of these genes in the various diseases. Comparing association data for gene families in different diseases may lead to better insight in the function of the genes in the onset and course of the disease.

Cytotoxic T Lymphocyte Antigen–4 +49A/G polymorphism does not affect susceptibility to autoimmune hepatitis

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.

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.

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

Background: Ulcerative colitis (UC) and Crohns 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.

Human pepsinogen A isozymogen patterns in serum and gastric mucosa.

The pepsinogen A isozymogen pattern in gastric mucosa is genetically determined and can be visualized in nondenaturating polyacrylamide gel electrophoresis of supernatants of sonified gastric mucosal biopsies by demonstrating proteolytic activity after converting pepsinogen into pepsin by acid. Pepsinogen isozymogens are present in very low concentrations in the blood but can now be demonstrated in serum by a newly developed immunoblotting procedure. This study investigated whether the serum pepsinogen A isozymogen pattern adequately reflects the pepsinogen A phenotype. Serum and gastric mucosal pepsinogen A isozymogen patterns were compared in 72 subjects from the routine endoscopy program. A close correlation was found between the relative intensities of the pepsinogen A isozymogens in the serum and the gastric mucosal patterns. Increasing the pepsinogen A release into the circulation by oral omeprazole did not affect the pepsinogen A patterns in the blood. It is concluded that the serum pepsinogen A pattern reflects the pepsinogen A phenotype in humans. In addition, no preferential release of a pepsinogen A isozymogen into the circulation was observed. Thus, immunoblotting of serum provides a new and reliable tool to study pepsinogen genetics in humans. Because a relationship was previously shown between specific pepsinogen A phenotypes and gastric malignancies in humans, serum pepsinogen A patterns may provide a tool to detect subjects who are at risk of gastric cancers.

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.

Immunoblot technique to visualise serum pepsinogen A isozymogen patterns.

Pepsinogen A (PGA) isozymogen patterns in urine and gastric mucosa can be visualised in non-denatured polyacrylamide gel electrophoresis by showing proteolytic activity after the conversion of pepsinogen into pepsin by acid. This method is not suitable for visualising PGA patterns in serum due to low PGA concentrations. To obtain a more sensitive visualisation method an immunoblotting technique was developed. PGA isozymogen patterns from urine and sonified gastric mucosa specimens obtained by immunoblotting were identical with those obtained by activity staining. The immunostaining method was at least 50 times more sensitive. PGA isozymogen patterns could be visualised in serum. Preliminary results suggest that the PGA patterns in serum and gastric mucosa are identical. As an association has been found between the genetically determined PGA isozymogen patterns in gastric mucosa and gastric malignancies in man, immunoblotting of PGA isozymogens in serum may provide a screening tool for subjects at risk of malignant gastric disease.

Sa1345 Increased Duodenal IL12A mRNA Expression in Celiac Disease Patients Might Lead to Increased IL-35 Production

BACKGROUND. Celiac disease (CD) is an autoimmune enteropathy that affects 1% of the general population. The association between CD and other autoimmune disorders is well established and increased prevalence of CD has been found in patients with type 1 diabetes (7%) and autoimmune thyroiditis (2%). Some reports on co-morbidity of CD and alopecia areata (AA), an autoimmune skin disorder that causes hair loss, have shown a variable hair re-growth upon institution of a gluten-free diet. No studies have addressed the association between CD and the different clinical subtypes of AA, which vary in duration and degree of hair loss. The aim of this study was to investigate the prevalence of CD across the phenotypic spectrum of AA using serological screening methods that have high sensitivity and specificity for CD diagnosis. METHODS. Sera obtained through the National Alopecia Areata Registry of the United States from 16 patients with transient AA (AAT), 24 with patchy persistent AA (AAP), 27 with total scalp hair loss (AT) and 32 with complete body hair loss (AU), were screened for CD using a commercial ELISA kit against combined antitissue transglutaminase and anti-deamidated gliadin peptide IgA and IgG antibodies (antitTG/DGP) from INOVA Diagnostics Inc. Titers >20 IU were considered positive and titers >30 IU were considered strongly positive and suggestive of CD. RESULTS. Of 99 samples (females=64, males=35), a total of 9 (9%) patients (all female) were positive for anti-tTG/ DGP antibodies (range 20.4 61.7 IU) and 4% (2 AT, 1 AU and 1 AAP) were strongly positive. By type of presentation, anti-tTG/DGP titers were elevated in 15% AT, 9% AU and 8% AAP patients but in none of AAT patients. CONCLUSIONS. These results demonstrate that AA patients have a higher prevalence of CD-associated antibodies (9%) than the general population (~3%). CD seropositivity is present in the more severe forms of AA, where hair loss is experienced for more than one year and/or results in total loss of hair from the scalp and/or body. Patients with the least severe form of the disease, transient hair loss lasting less than one year, do not appear to have an elevated risk for CD. Screening for CD is recommended in AA for early detection and institution of a gluten-free diet.