Expression analysis in intestinal mucosa reveals complex relations among genes under the association peaks in celiac disease.

Celiac disease is a chronic immune-mediated disorder with an important genetic component. To date, there are 57 independent association signals from 39 non-HLA loci, and a total of 66 candidate genes have been proposed. We aimed to scrutinize the functional implication of 45 of those genes by analyzing their expression in the disease tissue of celiac patients (at diagnosis/treatment) compared with non-celiac controls. Moreover, we investigated the SNP genotype effect in gene expression and performed coexpression analyses. Several genes showed differential expression among disease groups, most of them related to immune response. Multiple trans-eQTLs but only four cis-eQTLs were found, and surprisingly the genotype effect seems to be stimulus dependent as it differs among groups. Coexpression levels vary from higher to lower levels in active patients at diagnosis, treated patients and non-celiac controls respectively. A subset of 18 genes tightly correlated in both groups of patients but not in controls was identified. Interestingly, this subset of genes was influenced by the genotype of three SNPs. One of the SNPs, rs1018326 on chromosome two is on top of a known lincRNA whose function is not yet described, and whose expression seems to be upregulated in active disease when comparing biopsy pairs from the same individuals. Our results strongly suggest that the effects of disease-associated SNPs go far beyond the oversimplistic idea of transcriptional control at a nearby locus. Further investigations are needed to determine how each variant disrupts fine-tuning mechanisms in the genome that eventually lead to disease.

Methylation of the nonhomologous end joining repair pathway genes does not explain the increase of translocations with aging.

Chromosome translocations are especially frequent in human lymphomas and leukemias but are insufficient to drive carcinogenesis. Indeed, several of the so-called tumor specific translocations have been detected in peripheral blood of healthy individuals, finding a higher frequency of some of them with aging. The inappropriate repair of DNA double strand breaks by the nonhomologous end joining (NHEJ) pathway is one of the reasons for a translocation to occur. Moreover, fidelity of this pathway has been shown to decline with age. Although the mechanism underlying this inefficacy is unknown, other repair pathways are inactivated by methylation with aging. In this study, we analyzed the implication of NHEJ genes methylation in the increase of translocations with the age. To this aim, we determined the relationship between translocations and aging in 565 Spanish healthy individuals and correlated these data with the methylation status of 11 NHEJ genes. We found higher frequency of BCL2-JH and BCR-ABL (major) translocations with aging. In addition, we detected that two NHEJ genes (LIG4 and XRCC6) presented age-dependent promoter methylation changes. However, we did not observe a correlation between the increase of translocations and methylation, indicating that other molecular mechanisms are involved in the loss of NHEJ fidelity with aging.

A trichobezoar in a child with undiagnosed celiac disease: a case report.

Celiac disease is a chronic, immune-mediated enteropathy caused by a permanent sensitivity to ingested gluten cereals that develops in genetically susceptible individuals. The classic presentation of celiac disease includes symptoms of malabsorption but has long been associated with cognitive, emotional, and behavioral disorders. We describe an 8-year-old patient with non-scarring alopecia and diagnosed with trichotillomania. Furthermore, she presented with a 3-year history of poor appetite and two or three annual episodes of mushy, fatty stools. Laboratory investigations showed a normal hemoglobin concentration and a low ferritin level. Serologic studies showed an elevated tissue immunoglobulin G anti-tissue transglutaminase level. A duodenal biopsy showed subtotal villous atrophy and crypt hyperplasia, and a large gastric trichobezoar was found in the stomach. Immediately after beginning a gluten-free diet, complete relief of trichotillomania and trichophagia was achieved. In this report, we describe a behavioral disorder as a primary phenomenon of celiac disease, irrespective of nutritional status.

Coregulation and modulation of NFκB-related genes in celiac disease: uncovered aspects of gut mucosal inflammation.

It is known that the NFκB route is constitutively upregulated in celiac disease (CD), an immune-mediated disorder of the gut caused by intolerance to ingested gluten. Our aim was to scrutinize the expression patterns of several of the most biologically relevant components of the NFκB route in intestinal biopsies from active and treated patients and after in vitro gliadin challenge, and to assess normalization of the expression using an inhibitor of the MALT1 paracaspase. The expression of 93 NFκB genes was measured by RT-PCR in a set of uncultured active and treated CD and control biopsies, and in cultured biopsy series challenged with gliadin, the NFκB modulator, both compounds and none. Methylation of eight genes involved in NFκB signaling was analyzed by conventional pyrosequencing. Groups were compared and Pearson's correlation matrixes were constructed to check for coexpression and co-methylation. Our results confirm the upregulation of the NFκB pathway and show that constitutively altered genes usually belong to the core of the pathway and have central roles, whereas genes overexpressed only in active CD are more peripheral. Additionally, this is the first work to detect methylation level changes in celiac intestinal mucosa. Coexpression is very common in controls, whereas gliadin challenge and especially chronic inflammation present in untreated CD result in the disruption of the regulatory equilibrium. In contrast, co-methylation occurs more often in active CD. Importantly, NFκB modulation partially restores coregulation, opening the door to future therapeutic possibilities and targets.

Angiogenesis-related gene expression analysis in celiac disease.

Celiac disease (CD) involves disturbance of the small-bowel mucosal vascular network, and transglutaminase autoantibodies (TGA) have been related to angiogenesis disturbance, a complex phenomenon probably also influenced by common genetic variants in angiogenesis-related genes. A set of genes with "angiogenesis" GO term identified in a previous expression microarray experiment (SCG2, STAB1, TGFA, ANG, ERBB2, GNA13, PML, CASP8, ECGF1, JAG1, HIF1A, TNFSF13 and TGM2) was selected for genetic and functional studies. SNPs that showed a trend for association with CD in the first GWAS were genotyped in 555 patients and 541 controls. Gene expression of all genes was quantified in 15 pairs of intestinal biopsies (diagnosis vs. GFD) and in three-dimensional HUVEC and T84 cell cultures incubated with TGA-positive and negative serum. A regulatory SNP in TNFSF13 (rs11552708) is associated with CD (p = 0.01, OR = 0.7). Expression changes in biopsies pointed to TGM2 and PML as up-regulated antiangiogenic genes and to GNA13, TGFA, ERBB2 and SCG2 as down-regulated proangiogenic factors in CD. TGA seem to enhance TGM2 expression in both cell models, but PML expression was induced only in T84 enterocytes while GNA13 and ERBB2 were repressed in HUVEC endothelial cells, with several genes showing discordant effects in each model, highlighting the complexity of gene interactions in the pathogenesis of CD. Finally, cell culture models are useful tools to help dissect complex responses observed in human explants.

Upregulation of KIR3DL1 gene expression in intestinal mucosa in active celiac disease.

Killer cell immunoglobulin-like receptors (KIRs) modulate natural killer (NK) and T-cell function by human leukocyte antigen class I interaction and have been implicated in celiac disease (CD). Qualitative expression of 16 KIR genes was determined in biopsies from 22 CD patients at diagnosis and after >2 years on a gluten-free diet (GFD). Quantitative expression analysis of KIR2DL4, KIR3DL1, KIR3DL3, and KLRC2 (a marker of an NK-reprogrammed T-cell subpopulation augmented in CD) was performed in 35 additional CD biopsy pairs and 14 non-CD control biopsies. No specific KIR expression profile was observed in CD. KIR3DL1 was more frequently expressed in active CD compared with GFD (p = 0.0312) and controls (p = 0.0008), with slightly increased levels in active disease. KLRC2 was overexpressed in active (p = 0.0037) and GFD (p = 0.0469) patients compared with non-CD controls and coexpressed with KIR3DL1. Results suggest the participation of KIR3DL1 overexpression in the overall immune activation seen in CD mucosa, which could be partly explained by the NK-like T-cell subpopulation increase.

A regulatory single nucleotide polymorphism in the ubiquitin D gene associated with celiac disease.

An aberrant immune response triggered by dietary gluten is the main driving force underlying celiac disease (CD), but other biologic pathways that are dysregulated also participate in disease development. Genetic variation within these pathways might influence expression, contributing to susceptibility to CD. We have investigated the implication of ubiquitin D (UBD), a member of the ubiquitin-proteasome system that is strongly upregulated in the intestinal mucosa of active CD. Reverse transcriptase-polymerase chain reaction analysis of intestinal biopsy sample pairs (at diagnosis vs treated) from 30 CD patients confirmed overexpression of UBD in active disease tissue (fold change = 8.3; p = 0.0022). In silico prediction tools identified rs11724 as a putative regulatory single nucleotide polymorphism and association analysis of 468 CD patients and 459 controls revealed that the minor rs11724*C allele was more frequent among patients (minor allele frequency = 0.44 vs 0.39; odds ratio [OR] = 1.23; p = 0.028) and suggested a dominant allele effect (OR = 1.49; p = 0.0045). Correlation of the rs11724 genotype and UBD mRNA levels (OR = 0.76; p = 0.0021) further supports its implication in disease development.

Chromosome instability in lymphocytes of children with coeliac disease.

Undiagnosed individuals with celiac disease (CD) or those who do not comply with gluten-free diet (GFD) are at a higher risk of developing malignancies. A possible origin of chromosomal alteration in autoimmune reaction could be mistakes in the rearrangement of V(D)J of the IgH gene. Our aim was to verify whether higher genomic instability was found in coeliac individuals and whether GFD reduced it. As marker of genomic instability we analysed the frequency of 2 translocations, t(14;18) and t(11;14), in peripheral blood by nested PCR, in 37 patients with CD at diagnosis, 27 patients with CD after 2 years on GFD, and 36 control individuals. No significant differences were found.

Combined functional and positional gene information for the identification of susceptibility variants in celiac disease.

BACKGROUND & AIMS: Celiac disease is a complex, immune-mediated disorder of the intestinal mucosa with a strong genetic component. HLA-DQ2 is the major determinant of risk, but other minor genes, still to be identified, also are involved. METHODS: We designed a strategy that combines gene expression profiling of intestinal biopsy specimens, linkage region information, and different bioinformatics tools for the selection of potentially regulatory single-nucleotide polymorphisms (SNPs) involved in the disease. We selected 361 SNPs from 71 genes that fulfilled stringent functional (changes in expression level) and positional criteria (located in regions that have been linked to the disease, other than HLA). These polymorphisms were genotyped in 262 celiac patients and 214 controls. RESULTS: We detected strong evidence of association with several SNPs (the most significant were rs6747096, P = 2.38 x 10(-5); rs7040561, P = 6.55 x 10(-5); and rs458046, P = 1.35 x 10(-4)) that pinpoint novel candidate determinants of predisposition to the disease in previously identified linkage regions (eg, SERPINE2 in 2q33, and PBX3 or PPP6C in 9q34). CONCLUSIONS: Our study shows that the combination of function and position is a valid strategy for the genetic dissection of complex traits.

MICA response to gliadin in intestinal mucosa from celiac patients.

MHC class I chain-related gene A (MICA), a putative independent susceptibility gene in autoimmune diseases, encodes a surface protein present in epithelial cells that binds to NKG2D, an activating receptor of NK, alphabeta and gammadelta T cells, and could function as a stress-inducible activator of the innate immune response. There is no evidence of a long-term implication of MICA in the celiac autoimmune process. However, it could be that gliadin activation of MICA occurs only during the initial stages of the disease. In order to determine whether MICA is activated in response to gliadin in patients with celiac disease (CD), small intestinal mucosa biopsy samples from ten long-standing celiac patients on a gluten-free diet and from five non-celiac individuals were incubated with and without gliadin for 4 h. Total RNA was purified and MICA, IFNG and NKG2D mRNA were quantified by fluorescent real-time RT-PCR. Expression levels were calculated relative to GAPDH. MICA expression was detected in both patients and controls, but incubation with gliadin induced a strong increase in samples from the treated CD group compared with the non-CD controls (P=0.028), while no differences were observed for IFNG or NKG2D mRNA levels. The gliadin-provoked over-expression of MICA in "normalized" tissues from CD patients suggests a role for this stress-induced activator of the immune response in the early stages of organ-specific autoimmune destruction, probably preceding the onset of inflammation.

Analysis of the expression of MICA in small intestinal mucosa of patients with celiac disease.

The MHC class I chain-related A gene (MICA) is expressed in gastrointestinal epithelium and functions as an immune activation signal under stress conditions. MICA protein binds to NKG2D, a receptor of gamma delta T cells containing the TCR variable region V(delta)1, which are the most abundant subset of T cells in the intestinal epithelium. Ingested gluten in patients with celiac disease (CD) may function as a stress signal for the epithelial cells, and could enhance MICA expression on their surface. In this study, we have analyzed MICA expression in intestinal biopsy specimens from newly diagnosed and treated CD patients and controls. Quantitative RT-PCR analysis did not show differences in MICA expression among the three groups. With these results, we conclude that overexpression of MICA does not seem to play an important role in the pathogenesis of CD, at least at the time of diagnosis.