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.

Alteration of tight junction gene expression in celiac disease.

OBJECTIVE: The aim of the present study was to characterize the deregulation of epithelial tight junction genes and investigate its reversibility on removal of dietary gluten in small intestinal mucosa in celiac disease (CD). METHODS: The expression levels of 23 genes related to tight junctions were studied in biopsies from 16 patients with active CD and compared with biopsies from the same patients taken after 2 years on gluten-free diet (GFD) and with 16 non-CD controls. RESULTS: Nine genes showed altered expression levels in patients with active disease (CLDN2, PARD6A, ZAK, SYMPK, MYH14, and ACTB were upregulated, whereas MAGI1, TJP1, and PPP2R3A were downregulated). Alterations were reversible after 2 years on treatment, except for PPP2R3A, implicated in the negative control of cell growth and division. At the biological network level, important dysfunctions in several processes within the pathway were observed, including intestinal permeability, apicobasal polarity, and cell proliferation. CONCLUSIONS: Our work confirms the involvement of tight junction genes related to permeability, polarity, and cell proliferation in the epithelial destruction observed in CD. Coexpression patterns of several genes support the idea of a common regulatory mechanism that seems to be altered in active CD. In general, GFD normalization confirms the reversibility of the process, except for the constitutive downregulation of PPP2R3A suggestive of a genetic implication. Further studies in proteins and cells or tissues are necessary to confirm these findings.

Cow's-milk-free diet as a therapeutic option in childhood chronic constipation.

OBJECTIVES: It has been reported that a number of children with constipation respond to a diet free of cow's-milk (CM) proteins, although evidence is lacking to support an immunoglobulin E-mediated mechanism. PATIENTS AND METHODS: We performed an open-label crossover study comparing CM and rice milk in 69 children who fulfilled Rome III criteria for chronic constipation. Clinical, physical, and immunologic parameters of patients who responded (R) and who did not respond (NR) to a CM-free diet were compared. RESULTS: Thirty-five of the 69 children (51%) improved during the first CM-free diet phase, 8 of these did not develop constipation when CM was reintroduced, and 27 children (39%) developed constipation during the CM challenge and improved during the second CM-free diet phase (R group). Thirty-four children (49%) did not improve during the first CM-free diet phase (NR group). Bowel movements per week among R children significantly increased compared with NR children (R: 2.8-7.7 vs NR: 2.6-2.7) (P < 0.001). Seventy-eight percent of the children with developmental delay responded to the CM-free diet (P = 0.007). No significant statistical difference was found between the R and NR children in terms of fiber and milk consumption; atopic or allergic history; full-blood eosinophil count and percentage, and lymphocyte populations; immunoglobulins, immunoglobulin (Ig)G subclasses, total IgE; and serum-specific immunoglobulin E for CM proteins. CONCLUSIONS: A clear association between CM consumption and constipation has been found in more than one third of children. However, analytical parameters do not demonstrate an immunoglobulin E-mediated immunologic mechanism.

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.

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.

Celiac Male's Gluten-Free Diet Profile: Comparison to that of the Control Population and Celiac Women.

The aim of the present work was to analyze the body composition and dietary profile of Spanish celiac men and to compare them to control men and celiac women from our previous studies. Forty-two celiac men (31.5 ± 11.9 years) were recruited and anthropometric measurements were taken. Analysis of energy consumption, macro- and micronutrient intake and food frequency consumption was carried out. Celiac men were more overweight and obese than celiac women, but less than the control population, reporting the same energy intake and macronutrient distribution. Most micronutrient deficiencies in celiac men were not directly related to a gluten free diet; these were also observed for the entire population. The least adherence to Dietary Reference Intakes in women was reported for iron, iodine, potassium and selenium, whereas magnesium intake was higher than in men. Among celiac participants (both genders), cereal, vegetable and legume consumption was poor and meat intake was contrastingly excessive. In conclusion, the dietary profile of celiac men is as unbalanced as that of control men but slightly more than that of celiac women. General nutritional education should be given to both general and celiac populations, and specific advices to celiac men, in order to decrease the risk of celiac disease-related pathologies.

Two-year follow-up of anti-transglutaminase autoantibodies among celiac children on gluten-free diet: comparison of IgG and IgA.

OBJECTIVES: To investigate the evolution of IgA and IgG autoantibodies against tissue transglutaminase (tTGase) in celiac patients on gluten-free diet (GFD). METHODS: IgA and IgG anti-tTGAse autoantibodies was evaluated in 93 patients (58 girls and 35 boys; mean age 3.56 +/- 3.04 years; range 0.94-17.5 years) at diagnosis of celiac disease and after 1, 2, 4, 6, 12, 18, 24 months of follow-up on GFD. Autoantibodies were measured with a radioassay using in vitro transcribed-translated human recombinant tTGAse, and immune complexes were precipitated with protein A- or anti-IgA-agarose for IgG and IgA, respectively. RESULTS: Autoantibody titers started to decline very soon after removal of gluten, and no significant differences in the decrease rate between IgG and IgA antibodies were observed. After 6 months on GFD, 63 and 49% of the patients were negative for IgG and IgA, respectively. Patients who remained autoantibody-positive after 6 months of treatment initially presented with significantly higher titers at the time of diagnosis compared to patients that had lost their antibodies by that time. Children diagnosed before the age of two years presented lower autoantibody titers, while patients positive for HLA-DR7 had higher anti-tTGase levels, especially IgA. CONCLUSIONS: There are no differences in the performance of IgG and IgA class autoantibodies in the evolution of celiac patients. Between 3 and 6 months on GFD, almost half of the patients are negative for anti-tTGase antibodies. In our experience, they can be of help in evaluating compliance with diet, at least during the first two years of treatment.

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.

5'-Insulin gene VNTR polymorphism is specific for type 1 diabetes: no association with celiac or Addison's disease.

The VNTR region located at the 5'-end of the insulin gene on chromosome 11p15.5 is linked to susceptibility to type 1 diabetes mellitus (T1DM), and class I alleles have been associated with increased risk of disease, whereas class III alleles are considered to be protective. Although a potential effect on the expression level of thymic insulin and a consequent abnormal tolerance have been proposed as an explanation, it is still not clear whether the association is specific for T1DM or whether it is shared by other autoimmune disorders. To investigate the contribution of INS-VNTR to the genetic susceptibility to autoimmune disorders, we analyzed 102 autoantibody-positive T1DM patients, 59 patients with celiac disease (CD), and 57 patients with Addison's disease (ADD), as well as 111 unrelated healthy individuals from the general population. When analyzing the results, class I allele frequencies were 85.8% in the T1DM group, 77% among CD patients, 71% in the ADD group, and 76.1% in the general population. Association with increased risk was seen only in the T1DM group (pc = 0.015). Risk to T1DM was associated with the class I/class I homozygous genotype (RR, 1.92; 95% CI, 1.03-3.6). In conclusion, INS-VNTR does not seem to be involved in the susceptibility to autoimmune diseases other than T1DM and can be considered a diabetes-specific locus.

HLA-DRB1 and MICA in autoimmunity: common associated alleles in autoimmune disorders.

Autoimmune disorders such as type 1 diabetes (T1DM), celiac disease (CD), and Addison's disease (ADD) develop in individuals with genetic susceptibility that are exposed to environmental triggering factors not completely defined. Patients with an autoimmune disease (and their relatives) are at increased risk of developing another disorder, and this might be caused by a common genetic origin of autoimmunity; for example, HLA class II region in 6p21 shows a very strong association with most diseases. The aim of this study was to determine whether shared susceptibility markers extend from the central (DRB1) through the telomeric (MICA) HLA region. We analyzed three independent sets of families with one autoimmune disease, T1DM, CD, or ADD, and genotyped them for HLA-DRB1 and for the exon 5 GCT polymorphism of MICA. For HLA-DRB1, allele DRB1*0301 was the only one associated with risk for all three diseases; in the case of MICA, allele A9 was found to be the common protective allele. Haplotype analysis shows that haplotype A5.1-DRB1*0301 confers risk to autoimmunity. Our results show that there are common risk and protection alleles in both loci, suggesting a core of genetic association with autoimmunity (HLA-DRB1*0301 risk; A9 protection) that could be modulated by other alleles/loci or environmental factors toward one or another disease. Some alleles are part of conserved haplotypes (A5.1-DR3, A5.1-DR2), whereas others seem to have independent effect (A9) and support the idea of two independent loci in this region.

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.

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.

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.

Accuracy in copy number calling by qPCR and PRT: a matter of DNA.

The possible implication of copy number variation (CNV) in the genetic susceptibility to human disease needs to be assessed using robust methods that can be applied at a population scale. In this report, we analyze the performance of the two major techniques, quantitative PCR (qPCR) and paralog ratio test (PRT), and investigate the influence of input DNA amount and template integrity on the reliability of both methods. Analysis of three genes (PRELID1, SYNPO and DEFB4) in a large sample set showed that both methods are prone to false copy number assignments if sufficient attention is not paid to DNA concentration and quality. Accurate normalization of samples is essential for reproducible qPCR because it avoids the effect of differential amplification efficiencies between target and control assays, whereas PRT is generally more sensitive to template degradation due to the fact that longer amplicons are usually needed to optimize sensitivity and specificity of paralog sequence PCR. The use of normalized, high quality genomic DNA yields comparable results with both methods.

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.

Long-term and acute effects of gliadin on small intestine of patients on potentially pathogenic networks in celiac disease.

Celiac disease (CD) is a complex, immune-mediated intolerance to gliadin that develops in genetically susceptible individuals. Although the main driving force of the disease is an aberrant autoimmune response, several other pathogenic mechanisms, many still unidentified, are also involved. In order to describe at a network level the alterations provoked by a gliadin insult on the intestinal mucosa of patients, we compared the expression profiles of biopsies from 9 active and 9 treated patients (long-term effects of gliadin), and of 10 biopsies from gluten-free diet treated patients that were incubated in vitro with or without gliadin (acute effects) and integrated significantly altered transcripts into potentially pathogenic biological processes. Using information on Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology terms represented among the differentially expressed genes, we observed important dysfunction in several complex networks, including those related to cell-cell communication, intracellular signaling, ubiquitin-proteasome system, cell cycle/apoptosis and extracellular matrix. The reconstruction of the role of these biological networks in the development of the intestinal lesion in CD provides a comprehensive picture of key events that contribute to the disease, and could point towards novel functional candidates that might be potential therapeutic targets or responsible for genetic susceptibility.

Analysis of beta-defensin and Toll-like receptor gene copy number variation in celiac disease.

Celiac disease (CD) is an immune-mediated disorder of the gut in which innate and adaptive responses are involved. Toll-like receptor (TLR) 2 and TLR4 participate in host defense through antigen recognition, and show altered expression in CD gut mucosa. beta-defensins are inducible antimicrobial peptides, and DEFB gene copy number polymorphisms have been associated with autoimmune and inflammatory disorders. We performed copy number analysis of TLR2, TLR4, and the beta-defensin cluster (DEFB4, DEFB103 and DEFB104) by gene-specific, real-time polymerase chain reaction (PCR) in 376 CD patients and 376 controls. TLR genes did not show copy number variation, and all samples presented with two copies. beta-defensin clusters varied between 2 and 9 copies per genome, and when grouped into bins, high copy numbers (>4) were underrepresented among patients (p = 0.023; odds ratio = 0.69, 95% CI = 0.50-0.96), suggesting that increased copy numbers could protect from CD, possibly by impeding bacterial infiltration more efficiently and preserving gut epithelial integrity.

HLA-DQA1 and HLA-DQB1 genetic markers and clinical presentation in celiac disease.

BACKGROUND: Patients with celiac disease are diagnosed at any age and can exhibit a wide range of clinical manifestations. The reasons for this are unclear. The aim of this study was to investigate a possible correlation between the HLA-DQA1 and HLA-DQB1 genetic markers and clinical features of celiac disease. METHODS: A total of 133 patients with celiac disease were tested for the HLA-DQA1 and HLA-DQB1 genes. Their corresponding allele and haplotype frequency distributions were estimated from the phenotypes found. The results were correlated with data from the clinical records. RESULTS: The DQ2 molecule was found in 93% of the patients, and DQ2 or DQ8 was found in 98%. The DQA1*0201-DQB1*0202 haplotype showed strong linkage disequilibrium. DQ2 homozygosis was significantly associated with female sex, earlier age at diagnosis, and shorter delay between onset of symptoms and diagnosis. Double-dose DQB1*02 (01-02) allele was more frequent in patients with the classic presentation of the disease. CONCLUSIONS: The genetic markers investigated may prove useful for diagnosing and managing celiac disease. With some clinical variables, correlations not previously described were found. These correlations have a moderate strength and, therefore, must be confirmed by other studies.

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.