Maria Teresa Bardella

Multiple common variants for celiac disease influencing immune gene expression

We performed a second-generation genome-wide association study of 4,533 individuals with celiac disease (cases) and 10,750 control subjects. We genotyped 113 selected SNPs with PGWAS < 10−4 and 18 SNPs from 14 known loci in a further 4,918 cases and 5,684 controls. Variants from 13 new regions reached genome-wide significance (Pcombined < 5 × 10−8); most contain genes with immune functions (BACH2, CCR4, CD80, CIITA-SOCS1-CLEC16A, ICOSLG and ZMIZ1), with ETS1, RUNX3, THEMIS and TNFRSF14 having key roles in thymic T-cell selection. There was evidence to suggest associations for a further 13 regions. In an expression quantitative trait meta-analysis of 1,469 whole blood samples, 20 of 38 (52.6%) tested loci had celiac risk variants correlated (P < 0.0028, FDR 5%) with cis gene expression.

Genome Search in Celiac Disease

Celiac disease (CD), a malabsorption disorder of the small intestine, results from ingestion of gluten. The HLA risk factors involved in CD are well known but do not explain the entire genetic susceptibility. To determine the localization of other genetic risk factors, a systematic screening of the genome has been undertaken. The typing information of 281 markers on 110 affected sib pairs and their parents was used to test linkage. Systematic linkage analysis was first performed on 39 pairs in which both sibs had a symptomatic form of CD. Replication of the regions of interest was then carried out on 71 pairs in which one sib had a symptomatic form and the other a silent form of CD. In addition to the HLA loci, our study suggests that a risk factor in 5qter is involved in both forms of CD (symptomatic and silent). Furthermore, a factor on 11qter possibly differentiates the two forms. In contrast, none of the regions recently published was confirmed by the present screening.

Coeliac disease-associated risk variants in TNFAIP3 and REL implicate altered NF-κB signalling

Objective: Our previous coeliac disease genome-wide association study (GWAS) implicated risk variants in the human leucocyte antigen (HLA) region and eight novel risk regions. To identify more coeliac disease loci, we selected 458 single nucleotide polymorphisms (SNPs) that showed more modest association in the GWAS for genotyping and analysis in four independent cohorts. Design: 458 SNPs were assayed in 1682 cases and 3258 controls from three populations (UK, Irish and Dutch). We combined the results with the original GWAS cohort (767 UK cases and 1422 controls); six SNPs showed association with p<1×10−04 and were then genotyped in an independent Italian coeliac cohort (538 cases and 593 controls). Results: We identified two novel coeliac disease risk regions: 6q23.3 (OLIG3-TNFAIP3) and 2p16.1 (REL), both of which reached genome-wide significance in the combined analysis of all 2987 cases and 5273 controls (rs2327832 p = 1.3×10−08, and rs842647 p = 5.2×10−07). We investigated the expression of these genes in the RNA isolated from biopsies and from whole blood RNA. We did not observe any changes in gene expression, nor in the correlation of genotype with gene expression. Conclusions: Both TNFAIP3 (A20, at the protein level) and REL are key mediators in the nuclear factor kappa B (NF-κB) inflammatory signalling pathway. For the first time, a role for primary heritable variation in this important biological pathway predisposing to coeliac disease has been identified. Currently, the HLA risk factors and the 10 established non-HLA risk factors explain ∼40% of the heritability of coeliac disease.

Evolutionary and Functional Analysis of Celiac Risk Loci Reveals SH2B3 as a Protective Factor against Bacterial Infection

Celiac disease (CD) is an intolerance to dietary proteins of wheat, barley, and rye. CD may have substantial morbidity, yet it is quite common with a prevalence of 1%-2% in Western populations. It is not clear why the CD phenotype is so prevalent despite its negative effects on human health, especially because appropriate treatment in the form of a gluten-free diet has only been available since the 1950s, when dietary gluten was discovered to be the triggering factor. The high prevalence of CD might suggest that genes underlying this disease may have been favored by the process of natural selection. We assessed signatures of selection for ten confirmed CD-associated loci in several genome-wide data sets, comprising 8154 controls from four European populations and 195 individuals from a North African population, by studying haplotype lengths via the integrated haplotype score (iHS) method. Consistent signs of positive selection for CD-associated derived alleles were observed in three loci: IL12A, IL18RAP, and SH2B3. For the SH2B3 risk allele, we also show a difference in allele frequency distribution (Fst) between HapMap phase II populations. Functional investigation of the effect of the SH2B3 genotype in response to lipopolysaccharide and muramyl dipeptide revealed that carriers of the SH2B3 rs3184504*A risk allele showed stronger activation of the NOD2 recognition pathway. This suggests that SH2B3 plays a role in protection against bacteria infection, and it provides a possible explanation for the selective sweep on SH2B3, which occurred sometime between 1200 and 1700 years ago.

Analysis of HLA and Non-HLA Alleles Can Identify Individuals at High Risk for Celiac Disease

BACKGROUND & AIMS Celiac disease (CD) is a common chronic disorder of the small intestine, resulting from aberrant cellular responses to gluten peptides, and often remains undiagnosed. It is a complex genetic disorder, although 95% of the patients carry the risk heterodimer human leukocyte antigen (HLA)-DQ2. Genome-wide association studies on CD have identified 9 non-HLA loci that also contribute to CD risk, most of which are shared with other immune-related diseases. Our aim is to predict the genetic risk for CD using HLA and non-HLA risk alleles. METHODS We selected 10 independent polymorphisms in 2,308 cases and 4,585 controls from Dutch, UK, and Irish populations and categorized the individuals into 3 risk groups, based on their HLA-DQ2 genotype. We used the summed number of non-HLA risk alleles per individual to analyze their cumulative effect on CD risk, adjusting for gender and population group in logistic regression analysis. We validated our findings in 436 Italian cases and 532 controls. RESULTS CD cases carried more non-HLA risk alleles than controls: individuals carrying > or = 13 risk alleles had a higher CD risk (odds ratio, 6.2; 95% confidence interval, 4.1-9.3) compared with those carrying 0-5 risk alleles. Combining HLA and non-HLA risk genotypes in one model increases sensitivity by 6.2% compared with using only HLA for identification of high-risk individuals with slight decrease in specificity. CONCLUSIONS We can use non-HLA risk factors for CD to improve identification of high-risk individuals. Our risk model is a first step toward better diagnosis and prognosis in high-risk families and population-based screening.

Plasma chromogranin a in patients with inflammatory bowel disease.

Background: Circulating chromogranin A (CgA) levels, a marker for neuroendocrine tumors including carcinoids, have recently been found elevated in some patients with inflammatory bowel disease (IBD), although their significance is unclear. Therefore, we aimed to evaluate CgA levels and their possible relationship with clinical and biochemical disease activity indexes in 119 IBD patients. Methods: The study groups comprised 75 patients with ulcerative colitis, 44 with Crohns disease, in both active and quiescent phases, and 85 controls. Results: Mean CgA levels were significantly higher in IBD patients than in controls (20.4 ± 14.0 [SD] versus 11.3 ± 4.3 U/L, P < 0.001), without any statistical significant difference among the IBD subgroups. However, CgA levels were above the normal range (20 U/L) in 25/45 patients with active IBD (55%; 95% confidence interval [CI]: 40%–70%) and in 18/74 patients with remission IBD (24%; 95% CI: 15%–36%) (P < 0.001, Fishers test). Among biochemical parameters, CgA correlated with serum TNF‐&agr; levels (rs = 0.398, P < 0.001). Conclusions: High CgA levels can occur in IBD. The disease activity and TNF‐&agr; levels seem to influence the CgA pattern, which could reflect the neuroendocrine system activation in response to inflammation. From a clinical point of view, the possibility of high CgA levels in IBD should be taken into consideration when a carcinoid is suspected in such patients, since this event seems to be more frequent than previously considered. Indeed, revision of our 83 patients with gastrointestinal carcinoids, studied between 1997 and 2006, showed that 4 patients had IBD, with a prevalence of 4.8%, which is markedly higher than that of the general population.

Gluten intolerance: Gender- and age-related differences in symptoms

Objective Gluten intolerance is a common, immunologically mediated disorder with a widely variable clinical presentation that affects genetically predisposed subjects. Women seem to be more frequently affected although data on sex differences are poor. In this study the prevalence of different clinical pictures according to sex and age is analysed in a large series of patients. Material and methods A total of 1436 patients with gluten intolerance were retrospectively considered, diagnosed from January 1975 to August 2001 based on compatible small-bowel biopsy and response to a gluten-free diet, plus immunofluorescent detection of granular IgA in papillary derma for dermatitis herpetiformis. The clinical picture at onset (classic, non-classic, silent) and age at diagnosis (≤2 years, >2 and ≤14 years, >14 years) was recorded; 362 parents of coeliac probands undergoing a familial screening were also studied. The relations among sex, age class and symptoms were analysed using the χ2 test with Yatess correction. Results The overall female/male ratio was 2.3:1 but the inter-sex difference was significant only when the diagnosis was made in adulthood where a significant association between iron-deficiency anaemia as manifestation at onset in adult women (34% versus 7%) was found. Low weight, dyspepsia and hypertransaminasaemia were more common in adult men than women (20%, 14% and 7% versus 13%, 3% and 2%, respectively). Dermatitis herpetiformis was present more frequently in men (16% versus 9%). The prevalence of silent cases was 6% in men and 3% in women. Familial screening showed the same prevalence (9.3%) of current coeliac disease in fathers and mothers. Conclusions Diagnosis of coeliac disease is more frequent in women but physicians’ awareness of sex- and age-related differences in clinical presentation could improve diagnostic performances in men.

Six new coeliac disease loci replicated in an Italian population confirm association with coeliac disease

Background and aims: The first genome wide association study on coeliac disease (CD) and its follow-up have identified eight new loci that contribute significantly towards CD risk. Seven of these loci contain genes controlling adaptive immune responses, including IL2/IL21 (4q27), RGS1 (1q31), IL18RAP (2q11–2q12), CCR3 (3p21), IL12A (3q25–3q26), TAGAP (6q25) and SH2B3 (12q24). Methods: We selected the nine most associated single nucleotide polymorphisms to tag the eight new loci in an Italian cohort comprising 538 CD patients and 593 healthy controls. Results: Common variation in IL2/IL21, RGS1, IL12A/SCHIP and SH2B3 was associated with susceptibility to CD in our Italian cohort. The LPP and TAGAP regions also showed moderate association, whereas there was no association with CCR3 and IL18RAP. Conclusion: This is the first replication study of six of the eight new CD loci; it is also the first CD association study in a southern European cohort. Our results may imply there is a genuine population difference across Europe regarding the loci contributing to CD.

Tissue transglutaminase antibodies in patients with end-stage heart failure

OBJECTIVE:For celiac disease (CD), screening a trend has recently emerged to measure tissue transglutaminase antibodies (tTGA) by immunoassays instead of the more laborious endomysial antibodies (EmA), as they recognize the same target, tissue transglutaminase (tTG). However, a high rate of false-positive results has been reported in some patient series with diseases known to be associated with CD. Moreover, tTG is a ubiquitous, multifunctional enzyme, overexpressed in experimental models of heart failure. Therefore, we assessed the specificity of tTGA assays in a large series of EmA-negative patients with end-stage heart failure.METHODS:We studied 288 patients with end-stage heart failure and 60 blood donors. No subject had clinical evidence of CD or IgA deficiency, and all were EmA negative. Serum IgA and IgG tTGA were measured by means of commercial kits using as substrate, either guinea pig or recombinant human tTG. Blocking studies and Western blots were also performed using recombinant human tTG.RESULTS:All blood donor sera were IgA tTGA negative. IgA tTGA positivity was observed in 47.6% and 49.1% of patients with heart failure using, respectively, guinea pig tTG and recombinant human tTG as substrates. Preincubation of positive sera with recombinant human tTG resulted in 81% blocking of IgA tTGA in immunoassay. Western blot analysis confirmed the presence of antibodies against recombinant human tTG. IgA tTGA-positive sera were also IgG tTGA positive.CONCLUSION:IgA and IgG tTGA occur in a large number of EmA-negative patients with end-stage heart failure, and their presence is unlikely to be caused by concomitant CD.

Circulating ghrelin levels in celiac patients

OBJECTIVE:Ghrelin, the gut–brain peptide, recently identified as the natural endogenous ligand for growth hormone secretagogue receptors, exerts various endocrine and nonendocrine effects, including the control of energy homeostasis and food intake, but its possible relevance in malabsorption syndromes is unknown. Therefore, the aim of this study was to evaluate circulating ghrelin levels in adults with untreated and treated celiac disease (CD) and, for comparison, in healthy subjects.METHODS:Fasting serum ghrelin levels were measured in 30 consecutive patients with newly diagnosed CD, 13 celiac patients successfully treated with a gluten-free diet (GFD), and 30 healthy controls.RESULTS:Ghrelin levels were abnormally high in patients with active CD compared with controls (297 ± 17.6 vs 218 ± 15.2 pmol/L, p < 0.01) and correlated positively with intestinal mucosal lesion severity (rs= 0.444, p < 0.02). In the successfully GFD-treated patients, ghrelin values were normal compared with controls (233 ± 22.0 vs 218 ± 15.2 pmol/L, ns) and, moreover, correlated negatively with body mass index (r=−0.632, p = 0.02), unlike in the untreated patient group (r=−0.263, ns).CONCLUSION:High ghrelin levels characterized our series of adult patients with newly diagnosed CD and correlated significantly with the degree of severity of intestinal mucosal lesions. This is the first evidence of a relationship between ghrelin and inflammatory processes, but the mechanisms involved are still unclear. Furthermore, our findings suggest that an interplay of hormonal, metabolic, and nutritional factors could influence ghrelin secretion under pathophysiological circumstances.