Donatella Barisani

Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease.

Using variants from the 1000 Genomes Project pilot European CEU dataset and data from additional resequencing studies, we densely genotyped 183 non-HLA risk loci previously associated with immune-mediated diseases in 12,041 individuals with celiac disease (cases) and 12,228 controls. We identified 13 new celiac disease risk loci reaching genome-wide significance, bringing the number of known loci (including the HLA locus) to 40. We found multiple independent association signals at over one-third of these loci, a finding that is attributable to a combination of common, low-frequency and rare genetic variants. Compared to previously available data such as those from HapMap3, our dense genotyping in a large sample collection provided a higher resolution of the pattern of linkage disequilibrium and suggested localization of many signals to finer scale regions. In particular, 29 of the 54 fine-mapped signals seemed to be localized to single genes and, in some instances, to gene regulatory elements. Altogether, we define the complex genetic architecture of the risk regions of and refine the risk signals for celiac disease, providing the next step toward uncovering the causal mechanisms of the disease.

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.

A meta-analysis of genome-wide association scans identifies IL18RAP, PTPN2, TAGAP, and PUS10 as shared risk loci for Crohn's disease and celiac disease

Crohns disease (CD) and celiac disease (CelD) are chronic intestinal inflammatory diseases, involving genetic and environmental factors in their pathogenesis. The two diseases can co-occur within families, and studies suggest that CelD patients have a higher risk to develop CD than the general population. These observations suggest that CD and CelD may share common genetic risk loci. Two such shared loci, IL18RAP and PTPN2, have already been identified independently in these two diseases. The aim of our study was to explicitly identify shared risk loci for these diseases by combining results from genome-wide association study (GWAS) datasets of CD and CelD. Specifically, GWAS results from CelD (768 cases, 1,422 controls) and CD (3,230 cases, 4,829 controls) were combined in a meta-analysis. Nine independent regions had nominal association p-value <1.0×10−5 in this meta-analysis and showed evidence of association to the individual diseases in the original scans (p-value <1×10−2 in CelD and <1×10−3 in CD). These include the two previously reported shared loci, IL18RAP and PTPN2, with p-values of 3.37×10−8 and 6.39×10−9, respectively, in the meta-analysis. The other seven had not been reported as shared loci and thus were tested in additional CelD (3,149 cases and 4,714 controls) and CD (1,835 cases and 1,669 controls) cohorts. Two of these loci, TAGAP and PUS10, showed significant evidence of replication (Bonferroni corrected p-values <0.0071) in the combined CelD and CD replication cohorts and were firmly established as shared risk loci of genome-wide significance, with overall combined p-values of 1.55×10−10 and 1.38×10−11 respectively. Through a meta-analysis of GWAS data from CD and CelD, we have identified four shared risk loci: PTPN2, IL18RAP, TAGAP, and PUS10. The combined analysis of the two datasets provided the power, lacking in the individual GWAS for single diseases, to detect shared loci with a relatively small effect.

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.

Hepcidin and iron-related gene expression in subjects with dysmetabolic hepatic iron overload

BACKGROUND/AIMS Many patients with hepatic iron overload do not have identifiable mutations and often present with metabolic disorders and hepatic steatosis. Since the pathophysiology of Dysmetabolic Hepatic Iron Overload (DHIO) is still obscure, the aim of this study was to evaluate, in these patients, possible alterations in iron-related molecule expression. METHODS Iron-related gene mRNA levels were determined by quantitative-PCR in liver biopsies of subjects with NAFLD without iron overload and patients with HFE-hemochromatosis, beta-thalassemia major and DHIO. Urinary hepcidin was measured by immunoblotting. RESULTS No alterations in mRNA expression of either iron transporters or exporters were found in DHIO. mRNA and urinary hepcidin levels normalized for the amount of iron overload showed a significantly lower ratio than in controls, although not as low as in hemochromatosis or beta-thalassemia. Differently from what observed in hemochromatosis, hepcidin mRNA did not correlate with urinary hepcidin. CONCLUSIONS Patients with DHIO show appropriate regulation of mRNAs encoding proteins involved in iron uptake and efflux but dysregulation of hepcidin production. The relatively elevated urinary hepcidin can explain the iron phenotype in DHIO (more macrophage iron retention and low/normal transferrin saturation).

Improving coeliac disease risk prediction by testing non-HLA variants additional to HLA variants

Background The majority of coeliac disease (CD) patients are not being properly diagnosed and therefore remain untreated, leading to a greater risk of developing CD-associated complications. The major genetic risk heterodimer, HLA-DQ2 and DQ8, is already used clinically to help exclude disease. However, approximately 40% of the population carry these alleles and the majority never develop CD. Objective We explored whether CD risk prediction can be improved by adding non-HLA-susceptible variants to common HLA testing. Design We developed an average weighted genetic risk score with 10, 26 and 57 single nucleotide polymorphisms (SNP) in 2675 cases and 2815 controls and assessed the improvement in risk prediction provided by the non-HLA SNP. Moreover, we assessed the transferability of the genetic risk model with 26 non-HLA variants to a nested case–control population (n=1709) and a prospective cohort (n=1245) and then tested how well this model predicted CD outcome for 985 independent individuals. Results Adding 57 non-HLA variants to HLA testing showed a statistically significant improvement compared to scores from models based on HLA only, HLA plus 10 SNP and HLA plus 26 SNP. With 57 non-HLA variants, the area under the receiver operator characteristic curve reached 0.854 compared to 0.823 for HLA only, and 11.1% of individuals were reclassified to a more accurate risk group. We show that the risk model with HLA plus 26 SNP is useful in independent populations. Conclusions Predicting risk with 57 additional non-HLA variants improved the identification of potential CD patients. This demonstrates a possible role for combined HLA and non-HLA genetic testing in diagnostic work for CD.

Evidence for the presence of non-celiac gluten sensitivity in patients with functional gastrointestinal symptoms: Results from a multicenter randomized double-blind placebo-controlled gluten challenge

Non-celiac gluten sensitivity (NCGS) is characterized by the onset of symptoms after eating gluten-containing food. We aimed to single out NCGS subjects among subjects with functional gastrointestinal symptoms. Patients were enrolled in a multicenter double-blind placebo-controlled trial with crossover. Symptoms and quality of life were evaluated by means of 10-cm VAS and SF36. Iron parameters, transaminases and C reactive protein (CRP) were evaluated. After a three-week-long gluten-free diet (GFD), responsive patients were randomly assigned to gluten intake (5.6 g/day) or placebo for seven days, followed by crossover. The primary endpoint was the worsening of symptoms (VAS increase ≥3 cm) during gluten ingestion compared to placebo. One hundred and forty patients were enrolled and 134 (17 males, mean age 39.1 ± 11.7 years, BMI 22.4 ± 3.8) completed the first period. A total of 101 subjects (10 males, mean age 39.3 ± 11.0 years, BMI 22.3 ± 4.0) reported a symptomatic improvement (VAS score 2.3 ± 1.2 vs. 6.5 ± 2.2 before and after GFD, p = 0.001). 98 patients underwent the gluten challenge and 28 (all females, mean age 38.9 ± 12.7 years, BMI 22.0 ± 2.9) reported a symptomatic relapse and deterioration of quality of life. No parameters were found to be statistically associated with positivity to the challenge. However, 14 patients responded to the placebo ingestion. Taking into account this finding, about 14% of patients responding to gluten withdrawal showed a symptomatic relapse during the gluten challenge. This group is suspected to have NCGS.

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.

Cost-effective HLA typing with tagging SNPs predicts celiac disease risk haplotypes in the Finnish, Hungarian, and Italian populations.

Human leukocyte antigen (HLA) genes, located on chromosome 6p21.3, have a crucial role in susceptibility to various autoimmune and inflammatory diseases, such as celiac disease and type 1 diabetes. Certain HLA heterodimers, namely DQ2 (encoded by the DQA1*05 and DQB1*02 alleles) and DQ8 (DQA1*03 and DQB1*0302), are necessary for the development of celiac disease. Traditional genotyping of HLA genes is laborious, time-consuming, and expensive. A novel HLA-genotyping method, using six HLA-tagging single-nucleotide polymorphisms (SNPs) and suitable for high-throughput approaches, was described recently. Our aim was to validate this method in the Finnish, Hungarian, and Italian populations. The six previously reported HLA-tagging SNPs were genotyped in patients with celiac disease and in healthy individuals from Finland, Hungary, and two distinct regions of Italy. The potential of this method was evaluated in analyzing how well the tag SNP results correlate with the HLA genotypes previously determined using traditional HLA-typing methods. Using the tagging SNP method, it is possible to determine the celiac disease risk haplotypes accurately in Finnish, Hungarian, and Italian populations, with specificity and sensitivity ranging from 95% to 100%. In addition, it predicts homozygosity and heterozygosity for a risk haplotype, allowing studies on genotypic risk effects. The method is transferable between populations and therefore suited for large-scale research studies and screening of celiac disease among high-risk individuals or at the population level.