Agata Szperl

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.

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.

MEFV mutations affecting pyrin amino acid 577 cause autosomal dominant autoinflammatory disease

Objectives Autoinflammatory disorders are disorders of the innate immune system. Standard genetic testing provided no correct diagnosis in a female patient from a non-consanguineous family of British descent with a colchicine-responsive autosomal dominant periodic fever syndrome. We aimed to unravel the genetic cause of the symptoms. Methods Whole exome sequencing was used to screen for novel sequence variants, which were validated by direct Sanger sequencing. Ex vivo stimulation with peripheral blood mononuclear cells was performed to study the functional consequences of the mutation. mRNA and cytokine levels were measured by quantitative PCR and ELISA, respectively. Results Whole exome sequencing revealed a novel missense sequence variant, not seen in around 6800 controls, mapping to exon 8 of the MEFV gene (c.1730C>A; p.T577N), co-segregating perfectly with disease in this family. Other mutations at the same amino acid (c.1730C>G; p.T577S and c.1729A>T; p.T577S) were found in a family of Turkish descent, with autosomal dominant inheritance of familial Mediterranean fever (FMF)-like phenotype, and a Dutch patient, respectively. Moreover, a mutation (c.1729A>G; p.T577A) was detected in two Dutch siblings, who had episodes of inflammation of varying severity not resembling FMF. Peripheral blood mononuclear cells from one patient of the index family showed increased basal interleukin 1β mRNA levels and cytokine responses after lipopolysaccharide stimulation. Responses normalised with colchicine treatment. Conclusions Heterozygous mutations at amino acid position 577 of pyrin can induce an autosomal dominant autoinflammatory syndrome. This suggests that T577, located in front of the C-terminal B30.2/SPRY domain, is crucial for pyrin function.

Functional characterization of mutations in the myosin Vb gene associated with microvillus inclusion disease

Objectives: Microvillus inclusion disease (MVID) is a rare autosomal recessive enteropathy characterized by intractable diarrhea and malabsorption. Recently, various MYO5B gene mutations have been identified in patients with MVID. Interestingly, several patients with MVID showed only a MYO5B mutation in 1 allele (heterozygous) or no mutations in the MYO5B gene, illustrating the need to further functionally characterize the cell biological effects of the MYO5B mutations. Patients and Methods: The genomic DNA of 9 patients diagnosed as having MVID was screened for MYO5B mutations, and quantitative polymerase chain reaction and immunohistochemistry on the material of 2 patients was performed to investigate resultant cellular consequences. Results: We demonstrate for the first time that MYO5B mutations can be correlated with altered myosin Vb messenger RNA expression and with an aberrant subcellular distribution of the myosin Vb protein. Moreover, we demonstrate that the typical and myosin Vb–controlled accumulation of Rab11a- and FIP5-positive recycling endosomes in the apical cytoplasm of the cells is abolished in MVID enterocytes, which is indicative of altered myosin Vb function. Moreover, we report 8 novel MYO5B mutations in 9 patients of various ethnic backgrounds with MVID, including compound heterozygous mutations. Conclusions: Our functional analysis indicates that MYO5B mutations can be correlated with an aberrant subcellular distribution of the myosin Vb protein, and apical recycling endosomes, which, together with the additional compound heterozygous mutations, significantly strengthen the link between MYO5B and MVID.

Inflammatory bowel disease and celiac disease: overlaps in the pathology and genetics, and their potential drug targets.

Inflammatory bowel disease, which covers Crohns disease and ulcerative colitis, and celiac disease are both inflammatory diseases of the intestinal tract. In both diseases an antigen activates several inflammatory pathways, which cause extensive damage to the intestinal mucosa and lead to increased permeability of the intestinal epithelium. The causative antigen in inflammatory bowel disease is the microflora in the intestinal lumen, facilitated by an impaired innate immune system that is unable to halt the invasion of microbes into the lamina propria. These provoke T helper 1 and T helper 17 responses in Crohns disease and a T helper 2 response in ulcerative colitis. Pro-inflammatory cytokines and interleukins produced in these processes lead to impairment of tight junctions and increased permeability of the intestinal epithelial lining. In celiac disease, inflammation is caused by dietary gluten, a peptide present in wheat, barley and rye. In genetically predisposed people, gliadin peptides (derivatives of gluten) are presented on the Human Leukocyte Antigen DQ2 or DQ-8 molecules of antigen-presenting cells to T helper cells. This provokes a T helper 1 response, which leads to the production of pro-inflammatory cytokines and subsequent damage to, and increased permeability of the intestinal epithelium. We describe the details and overlaps in the pathomechanism and genetics of inflammatory bowel disease and celiac disease, and discuss potential drug targets for intervention.

Exome sequencing in a family segregating for celiac disease

Szperl AM, Ricaño‐Ponce I, Li JK, Deelen P, Kanterakis A, Plagnol V, van Dijk F, Westra HJ, Trynka G, Mulder CJ, Swertz M, Wijmenga C, Zheng H Ch. Exome sequencing in a family segregating for celiac disease.

Fine mapping of the celiac disease-associated LPP locus reveals a potential functional variant

Using the Immunochip for genotyping, we identified 39 non-human leukocyte antigen (non-HLA) loci associated to celiac disease (CeD), an immune-mediated disease with a worldwide frequency of ∼1%. The most significant non-HLA signal mapped to the intronic region of 70 kb in the LPP gene. Our aim was to fine map and identify possible functional variants in the LPP locus. We performed a meta-analysis in a cohort of 25 169 individuals from six different populations previously genotyped using Immunochip. Imputation using data from the Genome of the Netherlands and 1000 Genomes projects, followed by meta-analysis, confirmed the strong association signal on the LPP locus (rs2030519, P = 1.79 × 10−49), without any novel associations. The conditional analysis on this top SNP-indicated association to a single common haplotype. By performing haplotype analyses in each population separately, as well as in a combined group of the four populations that reach the significant threshold after correction (P < 0.008), we narrowed down the CeD-associated region from 70 to 2.8 kb (P = 1.35 × 10−44). By intersecting regulatory data from the ENCODE project, we found a functional SNP, rs4686484 (P = 3.12 × 10−49), that maps to several B-cell enhancer elements and a highly conserved region. This SNP was also predicted to change the binding motif of the transcription factors IRF4, IRF11, Nkx2.7 and Nkx2.9, suggesting its role in transcriptional regulation. We later found significantly low levels of LPP mRNA in CeD biopsies compared with controls, thus our results suggest that rs4686484 is the functional variant in this locus, while LPP expression is decreased in CeD.

Evaluation of European coeliac disease risk variants in a north Indian population.

Studies in European populations have contributed to a better understanding of the genetics of complex diseases, for example, in coeliac disease (CeD), studies of over 23 000 European samples have reported association to the HLA locus and another 39 loci. However, these associations have not been evaluated in detail in other ethnicities. We sought to better understand how disease-associated loci that have been mapped in Europeans translate to a disease risk for a population with a different ethnic background. We therefore performed a validation of European risk loci for CeD in 497 cases and 736 controls of north Indian origin. Using a dense-genotyping platform (Immunochip), we confirmed the strong association to the HLA region (rs2854275, P=8.2 × 10−49). Three loci showed suggestive association (rs4948256, P=9.3 × 10−7, rs4758538, P=8.6 × 10−5 and rs17080877, P=2.7 × 10−5). We directly replicated five previously reported European variants (P<0.05; mapping to loci harbouring FASLG/TNFSF18, SCHIP1/IL12A, PFKFB3/PRKCQ, ZMIZ1 and ICOSLG). Using a transferability test, we further confirmed association at PFKFB3/PRKCQ (rs2387397, P=2.8 × 10−4) and PTPRK/THEMIS (rs55743914, P=3.4 × 10−4). The north Indian population has a higher degree of consanguinity than Europeans and we therefore explored the role of recessively acting variants, which replicated the HLA locus (rs9271850, P=3.7 × 10−23) and suggested a role of additional four loci. To our knowledge, this is the first replication study of CeD variants in a non-European population.

Functional Polymorphism in IL12B Promoter Site Is Associated with Ulcerative Colitis

Ulcerative colitis (UC) is one of the two subtypes of inflammatory bowel disease (IBD). UC is a complex genetic disease that involves an abnormal immune response to the intestinal microflora causing ulceration of the epithelial barrier of the colon. UC symptoms include bloody diarrhea, anemia, and abdominal pain. Twin studies and clustering of IBD in families indicate the presence of genetic factors that account for the development of the disease. Genome-wide association studies (GWAS) for UC have identified 30 loci with mostly immune-related genes such as IL23R, JAK2, STAT3, and IL12B. However, these common variants explain only part of the heritability due to their low effect size (odds ratios [OR] <2). Part of the missing heritability may thus lie in rare causative variants with a larger effect size. One of the UC-associated loci is the IL12B gene, encoding the IL12p40 homodimer protein produced by monocytes and dendritic cells. IL12p40 is the common subunit of two interleukins: IL12 and IL23. IL12 is responsible for T helper (Th) 1 differentiation and induces the production of interferon-c (IFNc), whereas IL23 is responsible for stabilizing the Th17 phenotype by promoting IL-17 production. A common GWAS associated SNP (rs10045431) in IL12B was not associated to UC in a Dutch population. Hence, we hypothesized that other variants in the IL12B gene that were not detected in the replication study due to their low frequency (rare variants) or simply because they were not tagged by the replication single nucleotide polymorphism (SNP) (due to low linkage disequilibrium, LD) might be causative for UC. As the expression of cytokines is likely to be regulated by variants in their regulatory and coding sequence, we genotyped a functional IL12B promoter variant previously associated with two other autoimmune diseases, systemic lupus erythematosus (SLE) and asthma. Further, to search for rare variants, we sequenced the coding part (including splice-sites) of the IL12B gene in 350 UC cases of Dutch origin. Table 1 shows the genotype distribution, allele frequency, and statistics of the IL12B promoter polymorphism, an insertion/deletion of 4 basepairs (rs41292470, previously reported as rs17860508), in both a Dutch and a Finnish cohort and in a combined analysis. We found moderate association of the GC allele with UC in Dutch (OR 1⁄4 0.86; 95% confidence interval [CI]: 0.76–1.31, P 1⁄4 0.025), whereas in the Finish population we observed a borderline significant association in the same direction (Table 1). The signal became more significant in a combined analysis (OR 1⁄4 0.85; 95% CI: 0.76–0.95, P 1⁄4 0.003). In the recessive model of inheritance the GC/GC genotype was associated with UC in Dutch (OR 1⁄4 0.77; 95% CI: 0.63–0.93, P 1⁄4 0.008) and combined analysis (OR 1⁄4 0.79; 95% CI: 0.67–0.94, P 1⁄4 0.008). The promoter variant did not appear to be in LD with the previously reported GWAS SNP (pairwise LD statistics (PLINK v1.07, r 0.037 and D0 0.285), this may explain why this association signal was not identified in our earlier replication study. Different genotypes of the IL12B promoter polymorphism have been linked with a broad spectrum of phenotypes. The CTCTAA/CTCTAA insertion genotype has been associated with increased mortality in children with cerebral malaria. Recently, the GC/GC genotype was associated with susceptibility for SLE in a Bulgarian cohort, whereas the heterozygous genotype has been correlated with severity of asthma in children. These studies and the present one indicate that genetic variation in IL12B plays an important role in the immune response in many diseases and the genotype of the promoter variant may influence different phenotypes. The IL12B promoter genotype has also been associated with differences in IL12 production by stimulated dendritic cells and higher transcriptional activity of IL12B. In contrast to previous studies, we did not see any correlation between the IL12B genotype and IL12 peripheral blood mRNA expression in 350 Dutch controls (Supporting Fig. 1). In this new study we used unstimulated cells, so it is possible that the IL12b mRNA levels were not high enough to identify differential expression. We cannot exclude the possibility of measuring different isoforms from the one measured in previous studies. This might happen due to different platforms used for the mRNA quantification. In search of causative rare variants for UC, we sequenced the coding parts, including splice sites, of IL12B in 350 Dutch UC cases. We identified four known SNPs (two intronic and two nonsynonymous, all reported in the dbSNP database) and two heterozygous, unknown variants (one nonsense and one splice-site) (Supporting Fig. 2). We genotyped all the variants in our Dutch UC cohort and controls and found very low frequencies of the variants exclusively in UC cases (Supporting Fig. 2). To investigate if there is a significant enrichment of rare variants in the UC Dutch cohort, we performed a pooled association test (Fisher’s exact test) for rare, potentially functional polymorphisms: the two Additional supporting information may be found in the online version of this article. Supported by a grant from the Netherlands Organization for Scientific Research (NWO-VICI grant 918.66.620 to C.W.) and by EU Marie Curie Excellence Grant (MEXT-CT-2005-025270. R.W. is supported by a clinical fellow grant (90700281)

OR7-002 – Pyrin 577 mutations in dominant autoinflammation

Autoinflammatory disorders are disorders of the innate immune system. Standard genetic testing provided no correct diagnosis in a female patient from a non-consanguineous family of British descent with a colchicine-responsive autosomal dominant periodic fever syndrome.