Common polygenic variation in coeliac disease and confirmation of ZNF335 and NIFA as disease susceptibility loci.

Coeliac disease (CD) is a chronic immune-mediated disease triggered by the ingestion of gluten. It has an estimated prevalence of approximately 1% in European populations. Specific HLA-DQA1 and HLA-DQB1 alleles are established coeliac susceptibility genes and are required for the presentation of gliadin to the immune system resulting in damage to the intestinal mucosa. In the largest association analysis of CD to date, 39 non-HLA risk loci were identified, 13 of which were new, in a sample of 12,014 individuals with CD and 12 228 controls using the Immunochip genotyping platform. Including the HLA, this brings the total number of known CD loci to 40. We have replicated this study in an independent Irish CD case-control population of 425 CD and 453 controls using the Immunochip platform. Using a binomial sign test, we show that the direction of the effects of previously described risk alleles were highly correlated with those reported in the Irish population, (P=2.2 × 10(-16)). Using the Polygene Risk Score (PRS) approach, we estimated that up to 35% of the genetic variance could be explained by loci present on the Immunochip (P=9 × 10(-75)). When this is limited to non-HLA loci, we explain a maximum of 4.5% of the genetic variance (P=3.6 × 10(-18)). Finally, we performed a meta-analysis of our data with the previous reports, identifying two further loci harbouring the ZNF335 and NIFA genes which now exceed genome-wide significance, taking the total number of CD susceptibility loci to 42.

Quality Control Procedures for High-Throughput Genetic Association Studies.

Genome-wide association (GWA) studies provide an unbiased approach to discovering the role of genetic determinants of disease across the human genome. The case-control design, the most frequently used GWA study design employed to date, compares allele frequencies in affected patients to those of unaffected controls. Several large-scale GWA studies have identified numerous risk variants for celiac disease (CD). However, due to their low marker density, the early GWA arrays failed to adequately capture much of the genetic variance associated with CD. The Immunochip, a custom Illumina Infinium high-density array containing 196,524 common and rare polymorphisms, was developed to allow deep replication and fine mapping of the previously established GWA significant loci identified in 12 major autoimmune and inflammatory diseases, including CD. It has the advantage of allowing uniform sets of genetic markers to be compared across all diseases. This chapter describes the methods used to perform Immunochip genotyping and the bioinformatics steps necessary for quality control and analysis of the resulting data.

Transcriptome Analysis of CD4+ T Cells in Coeliac Disease Reveals Imprint of BACH2 and IFNγ Regulation.

Genetic studies have to date identified 43 genome wide significant coeliac disease susceptibility (CD) loci comprising over 70 candidate genes. However, how altered regulation of such disease associated genes contributes to CD pathogenesis remains to be elucidated. Recently there has been considerable emphasis on characterising cell type specific and stimulus dependent genetic variants. Therefore in this study we used RNA sequencing to profile over 70 transcriptomes of CD4+ T cells, a cell type crucial for CD pathogenesis, in both stimulated and resting samples from individuals with CD and unaffected controls. We identified extensive transcriptional changes across all conditions, with the previously established CD gene IFNy the most strongly up-regulated gene (log2 fold change 4.6; P(adjusted) = 2.40x10(-11)) in CD4+ T cells from CD patients compared to controls. We show a significant correlation of differentially expressed genes with genetic studies of the disease to date (P(adjusted) = 0.002), and 21 CD candidate susceptibility genes are differentially expressed under one or more of the conditions used in this study. Pathway analysis revealed significant enrichment of immune related processes. Co-expression network analysis identified several modules of coordinately expressed CD genes. Two modules were particularly highly enriched for differentially expressed genes (P<2.2x10(-16)) and highlighted IFNy and the genetically associated transcription factor BACH2 which showed significantly reduced expression in coeliac samples (log2FC -1.75; P(adjusted) = 3.6x10(-3)) as key regulatory genes in CD. Genes regulated by BACH2 were very significantly over-represented among our differentially expressed genes (P<2.2x10(-16)) indicating that reduced expression of this master regulator of T cell differentiation promotes a pro-inflammatory response and strongly corroborates genetic evidence that BACH2 plays an important role in CD pathogenesis.