Genetic characterisation of the CARD9-locus and Inflammatory Bowel Disease

INTRODUCTION: Inflammatory Bowel Disease (IBD) are common, chronic gastrointestinal inflammatory disorders. The major forms are Crohn’s disease (CD) and ulcerative colitis (UC). The pathogenesis consists of an aberrant immune response in reaction to normal gastrointestinal flora in a genetically susceptible host. The genetic factors are increasingly uncovered. Recently, locus 9q34.3 was found to be associated with IBD. There is strong linkage disequilibrium (LD) in this ~120 kb spanning locus, which complicates the identification of the candidate gene and the causal mutation. CARD9 is a likely candidate gene, because its encoded protein is involved in the differentiation of T cells in type 17 helper T cells, which are associated with immune related diseases. In this study several steps were taken to identify a common causal mutation which explains the association with IBD. MATERIAL AND METHODS: The correlation between the risk alleles of the associated SNPs and gene expression (eQTL-effect) was investigated. The region was finemapped by testing four additional single nucleotide polymorphisms (SNPs) in 534 IBD cases and 600 controls. Exon sequencing of CARD9 was performed on 300 IBD cases and 350 controls. The found mutation in CARD9 was genotyped in 826 Dutch IBD cases and 1264 controls. Several deletion polymorphisms are described in this region; Trityper was used to identify deletion polymorphisms in this region. RESULTS: The risk alleles of the associated SNPs are correlated with higher expression of CARD9. With finemapping we were able to narrow down the region. The region with strongest LD with the associated SNPs is the region ~16 kb upstream of CARD9. The highest association signal was found in CARD9. Sequencing results identified a rare splice site mutation, which protects against UC. This mutation was replicated in the Dutch cohort. No difference was seen in expression level of CARD9 between different genotypes for the splice site mutation. Several deletion polymorphisms were identified with Trityper. These deletions have the same eQTL-effect as the associated SNPs, but were not associated with IBD. DISCUSSION: This study indicates that CARD9 is the candidate gene for association with IBD, because a mutation which protects against UC was found. However, the splice site mutation turned out not to be the causal mutation. The identified deletion polymorphisms are also not plausible as causal mutation. Extensive finemapping of the region is necessary to further narrow down the region in order to find a causal mutation.