Gene-centric association mapping of chromosome 3p implicates MST1 in IBD pathogenesis

Abstract

Association mapping and candidate gene studies within inflammatory bowel diseases (IBD) linkage regions, as well as genome-wide association studies in Crohn's disease (CD) have led to the discovery of multiple risk genes, but these explain only a fraction of the genetic susceptibility observed in IBD. We have thus been pursuing a region on chromosome 3p21-22 showing linkage to CD and ulcerative colitis (UC) using a gene-centric association mapping approach. We identified 12 functional candidate genes by searching for literature cocitations with relevant keywords and for gene expression patterns consistent with immune/intestinal function. We then performed an association study composed of a screening phase, where tagging single nucleotide polymorphisms (SNPs) were evaluated in 1,020 IBD patients, and an independent replication phase in 745 IBD patients. These analyses identified and replicated significant association with IBD for four SNPs within a 1.2 Mb linkage disequilibrium region. We then identified a non-synonymous coding variant (rs3197999, R689C) in the macrophage-stimulating 1 (MST1) gene (P-value 3.62 x 10(-6)) that accounts for the association signal, and shows association with both CD and UC. MST1 encodes macrophage-stimulating protein (MSP), a protein regulating the innate immune responses to bacterial ligands. R689C is predicted to interfere with MSP binding to its receptor, suggesting a role for this gene in the pathogenesis of IBD.

Figure 1. Association signal and linkage disequilibrium patterns across the 1.2 Mb region of LD

A candidate-gene approach to association mapping of the linked region on chromosome 3p21-22 suggests a putative association signal located in a 1.2 Mb region of LD containing over 30 genes. The LD plot of pairwise D′ values, derived from HapMap project data, indicates strong LD across the region (lower panel). Loci in the region tagged by the original study design are indicated on the gene track. The associated region of 336 Kb, as defined by the correlation neighborhood at r2>0.8, along with the replicated SNPs in the region are mapped against the gene track. SNPs in bold were identified in this study, the MST1 coding SNP is boxed, and the reported WTCCC SNP (rs9858542, in BSN) is in normal type.

Figure 2. Sequence conservation of the arginine residue at position 689 of MSP across different species

Amino acid sequence is shown as a single letter code (uppercase). The amino acid modified by the associated coding variant at position 689 (R689C) is in bold and demonstrates conservation of the arginine across mammalian species.

Figure 3. Three-dimensional representation of MSP

(A) Ribbon diagram and (B) molecular surface representations of the MSP β chain. A merged superimposed image of the two representations is shown in the inset. The amino acid residues discussed in the text are highlighted in strong colors and labeled. The residues (Y661, Q522 and Q568) corresponding to the ‘catalytic triad’ of bona fide serine proteinases are colored red. The cluster of arginine residues (R689, R687 and R683) located within a positively charged patch on the L13 loop that forms a continuous region with the S1 specificity pocket, generating the putative primary (high-affinity) binding surface for its receptor, are colored blue. The two cysteine residues (C657 on loop L11 and C685 on loop L13) connected by a disulfide bond that lies in close proximity to the cluster of arginine residues, are colored green. The side-chains of the highlighted residues are displayed in the ribbon diagram as sticks. The structural representations were constructed with PyMOL (DeLano 2002) and the data was obtained from the Protein Data Bank (pdb: 2ASU) containing 3-D structure coordinates from X-ray crystallographic analysis of 225 residues of the MSP β chain, 4 residues of the α chain and 154 water molecules. Reference DeLano WL (2002). The PyMOL molecular graphics system. DeLano Scientific, Palo Alto, CA, USA.