Abstract
High throughput genome wide associations studies (GWAS) are now identifying a large number of genome loci related to risk of common human disease. Each such locus presents a challenge in identifying the relevant underlying mechanism. Here we report the experimental characterization of a proposed causal single nucleotide polymorphism (SNP) in a locus related to risk of Crohn's disease and ulcerative colitis. The SNP lies in the MST1 gene encoding Macrophage Stimulating Protein (MSP), and results in an R689C amino acid substitution within the β-chain of MSP (MSPβ). MSP binding to the RON receptor tyrosine kinase activates signaling pathways involved in the inflammatory response. We have purified wild-type and mutant MSPβ proteins and compared biochemical and biophysical properties that might impact the MSP/RON signaling pathway. Surface plasmon resonance (SPR) binding studies showed that MSPβ R689C affinity to RON is approximately 10-fold lower than that of the wild-type MSPβ and differential scanning fluorimetry (DSF) showed that the thermal stability of the mutant MSPβ was slightly lower than that of wild-type MSPβ, by 1.6 K. The substitution was found not to impair the specific Arg483-Val484 peptide bond cleavage by matriptase-1, required for MSP activation, and mass spectrometry of tryptic fragments of the mutated protein showed that the free thiol introduced by the R689C mutation did not form an aberrant disulfide bond. Together, the studies indicate that the missense SNP impairs MSP function by reducing its affinity to RON and perhaps through a secondary effect on in vivo concentration arising from reduced thermodynamic stability, resulting in down-regulation of the MSP/RON signaling pathway.
Highlights
Until recently, information on which variants within the human genome contribute to increased risk of common human disease was fragmentary and often statistically weak
For any given marker associated with increased disease risk, each of the single nucleotide polymorphism (SNP) in linkage disequilibrium (LD) is a candidate for involvement in the disease mechanism
The analyses show that about one third of all loci have at least one missense SNP predicted to have a large impact on protein function, and these represent possible molecular mechanisms for those loci
Summary
Information on which variants within the human genome contribute to increased risk of common human disease was fragmentary and often statistically weak. The current generation of GWAS typically includes several thousand individuals with the disease of interest and a similar number of control individuals without the disease (for example [1]). These studies, and more recently, meta-studies combining data from a number of individual experiments [2,3] have already led to identification of up to a 100 risk associated loci for some individual diseases. The range of LD varies widely across the genome, and the number of candidate SNPs does from approximately 100 to of the order of 10,000 in the highly linked MHC region of chromosome 6 (Pal and Moult, unpublished)
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