Functional characterization of a multiple sclerosis associated variant in IL7Ra. S. G. Gregory1, G. Galarza-Muņoz2, F. B. Briggs3, L. Bergamaschi1, S. Arvai1, X. Shao4, L. F. Barcellos4, M. A. Garcia-Blanco2 1) Medicine, Duke Molecular Physiology Institute, Durham, NC; 2) Molecular Genetics and Microbiology, Duke University medical Center, Durham, NC; 3) Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH; 4) School of Public Heath, University of California Berkeley, Berkeley, CA.

   PURPOSE: We previously identified a non-synonymous SNP, rs6897932, in the interleukin 7 receptor alpha chain (IL-7R) as genetically associated with multiple sclerosis (MS). In vitro functional analysis established that the C allele of rs6897932 increases exon 6 skipping altering the ratio of membrane and soluble receptor isoforms. We hypothesized that rs6897932 influences T cell IL-7 signaling in an allele specific manner, and that the trans-acting proteins that regulate IL-7R splicing could be MS susceptibility candidates. METHODS: Tobramycin affinity chromatography was used to identify exon 6 trans-acting proteins and their impact on splicing was validated by siRNA-based screening in depleted HeLa cells. These proteins were screened for genetic association in MS case/control cohorts using IMSGC data. Finally, we assessed allele specific signaling of rs6897932 by measuring phosphorylated STAT5 in primary T cell cultures from relapsing-remitting MS patients and in HEK293 cell lines transiently transfected with C or T alleles of rs6897932. RESULTS: We established that CPSF1, PTBP1, and DDX39B regulate alternative splicing of exon 6 of IL-7R and that SNPs within DDX39B are genetically associated with MS after adjusting for population stratification, gender, and known MS risk haplotypes. Analysis of soluble IL-7R in T-cells show dosage C allele dosage effects and IL-7 signaling was reduced in the presence of soluble IL-7R in C and T transiently transfected cell lines. Phospho-STAT5 analysis in T-cells and cell lines do not show differences in allele specific IL-7 signaling.
    CONCLUSIONS: We established that it is the level of soluble IL-7R and not genotype that are important for IL-7 signaling. We therefore focused on the etiological relationship between on the trans-acting protein regulation of IL-7R exon 6 skipping and MS. Our in vivo studies have established new mechanistic relationships between three proteins that regulate IL-7R splicing. After adjusting for multiple factors we also identified DDX39B as a novel genetically associated MS candidate gene. These data emphasize the significance of mRNA splicing in the development of autoimmune disease and illustrate the importance of establishing functional pathways to identify novel MS genes that would not have been identified using traditional genetic approaches.

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