Analisi genetica e caratterizzazione funzionale del geneCBL-Bassociato alla predisposizione nella sclerosi multipla

Aim:Multiple Sclerosis (MS) is a multifactorial autoimmune neurodegenerative disease with high frequency in Sardinia compared to the nearest European lands. Recently our research group identified variants inCBL-Bgene associated with MS in Sardinians1.We have subsequently refined the initial association results interrogating an ultra-dense sequencing-based map and found that rs9657904, the lead variant reported in along with an indel (rs147983726) in very strong linkage disequilibrium with it, are indeed the most associated variants in the region.CBL-B is a E3 ubiquitin ligase that plays a key role in the regulation of lymphocyte activity down regulating T and B cell-receptor activation.Methods:Hence, to understand the functional consequence of the disease association, we evaluated expression levels of differentCBL-Bisoforms by RT-qPCR in T and B-cells from volunteers homozygous for protective CC and predisposing TT genotypes of the top associated SNP (rs9657904) which perfectly predict the genotypes also at rs147983726. Cells were stimulated with specific inducers at different concentrations and time-points. We also assessed the effect of this SNP on the activation of T and B-cells in ELISA, monitoring respectively, IL-2 production and NF-kB activation in naïve and stimulated cells.Results:As described in mice where the lack ofCbl-bincreases the risk of experimental autoimmune encephalomyelitis2, we observed a small but significant decreased expression of the full length CBL-B isoform in T cells with the TT MS-risk genotype. No conclusive difference in isoform expression was instead detected in B-cells. No conclusive evidence about differences in IL-2 production and NF-kB activation was observed.Conclusion:Although many aspects about the specific role of risk variants at rs9657904 and rs147983726 and their downstream consequences remain unclear, the results suggest that one of these variants act finely at down regulatingCBL-Bexpression. To increase the resolving power of our functional studies we have planned to increase the sample size and to carry out studies in specific subpopulations of T and B cells to evaluate where these variants exert primarily their function.