We showed that genetic products coded in loci strongly associated with MS risk substantially interact with each other. Both direct and indirect interactions were significantly higher than what would be expected by chance only. When the PPI analysis was extended to suggestive SNPs, we found an increased number of total proteins participating in the network and direct interactions ( and ). The only parameter that did not reach significance was the number of indirect interactions. This finding could be explained by the possible lack of real MS association among several suggestive SNPs.
However, including suggestive SNPs in the PPI analysis increased the number of genes to prioritize from 22 to 43. Interestingly, more than half of these genes (n
23) were located within suggestive regions and many of them directly interacted with proteins coded within significant regions (e.g. CSF2RB-CBLB
, see Table S2
). Taken together the suggestive statistical evidence of genetic association and the functional evidence of protein-protein interaction support the hypothesis that these genes could play an important role in the pathogenesis of MS.
We validated our results looking at tissue specific expression of these candidate genes. Using the BioGPS database we were able to show that the suggestively associated genes identified by DAPPLE were largely and specifically expressed in immune cells as compared to other tissues. A gene ontology analysis also confirmed the immune-related functions of these genes. More generally, these findings provide additional support to the immunological nature of MS 
. Notably, candidate gene expression was particularly high among CD8+ and CD4+ T cells, B cells, NK cells and pDCs. Interestingly all these cell types have been implicated in the pathogenesis of MS.
Several immune specific genes are located within MS suggestive regions. For example a SNP located near the gene encoding the Spleen Tyrosine Kinase (SYK
) was found suggestive of association in Sawcer et al. Notably SYK
was particularly highly expressed in B-cells, DCs, monocytes, CD33+ myeloid cells and NK cells. This protein has a central role in adaptive immune receptor signalling by phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) 
. SYK mediated ITAMs phosphorylation determines activation of signalling intermediates such as NF-κB, JNK and PYK2 that ultimately lead to lymphocyte activation 
. ITAM signals mediated by SYK can also induce expansion of NK cells 
. Interestingly, the SYK-inhibitor R788 (fostamatinib) has beneficial effects in patients affected by RA, when compared to placebo 
is another gene particularly highly expressed in B-cells, DCs, monocytes, CD33+ myeloid cells and NK cells. It codes for the β-subunit (βc) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3 and IL-5 receptors that are expressed by peripheral leucocytes and blood DCs 
. This gene appears to play an important role in allergic inflammation 
. Interestingly, associations between CSF2RB
and schizophrenia 
and bipolar disorder 
have been recently found.
encodes the Eukaryotic Translation Initiation Factor 4E Binding Protein 2. The members of this family of proteins (4EBPs) can inhibit translation initiation through binding eIF4E 
. 4EBPs regulate cell proliferation by interaction with mTORC1 pathway 
. In addiction, EIF4EBP1
knock-out mice showed a type I IFN over production in pDCs 
. We found an over-expression of EIF4BP2
in pDCs, CD4 cells, CD8 cells and NK cells. CHST12
encodes the carbohydrate (chondroitin 4-O) sulfotransferase 2, a protein located in the membrane of the Golgi apparatus membrane and which is implicated in chondroitin and dermatan sulphate (DS) synthesis in different tissues 
. DS proteoglycans participate in various biological events such as extracellular matrix assembly, cell adhesion, migration and proliferation 
. We found high expression of CHST12
in pDCs, CD4 cells, CD8 cells and NK cells.
To conclude, a number of proteins coded by genes located within MS-associated genomic regions are implicated in the same PPI networks. The extent of this interaction substantially increases when genomic regions with suggestive evidence of association are included in the analysis. This suggests that at least some of these suggestive GWAS hits represent truly associated loci, and thus more common variants remain to be found to be associated to MS. Finally, we further confirmed the immunological nature of MS and show how a single cell type cannot explain the complexity of this disease. Future functional studies should investigate how and in which cell types the suggestive candidate genes are acting. This will improve our knowledge of this complex disease and hopefully provide future strategies of disease prevention and treatment.