Transcript specific regulation of expression influences susceptibility to multiple sclerosis.

Genome-wide association studies (GWAS) have identified over 100 loci containing single nucleotide variants (SNVs) that influence the risk of developing multiple sclerosis (MS). Most of these loci lie in non-coding regulatory regions of the genome that are active in immune cells and are therefore thought to modify risk by altering the expression of key immune genes. To explore this hypothesis we screened genes flanking MS-associated variants for evidence of allele specific expression (ASE) by quantifying the transcription of coding variants in linkage disequilibrium with MS-associated SNVs. In total, we were able to identify and successfully analyse 200 such coding variants (from 112 genes) in both CD4+ and CD8+ T cells from 106 MS patients and 105 controls. Fifty-six of these coding variants (from 43 genes) showed statistically significant evidence of ASE in one or both cell types. In the Lck interacting transmembrane adaptor 1 gene (LIME1), for example, we were able to show that in both cell types, the MS-associated variant rs2256814 increased the expression of some transcripts while simultaneously reducing the expression of other transcripts. In CD4+ cells from an additional independent set of 96 cases and 93 controls we were able to replicate the effect of this SNV on the balance of alternate LIME1 transcripts using qPCR (p = 5 × 10-24). Our data thus indicate that some of the MS-associated SNVs identified by GWAS likely exert their effects on risk by distorting the balance of alternate transcripts rather than by changing the overall level of gene expression.

Multiple sclerosis risk variants alter expression of co-stimulatory genes in B cells.

The increasing evidence supporting a role for B cells in the pathogenesis of multiple sclerosis prompted us to investigate the influence of known susceptibility variants on the surface expression of co-stimulatory molecules in these cells. Using flow cytometry we measured surface expression of CD40 and CD86 in B cells from 68 patients and 162 healthy controls that were genotyped for the multiple sclerosis associated single nucleotide polymorphisms (SNPs) rs4810485, which maps within the CD40 gene, and rs9282641, which maps within the CD86 gene. We found that carrying the risk allele rs4810485*T lowered the cell-surface expression of CD40 in all tested B cell subtypes (in total B cells P ≤ 5.10 × 10-5 in patients and ≤4.09 × 10-6 in controls), while carrying the risk allele rs9282641*G increased the expression of CD86, with this effect primarily seen in the naïve B cell subset (P = 0.048 in patients and 5.38 × 10-5 in controls). In concordance with these results, analysis of RNA expression demonstrated that the risk allele rs4810485*T resulted in lower total CD40 expression (P = 0.057) but with an increased proportion of alternative splice-forms leading to decoy receptors (P = 4.00 × 10-7). Finally, we also observed that the risk allele rs4810485*T was associated with decreased levels of interleukin-10 (P = 0.020), which is considered to have an immunoregulatory function downstream of CD40. Given the importance of these co-stimulatory molecules in determining the immune reaction that appears in response to antigen our data suggest that B cells might have an important antigen presentation and immunoregulatory role in the pathogenesis of multiple sclerosis.

Rab27a-mediated protease release regulates neutrophil recruitment by allowing uropod detachment.

Neutrophil migration is vital for immunity and precedes effector functions such as pathogen killing. Here, we report that this process is regulated by the Rab27a GTPase, a protein known to control granule exocytosis. Rab27a-deficient (Rab27a KO) neutrophils exhibit migration defects in vitro and in vivo, and live-cell microscopy suggests that delayed uropod detachment causes the migratory defect. Surface expression of CD11b, a key adhesion molecule, is increased in chemokine-stimulated Rab27a KO neutrophils compared with the control, suggesting a turnover delay caused by a defect in elastase secretion from azurophilic granules at the rear of bone marrow polymorphonuclear leukocytes (BM-PMNs). We suggest that Rab27a-dependent protease secretion regulates neutrophil migration through proteolysis-dependent de-adhesion of uropods, a mechanism that could be conserved in cell migration and invasion.