Novel approaches to detect serum biomarkers for clinical response to interferon-beta treatment in multiple sclerosis.

Interferon beta (IFNbeta) is the most common immunomodulatory treatment for relapsing-remitting multiple sclerosis (RRMS). However, some patients fail to respond to treatment. In this study, we identified putative clinical response markers in the serum and plasma of people with multiple sclerosis (MS) treated with IFNbeta. In a discovery-driven approach, we use 2D-difference gel electrophoresis (DIGE) to identify putative clinical response markers and apply power calculations to identify the sample size required to further validate those markers. In the process we have optimized a DIGE protocol for plasma to obtain cost effective and high resolution gels for effective spot comparison. APOA1, A2M, and FIBB were identified as putative clinical response markers. Power calculations showed that the current DIGE experiment requires a minimum of 10 samples from each group to be confident of 1.5 fold difference at the p<0.05 significance level. In a complementary targeted approach, Cytometric Beadarray (CBA) analysis showed no significant difference in the serum concentration of IL-6, IL-8, MIG, Eotaxin, IP-10, MCP-1, and MIP-1alpha, between clinical responders and non-responders, despite the association of these proteins with IFNbeta treatment in MS.

The multiple sclerosis whole blood mRNA transcriptome and genetic associations indicate dysregulation of specific T cell pathways in pathogenesis.

Multiple sclerosis (MS) is an autoimmune disease with a genetic component, caused at least in part by aberrant lymphocyte activity. The whole blood mRNA transcriptome was measured for 99 untreated MS patients: 43 primary progressive MS, 20 secondary progressive MS, 36 relapsing remitting MS and 45 age-matched healthy controls. The ANZgene Multiple Sclerosis Genetics Consortium genotyped more than 300 000 SNPs for 115 of these samples. Transcription from genes on translational regulation, oxidative phosphorylation, immune synapse and antigen presentation pathways was markedly increased in all forms of MS. Expression of genes tagging T cells was also upregulated (P < 10(-12)) in MS. A T cell gene signature predicts disease state with a concordance index of 0.79 with age and gender as co-variables, but the signature is not associated with clinical course or disability. The ANZgene genome wide association screen identified two novel regions with genome wide significance: one encoding the T cell co-stimulatory molecule, CD40; the other a region on chromosome 12q13-14. The CD40 haplotype associated with increased MS susceptibility has decreased gene expression in MS (P < 0.0007). The second MS susceptibility region includes 17 genes on 12q13-14 in tight linkage disequilibrium. Of these, only 13 are expressed in leukocytes, and of these the expression of one, FAM119B, is much lower in the susceptibility haplotype (P < 10(-14)). Overall, these data indicate dysregulation of T cells can be detected in the whole blood of untreated MS patients, and supports targeting of activated T cells in therapy for all forms of MS.

Association of common T cell activation gene polymorphisms with multiple sclerosis in Australian patients.

Susceptibility to multiple sclerosis (MS) may be influenced by the interaction of several genes within a biological pathway. T cell activation and costimulation may be potentially important in MS pathogenesis. We have therefore investigated associations between MS and polymorphisms in the CD152 (CTLA-4), CD28, CD80 and CD86 genes in Australian patients. We found no significant MS association with CTLA-4 exon 1 +49 alleles, and meta-analysis showed no significant association across nine comparable datasets (OR=1.04, p=0.54), nor with primary progressive MS across seven datasets (OR=1.19, p=0.21). Haplotype analysis showed a trend towards a decrease of the CTLA-4-1722C, -1577G, +49G haplotype in +49 G positive MS patients compared with controls (p=0.06). Screening of CD28, CD80 and CD86 genes identified novel polymorphisms in the putative promoter regions of CD28 (-372 G/A) and CD86 (exon 2 -359 deletionAAG). There was a significant increase of the CD28 -372 G allele frequency in MS patients vs. controls (p=0.045) and a trend towards a significant interaction between this allele and the CTLA-4 +49 G allele (OR=4.00, p=0.058). Our results suggest that the CTLA-4 +49 alone is not associated with overall susceptibility to MS, but may be important in clinical subsets of patients and/or may interact epistatically with other gene polymorphisms.