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Background: Agents promoting oligodendrocyte precursor cell differentiation have the potential to restore halted and/or delayed remyelination in patients with multiple sclerosis. However, few therapeutic targets have been identified. The objective of this study was to identify novel targets for promotion of remyelination and characterize their activity in vitro and in vivo., Methods: A high-content screening assay with differentiation of primary rat oligodendrocyte precursor cells was used to screen GSK-proprietary annotated libraries for remyelination-promoting compounds. Compounds were further validated in vitro and in vivo models; clinical relevance of target was confirmed in human post-mortem brain sections from patients with MS., Results: Of ~1000 compounds screened, 36 promoted oligodendrocyte precursor cell differentiation in a concentration-dependent manner; seven were histamine receptor-3 (H3R) antagonists. Inverse agonists of H3R but not neutral antagonists promoted oligodendrocyte precursor cell (OPC) differentiation. H3R was expressed throughout OPC differentiation; H3R expression was transiently upregulated on Days 3-5 and subsequently downregulated. H3R gene knockdown in OPCs increased the expression of differentiation markers and the number of mature oligodendrocytes. Overexpression of full-length H3R reduced differentiation marker expression and the number of mature cells. H3R inverse agonist GSK247246 reduced intracellular cyclic AMP (cAMP) and downstream cAMP response element-binding protein (CREB) phosphorylation in a dose-dependent manner. Histone deacetylase (HDAC-1) and Hes-5 were identified as key downstream targets of H3R during OPC differentiation. In the mouse cuprizone/rapamycin model of demyelination, systemic administration of brain-penetrable GSK247246 enhanced remyelination and subsequently protected axons. Finally, we detected high H3R expression in oligodendroglial cells from demyelination lesions in human samples of patients with MS, and validated a genetic association between an exonic single nucleotide polymorphism in HRH3 and susceptibility to multiple sclerosis., Conclusions: From phenotypic screening to human genetics, we provide evidence for H3R as a novel therapeutic target to promote remyelination in patients with multiple sclerosis.

Objective: To perform a 1-stage meta-analysis of genome-wide association studies (GWAS) of multiple sclerosis (MS) susceptibility and to explore functional consequences of new susceptibility loci., Methods: We synthesized 7 MS GWAS. Each data set was imputed using HapMap phase II, and a per single nucleotide polymorphism (SNP) meta-analysis was performed across the 7 data sets. We explored RNA expression data using a quantitative trait analysis in peripheral blood mononuclear cells (PBMCs) of 228 subjects with demyelinating disease., Results: We meta-analyzed 2,529,394 unique SNPs in 5,545 cases and 12,153 controls. We identified 3 novel susceptibility alleles: rs170934(T) at 3p24.1 (odds ratio [OR], 1.17; p = 1.6 × 10(-8)) near EOMES, rs2150702(G) in the second intron of MLANA on chromosome 9p24.1 (OR, 1.16; p = 3.3 × 10(-8)), and rs6718520(A) in an intergenic region on chromosome 2p21, with THADA as the nearest flanking gene (OR, 1.17; p = 3.4 × 10(-8)). The 3 new loci do not have a strong cis effect on RNA expression in PBMCs. Ten other susceptibility loci had a suggestive p < 1 × 10(-6) , some of these loci have evidence of association in other inflammatory diseases (ie, IL12B, TAGAP, PLEK, and ZMIZ1)., Interpretation: We have performed a meta-analysis of GWAS in MS that more than doubles the size of previous gene discovery efforts and highlights 3 novel MS susceptibility loci. These and additional loci with suggestive evidence of association are excellent candidates for further investigations to refine and validate their role in the genetic architecture of MS., (Copyright © 2011 American Neurological Association.)

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis.

Multiple sclerosis (MS) is a complex autoimmune disease characterised by demyelinating lesions in the central nervous system (CNS) and myelin oligodendrocyte glycoprotein (MOG), a CNS-restricted protein expressed on the outer cell membrane of oligodendrocytes, has been linked with disease pathogenesis. We have investigated whether expression of MOG in post-mortem human brain tissue is associated with genetic variations in the MOG gene that have previously been associated with genetic susceptibility to MS (520G>A, rs3130253, V145I and 511G>C, rs2857766, V142L). Using quantitative reverse transcriptase PCR (qPCR), we found that the haplotype containing the 520A (rs3130253A, I145) allele is associated with a 1.7-fold increase in splicing of exon 2 to exon 3, which encodes the extracellular and transmembrane domains of MOG. Using predictive algorithms, we found that the 520G>A variant also alters a putative exonic splicing enhancer (ESE) involving the SC35 and SRp55 RNA-binding proteins, supporting the notion that this variation has a regulatory effect. No consistent differences in allele-specific expression were observed for any of the SNPs using the SNaPshot® method. In this exploratory study we have observed that changes in splicing, but not expression levels, are associated with common genetic variation in the MOG gene. Further work is now required to confirm these data and determine whether this altered MOG expression profile, which is predicted to be over-represented in Northern Europeans with MS, is relevant to the pathophysiology of this debilitating disease., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We conducted an association study across the human leukocyte antigen (HLA) complex to identify loci associated with multiple sclerosis (MS). Comparing 1927 SNPs in 1618 MS cases and 3413 controls of European ancestry, we identified seven SNPs that were independently associated with MS conditional on the others (each P ≤ 4 x 10(-6)). All associations were significant in an independent replication cohort of 2212 cases and 2251 controls (P ≤ 0.001) and were highly significant in the combined dataset (P ≤ 6 x 10(-8)). The associated SNPs included proxies for HLA-DRB1*15:01 and HLA-DRB1*03:01, and SNPs in moderate linkage disequilibrium (LD) with HLA-A*02:01, HLA-DRB1*04:01 and HLA-DRB1*13:03. We also found a strong association with rs9277535 in the class II gene HLA-DPB1 (discovery set P = 9 x 10(-9), replication set P = 7 x 10(-4), combined P = 2 x 10(-10)). HLA-DPB1 is located centromeric of the more commonly typed class II genes HLA-DRB1, -DQA1 and -DQB1. It is separated from these genes by a recombination hotspot, and the association is not affected by conditioning on genotypes at DRB1, DQA1 and DQB1. Hence rs9277535 represents an independent MS-susceptibility locus of genome-wide significance. It is correlated with the HLA-DPB1*03:01 allele, which has been implicated previously in MS in smaller studies. Further genotyping in large datasets is required to confirm and resolve this association.

Recent association studies in multiple sclerosis (MS) have identified and replicated several single nucleotide polymorphism (SNP) susceptibility loci including CLEC16A, IL2RA, IL7R, RPL5, CD58, CD40 and chromosome 12q13-14 in addition to the well established allele HLA-DR15. There is potential that these genetic susceptibility factors could also modulate MS disease severity, as demonstrated previously for the MS risk allele HLA-DR15. We investigated this hypothesis in a cohort of 1006 well characterised MS patients from South-Eastern Australia. We tested the MS-associated SNPs for association with five measures of disease severity incorporating disability, age of onset, cognition and brain atrophy. We observed trends towards association between the RPL5 risk SNP and time between first demyelinating event and relapse, and between the CD40 risk SNP and symbol digit test score. No associations were significant after correction for multiple testing. We found no evidence for the hypothesis that these new MS disease risk-associated SNPs influence disease severity.

Multiple sclerosis (MS) is a genetically complex autoimmune disease. To dissect further the involvement of four recent identified MS susceptibility genes (KIAA0350, IL2RA, RPL5 and CD58) in disease pathogenesis, we genotyped 94 haplotype-tagging single nucleotide polymorphisms (SNPs) from these loci in 1146 MS cases and 1309 controls. Seven newly-typed SNP variants were nominally associated with risk of MS, and one SNP (rs791589) in the first intron of the IL2RA gene remained associated after adjustment for rs2104286 genotype, a previously reported SNP association. These data provide further evidence of allelic heterogeneity at the IL2RA locus and point to the existence of at least two independent MS susceptibility alleles.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system of unknown etiology with both genetic and environmental factors playing a role in susceptibility. To date, the HLA DR15/DQ6 haplotype within the major histocompatibility complex on chromosome 6p, is the strongest genetic risk factor associated with MS susceptibility. Additional alleles of IL7 and IL2 have been identified as risk factors for MS with small effect. Here we present two independent studies supporting an allelic association of MS with polymorphisms in the ST8SIA1 gene, located on chromosome 12p12 and encoding ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1. The initial association was made in a single three-generation family where a single-nucleotide polymorphism (SNP) rs4762896, was segregating together with HLA DR15/DQ6 in MS patients. A study of 274 family trios (affected child and both unaffected parents) from Australia validated the association of ST8SIA1 in individuals with MS, showing transmission disequilibrium of the paternal alleles for three additional SNPs, namely rs704219, rs2041906, and rs1558793, with p = 0.001, p = 0.01 and p = 0.01 respectively. These findings implicate ST8SIA1 as a possible novel susceptibility gene for MS.

Human leucocyte antigen (HLA)-DRB1*15 is associated with predisposition to multiple sclerosis (MS), although conjecture surrounds the possible involvement of an alternate risk locus in the class I region of the HLA complex. We have shown previously that an alternate MS risk allele(s) may be encompassed by the telomerically extended DRB1*15 haplotype, and here, we have attempted to map the putative variant. Thirteen microsatellite markers encompassing a 6.79-megabase (D6S2236-G51152) region, and the DRB1 and DQB1 genes, were genotyped in 166 MS simplex families and 104 control families from the Australian State of Tasmania and 153 narcolepsy simplex families (trios) from the USA. Complementary approaches were used to investigate residual predisposing effects of microsatellite alleles comprising the extended DRB1*15 haplotype taking into account the strong predisposing effect of DRB1*15: (1) Disease association of the extended DRB1*15 haplotype was compared for MS and narcolepsy families--predisposing effects were observed for extended class I microsatellite marker alleles in MS families, but not narcolepsy families; (2) disease association of the extended DRB1*15 haplotype was investigated after conditioning MS and control haplotypes on the absence of DRB1*15--a significant predisposing effect was observed for a 627-kb haplotype (D6S258 allele 8-MOGCA allele 4; MOG, myelin oligodendrocyte glycoprotein) spanning the extended class I region. MOGCA allele 4 displayed the strongest predisposing effect in DRB1*15-conditioned haplotypes (p = 0.0006; OR 2.83 [1.54-5.19]). Together, these data confirm that an alternate MS risk locus exists in the extended class I region in Tasmanian MS patients independent of DRB1*15.

We have developed a likelihood method to identify moderately distant genealogical relationships from genomewide scan data. The aim is to compare the genotypes of many pairs of people and identify those pairs most likely to be related to one another. We have tested the algorithm using the genotypes of 170 Tasmanians with multiple sclerosis recruited into a haplotype association study. It is estimated from genealogical records that approximately 65% of Tasmania's current population of 470,000 are direct descendants of the 13,000 female founders living in this island state of Australia in the mid-nineteenth century. All cases and four to five relatives of each case have been genotyped with microsatellite markers at a genomewide average density of 4 cM. Previous genealogical research has identified 51 pairwise relationships linking 56 of the 170 cases. Testing the likelihood calculation on these known relative pairs, we have good power to identify relationships up to degree eight (e.g. third cousins once removed). Applying the algorithm to all other pairs of cases, we have identified a further 61 putative relative pairs, with an estimated false discovery rate of 10%. The power to identify genealogical links should increase when the new, denser sets of SNP markers are used. Except in populations where there is a searchable electronic database containing virtually all genealogical links in the past six generations, the algorithm should be a useful aid for genealogists working on gene-mapping projects, both linkage studies and association studies.

Results are presented from a genomewide haplotype association study on multiple sclerosis (MS) cases from Tasmania, an island state of Australia. Cases were ascertained on strict clinical and radiological grounds and on the fact that they had at least one grandparent born in the state. This enriched for early settler chromosomes among present day Tasmanians with MS and increased the chances of finding common haplotype sharing at disease predisposition loci in distant relatives sharing common ancestral haplotypes. Four-to-five close relatives were also collected for each of 170 cases and 105 population-based controls. All were genotyped at a 5cM resolution, haplotypes reconstructed and sharing estimated using an empirical approach based on sorting haplotypes to find the most common at each locus and then generating a test statistic for excess sharing in the cases based on permutation testing. Five initial loci were found where there was an excess sharing in the cases. These were fine-mapped with 10-12 additional markers. Only loci on chromosomes 6 and 10 remained after fine mapping. These loci demonstrate an increase in sharing of multi-marker haplotypes in MS cases compared to both population control transmitted haplotypes and case non-transmitted haplotypes.

In order to resolve a multiple sclerosis (MS) susceptibility locus that we had identified in earlier work at the telomeric end of the HLA complex, we genotyped another 34 microsatellite markers (47 in total) across the class I/extended class I region in 166 Tasmanian MS case and 104 control families (D6S299-D6S265). Extended MS susceptibility haplotypes, up to 9 Mb in length, were observed in 11% of MS cases and 4% of controls. Direct comparison of the telomerically extended portion of the MS susceptibility haplotype in HFE-Cys282Tyr (C282Y)-homozygous haemochromatosis patients identified a common ancestry for this genomic segment, which translated into an increased frequency of the C282Y allele in 489 MS cases from Tasmania and Victoria (10.2%) compared with controls (6.7%). Six C282Y homozygotes (1.2%), a three-fold increased rate over the general population, and 88 heterozygotes (18%) were identified. One C282Y-homozygous female was identified who had MS and was being treated for symptoms of iron overload. Interestingly, for 71 Victorian MS cases not of north western European (NWE) ancestry, a DR15-independent reduction in the frequency of the C282Y allele was observed, supporting the theory of a NWE origin for the C282Y-variant of the DR15 ancestral haplotype (C282Y-HLA-A*0301-B*0702-DRB1*1501-DQB1*0602). The results of linkage disequilibrium (LD) and log linear modelling analyses suggest that C282Y is increased in MS cases of NWE ancestry because it is in LD with the ancestral DR15 susceptibility haplotype (7.1) and that it does not play an independent role in predisposition to MS. However, our findings provide the impetus for further investigations into the role of iron metabolism in the severity of MS.

Association of multiple sclerosis (MS) with the human leukocyte antigen (HLA) class II haplotype DRB1*1501-DQB1*0602 is the most consistently replicated finding of genetic studies of the disease. However, the high level of linkage disequilibrium (LD) in the HLA region has hindered the identification of other loci that single-marker tests for association are unlikely to resolve. In order to address this issue, we generated haplotypes spanning 14.754 Mb (5 cM) across the entire HLA region. The haplotypes, which were inferred by genotyping relatives of 152 patients with MS and 105 unaffected control subjects of Tasmanian ancestry, define a genomic segment from D6S276 to D6S291, including 13 microsatellite markers integrated with allele-typing data for DRB1 and DQB1. Association to the DRB1*1501-DQB1*0602 haplotype was replicated. In addition, we found that the class I/extended class I region, defined by a genomic segment of approximately 400 kb between MOGCA and D6S265, harbors genes that independently increase risk of, or provide protection from, MS. Log-linear modeling analysis of constituent haplotypes that represent genomic regions containing class I (MOGCA-D6S265), class III (TNFa-TNFd-D6S273), and class II (DRB1-DQB1) genes indicated that having class I and class II susceptibility variants on the same haplotype provides an additive effect on risk. Moreover, we found no evidence for a disease locus in the class III region defined by a 150-kb genomic segment containing the TNF locus and 14 other genes. A global overview of LD performed using GOLD identified two discrete blocks of LD in the HLA region that correspond well with previous findings. We propose that the analysis of haplotypes, by use of the types of approaches outlined in the present article, should make it possible to more accurately define the contribution of the HLA to MS.

Background: The role played by genetic factors in influencing the clinical course of multiple sclerosis (MS) is not yet well established.Objective: We aimed to identify genetic variants associated with progressive MS (PrMS).Methods: We conducted a genome-wide association study (GWAS) in 197 patients with PrMS and 234 controls of Italian origin. We tested the top 20 single nucleotide polymorphisms (SNPs) with suggestive evidence of association (p-value<10−4) in two independent sets of primary progressive MS cases and controls.Results: We identified a risk-associated SNP in the HLA region in linkage disequilibrium (LD) with DRB1*1501 and DQB*0602 loci, with genome-wide significance (rs3129934T, pcombined=6.7×10-16, OR=2.34, 95% CI=1.90–2.87), and a novel locus on chromosome 7q35 with suggestive evidence of association (rs996343G, pcombined=2.4×10-5, OR=0.70, 95% CI=0.59–0.83) which maps within a human endogenous retroviral (HERV) element. The new locus did not have a ‘cis’ effect on RNA expression in lymphoblastic cell lines, but pathway analyses of ‘trans’ effects point to an expression regulation of genes involved in neurodegeneration, including glutamate metabolism (p<0.01) and axonal guidance signalling (p<0.02).Conclusions: We have confirmed the established association with the HLA region and, despite the low statistical power of the study, we found suggestive evidence for association with a novel locus on chromosome 7, with a putative regulatory role. [ABSTRACT FROM PUBLISHER]

To identify multiple sclerosis (MS) susceptibility loci, we conducted a genome-wide association study (GWAS) in 1,618 cases and used shared data for 3,413 controls. We performed replication in an independent set of 2,256 cases and 2,310 controls, for a total of 3,874 cases and 5,723 controls. We identified risk-associated SNPs on chromosome 12q13–14 (rs703842, P = 5.4 × 10−11; rs10876994, P = 2.7 × 10−10; rs12368653, P = 1.0 × 10−7) and upstream of CD40 on chromosome 20q13 (rs6074022, P = 1.3 × 10−7; rs1569723, P = 2.9 × 10−7). Both loci are also associated with other autoimmune diseases. We also replicated several known MS associations (HLA-DR15, P = 7.0 × 10−184; CD58, P = 9.6 × 10−8; EVI5-RPL5, P = 2.5 × 10−6; IL2RA, P = 7.4 × 10−6; CLEC16A, P = 1.1 × 10−4; IL7R, P = 1.3 × 10−3; TYK2, P = 3.5 × 10−3) and observed a statistical interaction between SNPs in EVI5-RPL5 and HLA-DR15 (P = 0.001). [ABSTRACT FROM AUTHOR]

Abstract: Aberrant regulatory T cell populations, characterised by a wide array of CD markers, have been identified in many autoimmune diseases. CD127 has recently been identified as a specific marker for the CD4+CD25Hi (Tregs) subset. CD127 is the first non-HLA gene to have its association with multiple sclerosis widely replicated. We demonstrate that the regulatory or suppressor T cells CD4+CD25Hi (Tregs), CD8+CD28−, and CD3+CD56+ (NKT) all produce low levels of CD127, and so could be at a disadvantage in survival and/or proliferation where IL7 is limiting. The remissions seen in relapsing remitting multiple sclerosis (RRMS) could be driven by regulatory T cells, and the absence of remissions seen in primary progressive MS (PPMS) may point to a particularly reduced function of this cell subset. We found that the proportions of CD4+FoxP3+CD25Hi regulatory T cells were not aberrant in PPMS. There was, however, a trend towards reduced FoxP3 expression per cell in this fraction (p <0.083), which has been highly correlated with suppressor function. Notably, we found that the target of regulatory T cells, the CD4+CD25− cells, was in excess (p <0.009); and in PPMS a protective CD127 haplotype is correlated with higher CD127 expression (p <0.01). These data support further investigations into the regulatory T cell immunophenotype in MS. [Copyright &y& Elsevier]