Prudence N. Gatt

Antibodies to myelin oligodendrocyte glycoprotein in bilateral and recurrent optic neuritis.

Objective: We examined a cohort of adults with aquaporin-4 (AQP4) antibody–negative neuromyelitis optica/neuromyelitis optica spectrum disorder (NMO/NMOSD) for antibodies to myelin oligodendrocyte glycoprotein (MOG). Methods: We performed a flow cytometry cell-based assay using live human lentivirus–transduced cells expressing full-length surface MOG. Serum was tested in 23 AQP4 antibody–negative NMO/NMOSD patients with bilateral and/or recurrent optic neuritis (BON, n = 11), longitudinally extensive transverse myelitis (LETM, n = 10), and sequential BON and LETM (n = 2), as well as in patients with multiple sclerosis (MS, n = 76) and controls (n = 52). Results: MOG antibodies were detected in 9/23 AQP4 antibody–negative patients with NMO/NMOSD, compared to 1/76 patients with MS and 0/52 controls (p < 0.001). MOG antibodies were detected in 8/11 patients with BON, 0/10 patients with LETM, and 1/2 patients with sequential BON and LETM. Six of 9 MOG antibody–positive patients had a relapsing course. MOG antibody–positive patients had prominent optic disc swelling and were more likely to have a rapid response to steroid therapy and relapse on steroid cessation than MOG antibody–negative patients (p = 0.034 and p = 0.029, respectively). While 8/9 MOG antibody–positive patients had good follow-up visual acuity, one experienced sustained visual impairment, 3 had retinal nerve fiber layer thinning, and one had residual spinal disability. Conclusions: MOG antibodies have a strong association with BON and may be a useful clinical biomarker. MOG antibody–associated BON is a relapsing disorder that is frequently steroid responsive and often steroid dependent. Failure to recognize the disorder early and institute immunotherapy promptly may be associated with sustained impairment. Classification of evidence: This study provides Class II evidence that MOG antibodies are associated with AQP4 antibody–negative BON (sensitivity 69%, 95% confidence interval [CI] 42%–87%; specificity 99%, 95% CI 93.7%–99.8%).

The CYP27B1 variant associated with an increased risk of autoimmune disease is underexpressed in tolerizing dendritic cells

Genome-wide association studies have identified a linkage disequilibrium (LD) block on chromosome 12 associated with multiple sclerosis (MS), type 1 diabetes and other autoimmune diseases. This block contains CYP27B1, which catalyzes the conversion of 25 vitamin D3 (VitD3) to 1,25VitD3. Fine-mapping analysis has failed to identify which of the 17 genes in this block is most associated with MS. We have previously used a functional approach to identify the causal gene. We showed that the expression of several genes in this block in whole blood is highly associated with the MS risk allele, but not CYP27B1. Here, we show that CYP27B1 is predominantly expressed in dendritic cells (DCs). Its expression in these cells is necessary for their response to VitD, which is known to upregulate pathways involved in generating a tolerogenic DC phenotype. Here, we utilize a differentiation protocol to generate inflammatory (DC1) and tolerogenic (DC2) DCs, and show that for the MS risk allele CYP27B1 is underexpressed in DCs, especially DC2s. Of the other Chr12 LD block genes expressed in these cells, only METT21B expression was as affected by the genotype. Another gene associated with autoimmune diseases, CYP24A1, catabolizes 1,25 VitD3, and is predominantly expressed in DCs, but equally between DC1s and DC2s. Overall, these data are consistent with the hypothesis that reduced VitD pathway gene upregulation in DC2s of carriers of the risk haplotype of CYP27B1 contributes to autoimmune diseases. These data support therapeutic approaches aimed at targeting VitD effects on DCs.

The autoimmune disease-associated transcription factors EOMES and TBX21 are dysregulated in multiple sclerosis and define a molecular subtype of disease.

We have identified a marked over-representation of transcription factors controlling differentiation of T, B, myeloid and NK cells among the 110 MS genes now known to be associated with multiple sclerosis (MS). To test if the expression of these genes might define molecular subtypes of MS, we interrogated their expression in blood in three independent cohorts of untreated MS (from Sydney and Adelaide) or clinically isolated syndrome (CIS, from San Francisco) patients. Expression of the transcription factors (TF) controlling T and NK cell differentiation, EOMES, TBX21 and other TFs was significantly lower in MS/CIS compared to healthy controls in all three cohorts. Expression was tightly correlated between these TFs, with other T/NK cell TFs, and to another downregulated gene, CCL5. Expression was stable over time, but did not predict disease phenotype. Optimal response to therapy might be indicated by normalization of expression of these genes in blood.

Ribosomal protein S6 mRNA is a biomarker upregulated in multiple sclerosis, downregulated by interferon treatment, and affected by season

Background: Multiple Sclerosis (MS) is an immune-mediated disease of the central nervous system which responds to therapies targeting circulating immune cells. Objective: Our aim was to test if the T-cell activation gene expression pattern (TCAGE) we had previously described from whole blood was replicated in an independent cohort. Methods: We used RNA-seq to interrogate the whole blood transcriptomes of 72 individuals (40 healthy controls, 32 untreated MS). A cohort of 862 control individuals from the Brisbane Systems Genetics Study (BSGS) was used to assess heritability and seasonal expression. The effect of interferon beta (IFNB) therapy on expression was evaluated. Results: The MS/TCAGE association was replicated and rationalized to a single marker, ribosomal protein S6 (RPS6). Expression of RPS6 was higher in MS than controls (p<0.0004), and lower in winter than summer (p<4.6E-06). The seasonal pattern correlated with monthly UV light index (R=0.82, p<0.002), and was also identified in the BSGS cohort (p<0.0016). Variation in expression of RPS6 was not strongly heritable. RPS6 expression was reduced by IFNB therapy. Conclusions: These data support investigation of RPS6 as a potential therapeutic target and candidate biomarker for measuring clinical response to IFNB and other MS therapies, and of MS disease heterogeneity.

The low EOMES/TBX21 molecular phenotype in multiple sclerosis reflects CD56+ cell dysregulation and is affected by immunomodulatory therapies

Multiple Sclerosis (MS) is an autoimmune disease treated by therapies targeting peripheral blood cells. We previously identified that expression of two MS-risk genes, the transcription factors EOMES and TBX21 (ET), was low in blood from MS and stable over time. Here we replicated the low ET expression in a new MS cohort (p<0.0007 for EOMES, p<0.028 for TBX21) and demonstrate longitudinal stability (p<10(-4)) and high heritability (h(2)=0.48 for EOMES) for this molecular phenotype. Genes whose expression correlated with ET, especially those controlling cell migration, further defined the phenotype. CD56+ cells and other subsets expressed lower levels of Eomes or T-bet protein and/or were under-represented in MS. EOMES and TBX21 risk SNP genotypes, and serum EBNA-1 titres were not correlated with ET expression, but HLA-DRB1*1501 genotype was. ET expression was normalised to healthy control levels with natalizumab, and was highly variable for glatiramer acetate, fingolimod, interferon-beta, dimethyl fumarate.

IL7Rα Expression and Upregulation by IFNβ in Dendritic Cell Subsets Is Haplotype-Dependent

The IL7Rα gene is unequivocally associated with susceptibility to multiple sclerosis (MS). Haplotype 2 (Hap 2) confers protection from MS, and T cells and dendritic cells (DCs) of Hap 2 exhibit reduced splicing of exon 6, resulting in production of relatively less soluble receptor, and potentially more response to ligand. We have previously shown in CD4 T cells that IL7Rα haplotypes 1 and 2, but not 4, respond to interferon beta (IFNβ), the most commonly used immunomodulatory drug in MS, and that haplotype 4 (Hap 4) homozygotes have the highest risk of developing MS. We now show that IL7R expression increases in myeloid cells in response to IFNβ, but that the response is haplotype-dependent, with cells from homozygotes for Hap 4 again showing no response. This was shown using freshly derived monocytes, in vitro cultured immature and mature monocyte-derived dendritic cells, and by comparing homozygotes for the common haplotypes, and relative expression of alleles in heterozygotes (Hap 4 vs not Hap 4). As for T cells, in all myeloid cell subsets examined, Hap 2 homozygotes showed a trend for reduced splicing of exon 6 compared to the other haplotypes, significantly so in most conditions. These data are consistent with increased signaling being protective from MS, constitutively and in response to IFNβ. We also demonstrate significant regulation of immune response, chemokine activity and cytokine biosynthesis pathways by IL7Rα signaling in IFNβ -treated myeloid subsets. IFNβ-responsive genes are over-represented amongst genes associated with MS susceptibility. IL7Rα haplotype may contribute to MS susceptibility through reduced capacity for IL7Rα signalling in myeloid cells, especially in the presence of IFNβ, and is currently under investigation as a predictor of therapeutic response.

Response to interferon-beta treatment in multiple sclerosis patients: A genome-wide association study.

Up to 50% of multiple sclerosis (MS) patients do not respond to interferon-beta (IFN-β) treatment and determination of response requires lengthy clinical follow-up of up to 2 years. Response predictive genetic markers would significantly improve disease management. We aimed to identify IFN-β treatment response genetic marker(s) by performing a two-stage genome-wide association study (GWAS). The GWAS was carried out using data from 151 Australian MS patients from the ANZgene/WTCCC2 MS susceptibility GWAS (responder (R)=51, intermediate responders=24 and non-responders (NR)=76). Of the single-nucleotide polymorphisms (SNP) that were validated in an independent group of 479 IFN-β-treated MS patients from Australia, Spain and Italy (R=273 and NR=206), eight showed evidence of association with treatment response. Among the replicated associations, the strongest was observed for FHIT (Fragile Histidine Triad; combined P-value 6.74 × 10−6) and followed by variants in GAPVD1 (GTPase activating protein and VPS9 domains 1; combined P-value 5.83 × 10−5) and near ZNF697 (combined P-value 8.15 × 10−5).

Data characterizing the ZMIZ1 molecular phenotype of multiple sclerosis

The data presented in this article are related to the research article entitled “The autoimmune risk gene ZMIZ1 is a vitamin D responsived marker of a molecular phenotype of multiple sclerosis” Fewings et al. (2017) [1]. Here we identify the set of genes correlated with ZMIZ1 in multiple cohorts, provide phenotypic details on those cohorts, and identify the genes negatively correlated with ZMIZ1 and the cells predominantly expressing those genes. We identify the metabolic pathways in which the molecular phenotype genes are over-represented. Finally, we present the flow cytometry gating strategy we have used to identify the immune cells from blood which are producing ZMIZ1 and RPS6.

Characterising the Molecular Phenotypes of MS: heredity, gene expression modules, dysregulated immune cell subsets and response to therapy

Background: Current MS therapies target the systemic circulation. We previously identified that genes encoding transcription factors were over-represented amongst MS risk factors, and that expression of several of these were altered in MS blood, including EOMES, TBX21 and ZMIZ1. Expression was stable over time, so that it defines a molecular phenotype. Objectives: Here we tested if this finding could be replicated in independent cohorts, if expression was heritable, longitudinally stable, affected by therapy, and associated with genetic and environmental risk factors. We sought the immune cell subsets expressing these transcription factors, and tested if protein expression was also altered. Methods: Whole blood mRNA expression was determined in new cohorts of untreated MS (n = 23, Sydney; n = 47, Perth) and healthy controls (n = 23) and protein expression determined by flow cytometry in PBMCs of untreated MS and healthy controls (n = 28, 30 respectively). Effect of major therapies and correlation of gene expression with risk SNP genotypes, and anti-Epstein Barr Virus EBNA-1 titres was assessed, and heritability tested in a large twin cohort. Results: MS Molecular Phenotypes were replicated in new cohorts. Modules of genes, whose expression correlated with EOMES/TBX21 or ZMIZ1, further defined the phenotypes. CD56+ cells, inflammatory monocytes and plasmacytoid dendritic cells expressed lower levels of Molecular Phenotype transcription factors. EOMES and TBX21 risk SNP genotypes, and serum EBNA-1 titres were not correlated with gene expression, but HLA-DRB1*1501 genotype was. Therapies altered expression levels (e.g. TBX21 returned to control levels on natalizumab) with significant variability between individuals. Conclusions: EOMES/TBX21 and ZMIZ1 tag molecular phenotypes of MS that are affected by therapy. The phenotypes are due to under-representation and altered state of CD56+ and inflammatory monocytes/pDCs respectively. The dysregulated immune cells are a novel target for therapy and, together with gene expression, represent potential biomarkers of therapeutic response.Background: Population based genome wide association studies have identified 110 single nucleotide polymorphisms (SNPs) that are associated with an increase in MS risk. These SNPs are all common, and have odds ratios of between 1.1 and 1.4. Most are found in non-protein coding regions, and their functions are largely unknown. Objectives: Importantly, recent work has shown that some non-coding SNPs can function by changing immune gene expression levels as a quantitative trait, termed expression quantitative trait loci (eQTL). We conducted studies to evaluate the effects of MS risk SNPs on gene expression in four main immune cell types. Methods: We isolated monocytes, B-cells, CD4- and CD8- T-cells from untreated relapsing MS cases (n=79) and healthy controls (n=101). To test for cis-eQTL associations, we selected all genes within +/-500kb of an MS risk SNP (2500 pairs in total). The Illumina Immunochip was used to genotype for MS risk SNPs, and gene expression was measured for each cell type by microarray. Results: We have identified MS risk eQTL associations in each immune cell type, some of which are cell type specific. We also present preliminary data showing that some MS risk SNPs could exert differential effects on gene expression in cases compared to controls. Here we report likely disease state specific eQTLs for all cell types with the top associations being: RNF26/rs9736016, B cells; MACROD1/rs694739, CD8 cells; SLC25A41/rs1077667, CD4 cells; GPR18/rs4772201, monocyte cells. Conclusions: We have shown that MS risk SNPs contribute to immune heterogeneity. It is hoped that through an understanding of the functions of individual common risk variants, it may be possible to uncover the processes and cell types that are most important for conveying the genetic risk of MS.

Antibodies to myelin oligodendrocyte glycoprotein in bilateral and recurrent optic neuritis

Background: Cerebrospinal fluid (CSF) levels of inflammatory biomarkers have been suggested to predictmultiple sclerosis development after clinically isolated syndromes, but there is a need for studies looking at the long-term prognosis. Aim: To assess the predictive ability of CSF biomarkers of inflammation and tissue-damage with regard to development of clinically definite multiple sclerosis (CDMS) and long-term disability. Methods: 86 patients with acute optic neuritis (ON) as a first demyelinating event seen in our clinic between 1993 and 2002 were included retrospectively. Patients had undergone MRI and a lumbar puncture b 3 months from onset; CSF was stored at −80 °C. Established MS-risk parameters (MRI, CSF leukocyte count, IgG-index and oligoclonal bands) were registered and CSF levels of chitinase-3-like-1 (CHI3L1), osteopontin, neurofilament light chain (NFL), myelin basic protein, CCL2, CXCL10, CXCL13 and matrix metalloproteinase-9 were measured by ELISA. Patients were followed up after amedian time of 13.6 (range: 9.6– 19.4) years. 81.4%were examined by the corresponding author including an EDSS and multiple sclerosis functional composite (MSFC) evaluation. 18.6% were interviewed by phone, incl. an EDSS-questionnaire. Time to CDMS was confirmed by collection of original charts from treating hospitals. Cox regression was used for time to CDMS, multiple regression for multiple sclerosis severity score (MSSS) and MSFC. Results: 46 of 86 (53.5%) developed CDMS between ON and followup. Of the established MS-risk parameters, MRI (p = 0.0008) and leukocyte count (p = 0.036) combined predicted MS development best. These two were then tested in a second model with the other CSF biomarkers, where MRI (p = 0.0001) and CHI3L1 (p = 0.017) combined predicted MS development best. NFL predicted long-term disability as assessed by the MSSS (p = 0.0135), nine-hole peg test (p = 0.0137) and visual acuity (p = 0.0005). CHI3L1 predicted longterm cognitive impairment as assessed by the paced auditory serial addition test (p = 0.0142). Conclusions: NFL and CHI3L1 were significant predictors of long-term physical and cognitive disability. We have previously suggested these to be markers of tissue damage-related inflammation in ON, supporting these findings. Furthermore, CHI3L1 appeared to predict CDMS better than the CSF parameters commonly used today. Thus, the two hold potential as clinically valuable biomarkers of future MS disease and disability after a first demyelinating event.