Sunny Malhotra

NLRP3 inflammasome is associated with the response to IFN-β in patients with multiple sclerosis

Evidence exists for a potential modulation of inflammasome activity by interferon beta. Here, we investigated the roles of inflammasomes [absent in melanoma 2 (AIM2); NLR family, CARD domain containing 4 (NLRC4); NLR family, pyrin domain containing 1 and 3 (NLRP1 and NLRP3)] and related cytokines (IL1B, IL10, IL18) in the response to interferon beta in patients with relapsing-remitting multiple sclerosis. Ninety-seven patients treated with interferon beta were classified into responders and non-responders according to clinical criteria after 24 months and clinical-radiological criteria after 12 months of treatment. Messenger RNA expression levels of inflammasomes and cytokines were determined by real-time polymerase chain reaction in peripheral blood mononuclear cells collected before treatment with interferon beta. In a subgroup of patients, NLRP3 and IL1B expression was also determined after 3 months (n = 32) and 12 months (n = 20) of interferon beta treatment. A polymorphism located in the NLRP3 gene, rs35829419, was genotyped in 789 multiple sclerosis patients treated with interferon beta. Baseline mRNA expression levels for NLRP3 and IL1B were increased in peripheral blood mononuclear cells from non-responders compared to responders classified according to clinical criteria after 24 months (P = 0.02 and P = 0.001, respectively). No significant differences were observed for other inflammasomes and related cytokines. Differences in NLRP3 and IL1B expression remained significant following a clinical-radiological classification after 12 months (P = 0.007 and P = 0.02, respectively). After treatment with interferon beta, NLRP3 and IL1B expression was increased in responders but unchanged in non-responders. A trend for association was observed between rs35829419 and interferon beta response (pM-H = 0.08). These results point to a role of the NLRP3 inflammasome and its related cytokine IL1B in the response to interferon beta in patients with relapsing-remitting multiple sclerosis.

MANBA, CXCR5, SOX8, RPS6KB1 and ZBTB46 are genetic risk loci for multiple sclerosis

A recent genome-wide association study reported five loci for which there was strong, but sub-genome-wide significant evidence for association with multiple sclerosis risk. The aim of this study was to evaluate the role of these potential risk loci in a large and independent data set of ≈ 20,000 subjects. We tested five single nucleotide polymorphisms rs228614 (MANBA), rs630923 (CXCR5), rs2744148 (SOX8), rs180515 (RPS6KB1), and rs6062314 (ZBTB46) for association with multiple sclerosis risk in a total of 8499 cases with multiple sclerosis, 8765 unrelated control subjects and 958 trios of European descent. In addition, we assessed the overall evidence for association by combining these newly generated data with the results from the original genome-wide association study by meta-analysis. All five tested single nucleotide polymorphisms showed consistent and statistically significant evidence for association with multiple sclerosis in our validation data sets (rs228614: odds ratio = 0.91, P = 2.4 × 10(-6); rs630923: odds ratio = 0.89, P = 1.2 × 10(-4); rs2744148: odds ratio = 1.14, P = 1.8 × 10(-6); rs180515: odds ratio = 1.12, P = 5.2 × 10(-7); rs6062314: odds ratio = 0.90, P = 4.3 × 10(-3)). Combining our data with results from the previous genome-wide association study by meta-analysis, the evidence for association was strengthened further, surpassing the threshold for genome-wide significance (P < 5 × 10(-8)) in each case. Our study provides compelling evidence that these five loci are genuine multiple sclerosis susceptibility loci. These results may eventually lead to a better understanding of the underlying disease pathophysiology.

Genetic variants are major determinants of CSF antibody levels in multiple sclerosis

Immunological hallmarks of multiple sclerosis include the production of antibodies in the central nervous system, expressed as presence of oligoclonal bands and/or an increased immunoglobulin G index-the level of immunoglobulin G in the cerebrospinal fluid compared to serum. However, the underlying differences between oligoclonal band-positive and -negative patients with multiple sclerosis and reasons for variability in immunoglobulin G index are not known. To identify genetic factors influencing the variation in the antibody levels in the cerebrospinal fluid in multiple sclerosis, we have performed a genome-wide association screen in patients collected from nine countries for two traits, presence or absence of oligoclonal bands (n = 3026) and immunoglobulin G index levels (n = 938), followed by a replication in 3891 additional patients. We replicate previously suggested association signals for oligoclonal band status in the major histocompatibility complex region for the rs9271640*A-rs6457617*G haplotype, correlated with HLA-DRB1*1501, and rs34083746*G, correlated with HLA-DQA1*0301 (P comparing two haplotypes = 8.88 × 10(-16)). Furthermore, we identify a novel association signal of rs9807334, near the ELAC1/SMAD4 genes, for oligoclonal band status (P = 8.45 × 10(-7)). The previously reported association of the immunoglobulin heavy chain locus with immunoglobulin G index reaches strong evidence for association in this data set (P = 3.79 × 10(-37)). We identify two novel associations in the major histocompatibility complex region with immunoglobulin G index: the rs9271640*A-rs6457617*G haplotype (P = 1.59 × 10(-22)), shared with oligoclonal band status, and an additional independent effect of rs6457617*G (P = 3.68 × 10(-6)). Variants identified in this study account for up to 2-fold differences in the odds of being oligoclonal band positive and 7.75% of the variation in immunoglobulin G index. Both traits are associated with clinical features of disease such as female gender, age at onset and severity. This is the largest study population so far investigated for the genetic influence on antibody levels in the cerebrospinal fluid in multiple sclerosis, including 6950 patients. We confirm that genetic factors underlie these antibody levels and identify both the major histocompatibility complex and immunoglobulin heavy chain region as major determinants.

Search for specific biomarkers of IFNβ bioactivity in patients with multiple sclerosis.

Myxovirus A (MxA), a protein encoded by the MX1 gene with antiviral activity, has proven to be a sensitive measure of IFNβ bioactivity in multiple sclerosis (MS). However, the use of MxA as a biomarker of IFNβ bioactivity has been criticized for the lack of evidence of its role on disease pathogenesis and the clinical response to IFNβ. Here, we aimed to identify specific biomarkers of IFNβ bioactivity in order to compare their gene expression induction by type I IFNs with the MxA, and to investigate their potential role in MS pathogenesis. Gene expression microarrays were performed in PBMC from MS patients who developed neutralizing antibodies (NAB) to IFNβ at 12 and/or 24 months of treatment and patients who remained NAB negative. Nine genes followed patterns in gene expression over time similar to the MX1, which was considered the gold standard gene, and were selected for further experiments: IFI6, IFI27, IFI44L, IFIT1, HERC5, LY6E, RSAD2, SIGLEC1, and USP18. In vitro experiments in PBMC from healthy controls revealed specific induction of selected biomarkers by IFNβ but not IFNγ, and several markers, in particular USP18 and HERC5, were shown to be significantly induced at lower IFNβ concentrations and more selective than the MX1 as biomarkers of IFNβ bioactivity. In addition, USP18 expression was deficient in MS patients compared with healthy controls (p = 0.0004). We propose specific biomarkers that may be considered in addition to the MxA to evaluate IFNβ bioactivity, and to further explore their implication in MS pathogenesis.

Genome-wide significant association with seven novel multiple sclerosis risk loci

Objective A recent large-scale study in multiple sclerosis (MS) using the ImmunoChip platform reported on 11 loci that showed suggestive genetic association with MS. Additional data in sufficiently sized and independent data sets are needed to assess whether these loci represent genuine MS risk factors. Methods The lead SNPs of all 11 loci were genotyped in 10 796 MS cases and 10 793 controls from Germany, Spain, France, the Netherlands, Austria and Russia, that were independent from the previously reported cohorts. Association analyses were performed using logistic regression based on an additive model. Summary effect size estimates were calculated using fixed-effect meta-analysis. Results Seven of the 11 tested SNPs showed significant association with MS susceptibility in the 21 589 individuals analysed here. Meta-analysis across our and previously published MS case-control data (total sample size n=101 683) revealed novel genome-wide significant association with MS susceptibility (p<5×10−8) for all seven variants. This included SNPs in or near LOC100506457 (rs1534422, p=4.03×10−12), CD28 (rs6435203, p=1.35×10−9), LPP (rs4686953, p=3.35×10−8), ETS1 (rs3809006, p=7.74×10−9), DLEU1 (rs806349, p=8.14×10−12), LPIN3 (rs6072343, p=7.16×10−12) and IFNGR2 (rs9808753, p=4.40×10−10). Cis expression quantitative locus effects were observed in silico for rs6435203 on CD28 and for rs9808753 on several immunologically relevant genes in the IFNGR2 locus. Conclusions This study adds seven loci to the list of genuine MS genetic risk factors and further extends the list of established loci shared across autoimmune diseases.

Ancient and Recent Selective Pressures Shaped Genetic Diversity at AIM2-Like Nucleic Acid Sensors

AIM2-like receptors (ALRs) are a family of nucleic acid sensors essential for innate immune responses against viruses and bacteria. We performed an evolutionary analysis of ALR genes (MNDA, PYHIN1, IFI16, and AIM2) by analyzing inter- and intraspecies diversity. Maximum-likelihood analyses indicated that IFI16 and AIM2 evolved adaptively in primates, with branch-specific selection at the catarrhini lineage for IFI16. Application of a population genetics–phylogenetics approach also allowed identification of positive selection events in the human lineage. Positive selection in primates targeted sites located at the DNA-binding interface in both IFI16 and AIM2. In IFI16, several sites positively selected in primates and in the human lineage were located in the PYD domain, which is involved in protein–protein interaction and is bound by a human cytomegalovirus immune evasion protein. Finally, positive selection was found to target nuclear localization signals in IFI16 and the spacer region separating the two HIN domains. Population genetic analysis in humans revealed that an IFI16 genic region has been a target of long-standing balancing selection, possibly acting on two nonsynonymous polymorphisms located in the spacer region. Data herein indicate that ALRs have been repeatedly targeted by natural selection. The balancing selection region in IFI16 carries a variant with opposite risk effect for distinct autoimmune diseases, suggesting antagonistic pleiotropy. We propose that the underlying scenario is the result of an ancestral and still ongoing host–pathogen arms race and that the maintenance of susceptibility alleles for autoimmune diseases at IFI16 represents an evolutionary trade-off.

TNFRSF1A polymorphisms rs1800693 and rs4149584 in patients with multiple sclerosis.

Objectives: To investigate the roles of 2 polymorphisms of the tumor necrosis factor (TNF) receptor superfamily member 1A (TNFRSF1A) gene, rs1800693 (a common variant) and rs4149584 (a coding polymorphism that results in an amino acid substitution—R92Q), as genetic modifiers of multiple sclerosis (MS), and to evaluate their potential functional implications in the disease. Methods: The effects of rs1800693 and rs4149584 on 2 measures of disease severity, age at disease onset and Multiple Sclerosis Severity Score, were analyzed in 2,032 patients with MS. In a subgroup of patients, serum levels of the soluble form of TNF-R1 (sTNF-R1) were measured by ELISA; mRNA expression levels of the full-length TNF-R1 and Δ6-TNF-R1 isoform were investigated in peripheral blood mononuclear cells (PBMC) by real-time PCR; cell surface expression of the TNF-R1 was determined in T cells by flow cytometry. Results: For rs4149584, R92Q carriers were younger at disease onset and progressed slower compared to noncarriers. However, no association with disease severity was observed for rs1800693. Serum levels of sTNF-R1 and mRNA expression levels of the full-length receptor were significantly increased in patients with MS carrying the R92Q mutation (p = 0.003 and p = 0.011, respectively), but similarly distributed among rs1800693 genotypes; cell surface TNF-R1 expression in T cells did not differ between rs4149584 and rs1800693 genotypes. The truncated soluble Δ6-TNF-R1 isoform was identified in PBMC from patients carrying the risk allele for rs1800693. Conclusions: These findings suggest that both rs1800693 and rs4149584 TNFRSF1A polymorphisms have functional consequences in the TNF-R1.

A functional variant that affects exon-skipping and protein expression of SP140 as genetic mechanism predisposing to multiple sclerosis

Several variants in strong linkage disequilibrium (LD) at the SP140 locus have been associated with multiple sclerosis (MS), Crohns disease (CD) and chronic lymphocytic leukemia (CLL). To determine the causal polymorphism, we have integrated high-density data sets of expression quantitative trait loci (eQTL), using GEUVADIS RNA sequences and 1000 Genomes genotypes, with MS-risk variants of the high-density Immunochip array performed by the International Multiple Sclerosis Genetic Consortium (IMSGC). The variants most associated with MS were also correlated with a decreased expression of the full-length RNA isoform of SP140 and an increase of an isoform lacking exon 7. By exon splicing assay, we have demonstrated that the rs28445040 variant was the causal factor for skipping of exon 7. Western blots of peripheral blood mononuclear cells from MS patients showed a significant allele-dependent reduction of the SP140 protein expression. To confirm the association of this functional variant with MS and to compare it with the best-associated variant previously reported by GWAS (rs10201872), a case-control study including 4384 MS patients and 3197 controls was performed. Both variants, in strong LD (r(2) = 0.93), were found similarly associated with MS [P-values, odds ratios: 1.9E-9, OR = 1.35 (1.22-1.49) and 4.9E-10, OR = 1.37 (1.24-1.51), respectively]. In conclusion, our data uncover the causal variant for the SP140 locus and the molecular mechanism associated with MS risk. In addition, this study and others previously reported strongly suggest that this functional variant may be shared with other immune-mediated diseases as CD and CLL.

IL28B polymorphisms are not associated with the response to interferon-beta in multiple sclerosis

Recent studies have revealed an association between interleukin 28B (IL28B) and response to IFN-alpha treatment in hepatitis C patients. Here we investigated the influence of IL28B polymorphisms in the response to interferon-beta (IFNβ) in multiple sclerosis (MS) patients. We genotyped two SNPs of the IL28B gene (rs8099917 and rs12979860) in 588 MS patients classified into responders (n=281) and non-responders (n=307) to IFNβ. Combined analysis of the study cohorts showed no significant associations between SNPs rs8099917 and rs12979860 and the response to treatment. These findings do not support a role of IL28B polymorphisms in the response to IFNβ in MS patients.

Role of high mobility group box protein 1 (HMGB1) in peripheral blood from patients with multiple sclerosis

BackgroundHigh mobility group box protein 1 (HMGB1) is a transcriptional regulator that is receiving increasing attention in autoimmune disorders including multiple sclerosis (MS). Here, we investigated the role of HMGB1 in the peripheral blood compartment from MS patients.MethodsHMGB1 mRNA expression levels were determined by PCR in peripheral blood mononuclear cells (PBMC) of 29 healthy controls and 57 untreated MS patients (26 with relapsing-remitting MS - RRMS, 13 with secondary progressive MS - SPMS, and 18 with primary progressive MS - PPMS). HMGB1 protein levels were measured by ELISA in serum samples from 18 HC and 37 untreated MS patients (13 with RRMS, 14 with SPMS, and 10 with PPMS).ResultsHMGB1 expression levels were increased in PBMC from the whole MS group compared with controls (P = 0.03). Further stratification of the MS group revealed higher expression levels in PBMC from patients with relapse-onset MS, and differences were statistically significant for RRMS patients compared with PPMS patients and controls (P = 4 × 10−5 and P = 0.005, respectively) and also for SPMS patients compared with PPMS patients (P = 0.001). HMGB1 serum levels were increased in the whole MS group compared with controls (P = 2 × 10−4). In MS clinical forms, the highest HMGB1 serum levels were observed in RRMS patients, and differences were statistically significant compared to PPMS patients (P = 5 × 10−5), SPMS patients (P = 0.001), and controls (P = 0.001).ConclusionsThese results point to a role of HMGB1 mRNA and protein levels as disease activity biomarkers to discriminate the more inflammatory relapse-onset MS forms, particularly RRMS, from the less inflammatory PPMS form of the disease.