Association of Genetic Markers with CSF Oligoclonal Bands in Multiple Sclerosis Patients

Objective:to explore the association between genetic markers and Oligoclonal Bands (OCB) in the Cerebro Spinal Fluid (CSF) of Italian Multiple Sclerosis patients.Methods:We genotyped 1115 Italian patients for HLA-DRB1*15 and HLA-A*02. In a subset of 925 patients we tested association with 52 non-HLA SNPs associated with MS susceptibility and we calculated a weighted Genetic Risk Score. Finally, we performed a Genome Wide Association Study (GWAS) with OCB status on a subset of 562 patients. The best associated SNPs of the Italian GWAS were replicated in silico in Scandinavian and Belgian populations, and meta-analyzed.Results:HLA-DRB1*15 is associated with OCB+: p = 0.03, Odds Ratio (OR) = 1.6, 95% Confidence Limits (CL) = 1.1-2.4. None of the 52 non-HLA MS susceptibility loci was associated with OCB, except one SNP (rs2546890) near IL12B gene (OR: 1.45; 1.09-1.92). The weighted Genetic Risk Score mean was significantly (p = 0.0008) higher in OCB+ (7.668) than in OCB- (7.412) patients. After meta-analysis on the three datasets (Italian, Scandinavian and Belgian) for the best associated signals resulted from the Italian GWAS, the strongest signal was a SNP (rs9320598) on chromosome 6q (p = 9.4×10-7) outside the HLA region (65 Mb).Discussion:genetic factors predispose to the development of OCB

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

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

Low-Frequency and Rare-Coding Variation Contributes to Multiple Sclerosis Risk

Multiple sclerosis is a complex neurological disease, with 3c20% of risk heritability attributable to common genetic variants, including >230 identified by genome-wide association studies. Multiple strands of evidence suggest that much of the remaining heritability is also due to additive effects of common variants rather than epistasis between these variants or mutations exclusive to individual families. Here, we show in 68,379 cases and controls that up to 5% of this heritability is explained by low-frequency variation in gene coding sequence. We identify four novel genes driving MS risk independently of common-variant signals, highlighting key pathogenic roles for regulatory T cell homeostasis and regulation, IFN\u3b3 biology, and NF\u3baB signaling. As low-frequency variants do not show substantial linkage disequilibrium with other variants, and as coding variants are more interpretable and experimentally tractable than non-coding variation, our discoveries constitute a rich resource for dissecting the pathobiology of MS. In a large multi-cohort study, unexplained heritability for multiple sclerosis is detected in low-frequency coding variants that are missed by GWAS analyses, further underscoring the role of immune genes in MS pathology

Characterization of <i>SH2D2A</i>-deficient Id-specific TCR-transgenic CD4+ T cells.

<p>(A) Thymocytes from Id-specific TCR-transgenic mice with indicated genotypes (9–15 weeks old) were labeled with anti-transgenic TCR (GB113), anti-CD4 and -CD8 mAbs. Expression of CD4 and CD8 on GB113+ thymocytes was monitored by flow cytometry. The mean average percentage with SD of double positive (DP), double negative (DN), single positive (SP) GB113+ CD4+ and CD8+ thymocytes is indicated for each genotype (n = 3). (B) Splenocytes from normal (<i>SH2D2A</i>+/+) and age-matched <i>SH2D2A-</i>deficient (<i>SH2D2A</i>−/−) mice (6–14 weeks old) were labeled with anti-CD4 and GB113 and expression was monitored by flow cytometry. Diagrams show the frequency of CD4+ T cells (left diagram) and the percentage of CD4+ T cells that express the Id-specific transgenic TCR (GB113+) (right diagram). The median values are shown as lines in the diagrams. (C) CFSE labeled CD4+ T cells from TCR transgenic <i>SH2D2A</i>+/+ (upper diagram) and <i>SH2D2A</i>−/− (lower diagram) were harvested after 24, 48 and 72 hours in culture with Id-positive F9, labeled with GB113 antibody. The CFSE dilution in GB113+ CD4+ T cells was measured by flow cytometry; grey area - CFSE profiles after 24 hours, open area (black line) - CFSE profiles after 48 hours and open area (stippled line) - CFSE profiles after 72 hours. One representative experiment out of four is shown. (D) Unlabeled CD4+ T cells from <i>SH2D2A</i>+/+ and <i>SH2D2A</i>−/− mice were cultivated as in C. Prior to and after 24, 48 and 72 hours, cells were stained with GB113 in addition to CD69, CD44, CD62L or CD25 mAbs to assess T cell activation by flow cytometry. FACS plots gated on GB113+ CD4+ T cells from one representative experiment of at least three are shown.</p

TCR transgenic <i>SH2D2A</i> −<b>/</b>− mice are resistant towards transplanted myeloma.

<p>Non-transgenic BALB/c and Id-specific TCR-transgenic (Id-TCR) BALB/c mice with (+/+) or without (−/−) <i>SH2D2A</i> expression were injected subcutaneously with low dose MOPC315 cells (160 000 cells). Tumor development was followed by palpation. (A, B) A tumor-free mouse was defined as a mouse that did not have a palpable tumor during the course of the experiment. The plots display tumor-take for (A) non-transgenic BALB/c mice with (n = 9) or without (n = 11) <i>SH2D2A</i> expression and (B) Id-specific TCR-transgenic BALB/c mice with (n = 23) or without (n = 23) <i>SH2D2A</i> expression. P values were calculated with two-tailed log rank test. Ns = non-significant.(C, D) Splenic CD4+ T cells were isolated from surviving tumor-free mice and stimulated <i>in vitro</i> with Id-positive F9 cells. Cells were labeled with GB113, recognizing the Id-specific transgenic TCR, and anti-CD69, CD44, CD62L or CD25 mAbs, prior to and after 24, 48 and 72 hours in culture with Id-positive cells. (C) FACS plots gated on GB113+ CD4+ T cells from one representative experiment are shown. (D) The diagram show the median value of the MFI (median fluorescent intensity) of CD69 at the indicated time points (n = 11) with SD. P-values were calculated with two-tailed, unpaired student t test, * indicate significant differences (at 48 hours: p = 0,003; at 72 hours: p = 0,03).</p

<i>SH2D2A</i>-deficient mice have increased numbers of Id-specific TCR–transgenic SP CD4+ thymocytes.

<p>The graphs represents from the left; “UC” –unchallenged mice, i.e. control Id-specific TCR-transgenic mice that did not receive tumor cells, wild-type (<i>SH2D2A</i>+/+) and <i>SH2D2A</i> deficient (<i>SH2D2A</i>−/−) mice that had to be sacrificed due to development of large tumors (tumor volume > 1 cm³) (T = tumor) or that was tumor free at the end of the experiment 70 days after tumor inoculation (TF = tumor free). These tumor-free mice never had a palpable tumor during the course of the experiment, or they had a tumor that never reached 1 cm³ and that disappeared prior to the end of the experiment. The median values are shown as lines in the diagrams. (A, B) Thymocytes from mice with indicated genotypes were labeled with anti Id-TCR (GB113), anti-CD4 and anti-CD8 mAbs and their expressions were monitored by flow cytometry. The total number of Id-specific TCR-transgenic (GB113+) double positive (DP, CD4+ CD8+) and single positive CD4+ (SP, CD4+ CD8−) thymocytes is indicated for each genotype. (C) The diagram display the number of GB113+ CD4+ T cells in the tumor-draining lymph node of the same mice as in A and B. Of note is that the number of GB113+ CD4+ T cells in unchallenged mice is very low due to the small size of the draining lymph node in the absence of tumor challenge. (D) The GB113+ CD4+ T cells from the draining lymph node were labeled with anti-CD69 mAb, and their expression was monitored by flow cytometry. The diagram shows the median fluorescence intensity (MFI) of CD69 divided by the MFI of isotype control in GB113+ CD4+ T cells. P values were calculated with two-tailed Mann-Whitney U test. Ns = non-significant.</p

<i>SH2D2A</i>-deficiency does not have a major effect on anti-CD3/CD28 induced CD4+ T cell proliferation or expression of activation markers.

<p>(A) CFSE labeled CD4+ T cells from wild-type (<i>SH2D2A</i>+/+) and <i>SH2D2A</i>-deficient (<i>SH2D2A</i>-/-) C57BL/6 mice were cultivated with anti-CD3/CD28 beads as described in materials and methods. After 24, 48 and 72 hours, cells were harvested and the CFSE dilutions of <i>SH2D2A</i>+/+ CD4+ T cells (left diagram) and <i>SH2D2A</i>−/− CD4+ T cells (right diagram) were measured by flow cytometry. Grey area - CFSE profiles after 24 hours, open area (black line) - CFSE profiles after 48 hours and open area (stippled line) - CFSE profiles after 72 hours in culture with anti-CD3/CD28 beads. (B, C) Unlabeled CD4+ T cells from <i>SH2D2A</i>+/+ and <i>SH2D2A</i>−/− C57BL/6 (C, left side) and BALB/c (B and right side of C) mice were cultivated as in A. Prior to and after 24, 48 and 72 hours, cells were stained with CD69, CD44, CD62L or CD25 mAbs to assess T cell activation. (B) The percentage of CD62L positive CD4+ T cells from BALB/c mice is shown after indicated time points of anti-CD3/28 bead stimulation. P values were calculated with unpaired t-test, *p < 0.05. (C) FACS plots gated on CD4+ T cells from one representative experiment of at least three is shown. Solid line – <i>SH2D2A</i>+/+ CD4+ T cells, dashed line - <i>SH2D2A</i>−/− CD4+ T cells. (D) CD4+ T cells from <i>SH2D2A</i>+/+ and <i>SH2D2A</i>−/− C57BL/6 mice were cultivated with 1 µg/ml soluble αCD28 mAb and either 1, 5 or 10 µg/ml plate-bound αCD3 mAb for 24 hours prior to staining with the indicated mAbs. The percentage of positive cells is shown as a function of µg/ml of stimulating αCD3 mAb. NS – non-stimulated.</p

Schematic depiction of the MOPC315 model.

<p>(A) The MHCII negative MOPC315 cells secrete the IgA myeloma protein M315. This protein is endocytosed and processed by antigen-presenting cells (APCs), and CD4+ T cells with transgenic TCRs recognize a myeloma-derived idiotypic (Id³¹⁵) peptide in the context of the MHC class II I-E^d molecule. (B) Normal BALB/c mice are susceptible to tumor development upon injection with MOPC315 cells, while mice with the Id³¹⁵-TCR transgene are resistant towards tumor development. Resistance is mediated by tumor specific (Id-specific) TCR transgenic CD4+ T cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048239#pone.0048239-Bogen3" target="_blank">[16]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048239#pone.0048239-Corthay2" target="_blank">[19]</a>.</p