Methotrexate Treatment of Newly Diagnosed RA Patients Is Associated With DNA Methylation Differences at Genes Relevant for Disease Pathogenesis and Pharmacological Action

Background: Methotrexate (MTX) is the fi rst line treatment of rheumatoid arthritis (RA), and methylation changes in bulk T cells have been reported after treatment with MTX. We have investigated cell-type speci fi c DNA methylation changes across the genome in naïve and memory CD4 + T cells before and after MTX treatment of RA patients. DNA methylation pro fi les of newly diagnosed RA patients (N=9) were assessed by reduced representation bisul fi te sequencing. Results: We found that MTX treatment signi fi cantly in fl uenced DNA methylation levels at multiple CpG sites in both cell populations. Interestingly, we identi fi ed differentially methylated sites annotated to two genes; TRIM15 and SORC2, previously reported to predict treatment outcome in RA patients when measured in bulk T cells. Furthermore, several of the genes, including STAT3, annotated to the signi fi cant CpG sites are relevant for RA susceptibility or the action of MTX. Conclusion: We detected CpG sites that were associated with MTX treatment in CD4 + naïve and memory T cells isolated from RA patients. Several of these sites overlap genetic regions previously associated with RA risk and MTX treatment outcome

Late Onset Myasthenia Gravis Is Associated with HLA DRB1*15:01 in the Norwegian Population

BACKGROUND: Acquired myasthenia gravis (MG) is a rare antibody-mediated autoimmune disease caused by impaired neuromuscular transmission, leading to abnormal muscle fatigability. The aetiology is complex, including genetic risk factors of the human leukocyte antigen (HLA) complex and unknown environmental factors. Although associations between the HLA complex and MG are well established, not all involved components of the HLA predisposition to this heterogeneous disease have been revealed. Well-powered and comprehensive HLA analyses of subgroups in MG are warranted, especially in late onset MG. METHODOLOGY/PRINCIPAL FINDINGS: This case-control association study is of a large population-based Norwegian cohort of 369 MG patients and 651 healthy controls. We performed comprehensive genotyping of four classical HLA loci (HLA-A, -B, -C and -DRB1) and showed that the DRB1*15:01 allele conferred the strongest risk in late onset MG (LOMG; onset ≥ 60 years) (OR 2.38, p(c)7.4 × 10(-5)). DRB1*13:01 was found to be a protective allele for both early onset MG (EOMG) and LOMG (OR 0.31, p(c) 4.71 × 10(-4)), a finding not previously described. No significant association was found to the DRB1*07:01 allele (p(nc) = 0.18) in a subset of nonthymomatous anti-titin antibody positive LOMG as reported by others. HLA-B*08 was mapped to give the strongest contribution to EOMG, supporting previous studies. CONCLUSION: The results from this study provide important new information concerning the susceptibility of HLA alleles in Caucasian MG, with highlights on DRB1*15:01 as being a major risk allele in LOMG

Transcriptome profiling of human thymic CD4+ and CD8+ T cells compared to primary peripheral T cells

Background The thymus is a highly specialized organ of the immune system where T cell precursors develop and differentiate into self-tolerant CD4+ or CD8+ T cells. No studies to date have investigated how the human transcriptome profiles differ, between T cells still residing in the thymus and T cells in the periphery. Results We have performed high-throughput RNA sequencing to characterize the transcriptomes of primary single positive (SP) CD4+ and CD8+ T cells from infant thymic tissue, as well as primary CD4+ and CD8+ T cells from infant and adult peripheral blood, to enable the comparisons across tissues and ages. In addition, we have assessed the expression of candidate genes related to autoimmune diseases in thymic CD4+ and CD8+ T cells. The thymic T cells showed the largest number of uniquely expressed genes, suggesting a more diverse transcription in thymic T cells. Comparing T cells of thymic and blood origin, revealed more differentially expressed genes, than between infant and adult blood. Functional enrichment analysis revealed an over-representation of genes involved in cell cycle and replication in thymic T cells, whereas infant blood T cells were dominated by immune related terms. Comparing adult and infant blood T cells, the former was enriched for inflammatory response, cytokine production and biological adhesion, while upregulated genes in infant blood T cells were associated with cell cycle, cell death and gene expression. Conclusion This study provides valuable insight into the transcriptomes of the human primary SP T cells still residing within the thymus, and offers a unique comparison to primary blood derived T cells. Interestingly, the majority of autoimmune disease associated genes were expressed in one or more T cell subset, however ~ 11% of these were not expressed in frequently studied adult peripheral blood

Clinical characteristics of the Norwegian myasthenia gravis study cohort (n = 369).

1<p>According to MGFA classification <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036603#pone.0036603-JaretzkiA1" target="_blank">[39]</a>: ocular MG (MGFA grade I), generalised MG (MGFA grade II-V), not available in 12 cases.</p>2<p>Concomitant immune-mediated diseases include thyroid disease (hypo-, hyperthyroidsm, thyroiditis), type 1-diabetes, rheumatic diseases, systemic lupus erythematosus (SLE), celiac disease, inflammatory bowel diseases (Crohńs disease or ulcerative colitits). EOMG = early onset MG; LOMG = late onset MG; AChR-ab+  =  acetylcholine receptor-antibody positive.</p

The most strongly associated HLA alleles in early onset myasthenia gravis (EOMG).

<p>Odds Ratio (OR) and 95% confidence interval (CI) are shown for nominal p-values.</p

Conditional analyses of the HLA-A, -B, -C and DRB1 loci (p-values) in early onset myasthenia gravis (EOMG).

<p>P-values are not corrected, p<0.01 is considered significant after correction for multiple comparisons (n = 4).</p

The association between HLA loci and myasthenia gravis subgroups (p-values).

<p>EOMG = early onset MG, LOMG = late onset MG.</p><p>P-values are not corrected, p<0.01 is considered significant after correction for multiple comparisons (n = 4).</p

Associated alleles in late onset myasthenia gravis (LOMG).

<p>Odds Ratio (OR) and 95% confidence interval (CI) are shown for nominal p-values.</p

Assessment of the strongest association on the HLA-B*08-DRB1*03:01 haplotype in early onset myasthenia gravis (EOMG).

<p>+  =  positives, −  =  negatives.</p