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1.  Autoimmune disease in the epigenetic era: how has epigenetics changed our understanding of disease and how can we expect the field to evolve? 
Autoimmune diseases are complex and enigmatic, and have presented particular challenges to researchers seeking to define their etiology and explain progression. Previous studies have implicated epigenetic influences in the development of autoimmunity. Epigenetics describes changes in gene expression related to environmental influences without alterations in the underlying genomic sequence, generally classified into three main groups: cytosine genomic DNA methylation, modification of various sidechain positions of histone proteins, and noncoding RNAs. The purpose of this article is to review the most relevant literature describing alterations of epigenetic marks in the development and progression of four common autoimmune diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), and Sjogren’s syndrome (SS). The contribution of DNA methylation, histone modification, and noncoding RNA for each of these disorders is discussed, including examples both of candidate studies and larger epigenomics surveys, and in various tissue types important for the pathogenesis of each. The future of the field is speculated briefly, as is the possibility of therapeutic interventions targeting the epigenome.
doi:10.1586/1744666X.2015.994507
PMCID: PMC4636192  PMID: 25534978
epigenetics; methylation; histone modification; microRNA; autoimmune disease; systemic lupus erythematosus; rheumatoid arthritis; systemic sclerosis; Sjögren’s syndrome
2.  DNA methylation patterns in naïve CD4+ T cells identify epigenetic susceptibility loci for malar rash and discoid rash in systemic lupus erythematosus 
Lupus Science & Medicine  2015;2(1):e000101.
Objective
Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterised by heterogeneous clinical manifestations, autoantibody production and epigenetic dysregulation in T cells. We sought to investigate the epigenetic contribution to the development of cutaneous manifestations in SLE.
Methods
We performed genome-wide DNA methylation analyses in patients with SLE stratified by a history of malar rash, discoid rash or neither cutaneous manifestation, and age, sex and ethnicity matched healthy controls. We characterised differentially methylated regions (DMRs) in naïve CD4+ T cells unique to each disease subset, and assessed functional relationships between DMRs using bioinformatic approaches.
Results
We identified 36 and 37 unique DMRs that contribute to the epigenetic susceptibility to malar rash and discoid rash, respectively. These DMRs were primarily localised to genes mediating cell proliferation and apoptosis. Hypomethylation of MIR886 and TRIM69, and hypermethylation of RNF39 were specific to patients with SLE with a history of malar rash. Hypomethylation of the cytoskeleton-related gene RHOJ was specific to patients with SLE with a history of discoid rash. In addition, discoid rash-specific hypomethylated DMRs were found in genes involved in antigen-processing and presentation such as TAP1 and PSMB8. Network analyses showed that DMRs in patients with SLE with but not without a history of cutaneous manifestations are associated with TAP-dependent processing and major histocompatibility-class I antigen cross-presentation (p=3.66×10−18 in malar rash, and 3.67×10−13 in discoid rash).
Conclusions
We characterised DNA methylation changes in naïve CD4+ T cells specific to malar rash and discoid rash in patients with SLE. These data suggest unique epigenetic susceptibility loci that predispose to or are associated with the development of cutaneous manifestations in SLE.
doi:10.1136/lupus-2015-000101
PMCID: PMC4577980  PMID: 26405558
Systemic Lupus Erythematosus; Gene Polymorphism; Autoimmune Diseases
3.  Epigenome-wide scan identifies a treatment-responsive pattern of altered DNA methylation among cytoskeletal remodeling genes in monocytes and CD4+ T cells in Behçet’s disease 
Objective
Behçet’s disease (BD) is an inflammatory disease characterized by multi-system involvement including recurrent oral and genital ulcers, cutaneous lesions, and uveitis. The pathogenesis of BD remains poorly understood. We performed a genome-wide DNA methylation study in BD before and after disease remission, and in healthy matched controls.
Methods
We examined genome-wide DNA methylation in monocytes and CD4+ T cells from a set of 16 untreated male BD patients and age, sex, and ethnicity-matched controls. Additional samples were collected from 12 of the same BD patients after treatment and disease remission. Genome-wide DNA methylation patterns were assessed using the HumanMethylation450 DNA Analysis BeadChip array which includes over 485,000 individual methylation sites across the genome.
Results
We identified 383 differentially methylated CpG sites between BD patients and controls in monocytes and 125 differentially methylated CpG sites in CD4+ T cells. Bioinformatic analysis revealed a pattern of aberrant DNA methylation among genes that regulate cytoskeletal dynamics suggesting that aberrant DNA methylation of multiple classes of structural and regulatory proteins of the cytoskeleton might contribute to the pathogenesis of BD. Further, DNA methylation changes associated with treatment act to restore methylation differences observed between patients and controls. Indeed, among CpG sites differentially methylated before and after disease remission, there was almost exclusive reversal of the direction of aberrant DNA methylation observed between patients and healthy controls.
Conclusions
We performed the first epigenome-wide study in BD and provide strong evidence that epigenetic modification of cytoskeletal dynamics underlies the pathogenesis and therapeutic response in BD.
doi:10.1002/art.38409
PMCID: PMC4096298  PMID: 24574333
Behçet’s Disease; Cytoskeleton; Epigenetics; DNA Methylation
4.  Genome-wide DNA methylation patterns in naïve CD4+ T cells from patients with primary Sjögren’s syndrome 
Objective
Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease with incompletely understood etiology. Very little is known about the role of epigenetic dysregulation in the pathogenesis of pSS.
Methods
We performed a genome-wide DNA methylation study in naïve CD4+ T cells in eleven pSS patients compared to age-, sex-, and ethnicity-matched healthy controls. Cytosine methylation was quantified using the Illumina Infinium HumanMethylation450 BeadChip array and validated using bisulfite sequencing.
Results
We identified 553 hypomethylated and 200 hypermethylated CpG sites in naïve CD4+ T cells from pSS patients compared to healthy matched controls, representing 311 hypomethylated and 115 hypermethylated gene regions. Hypomethylated genes in pSS include LTA, coding for Lymphotoxin α. Other relevant genes such as CD247, TNFRSF25, PTPRC, GSTM1 and PDCD1 were also hypomethylated. The interferon signature pathway was represented by hypomethylation of STAT1, IFI44L, USP18 and IFITM1. A group of genes encoding for members of the solute carrier proteins were differentially methylated. In addition, the transcription factor RUNX1 was hypermethylated in patients, suggesting a possible connection to lymphoma predisposition. Gene ontology (GO) analysis of hypomethylated genes demonstrated enrichment of genes involved in lymphocyte activation and immune response. GO terms for hypermethylated genes included antigen processing and presentation.
Conclusion
This is the first epigenome-wide DNA methylation study in pSS. Our data highlight a role for DNA methylation in pSS and identify disease-associated DNA methylation changes in several genes and pathways in naïve CD4+ T cells in pSS that may be involved in the pathogenesis of this disease.
doi:10.1002/art.38264
PMCID: PMC4009982  PMID: 24574234
5.  Overexpression of methyl-CpG-binding protein 2 and autoimmunity: Evidence from MECP2 duplication syndrome, lupus, MECP2 transgenic and Mecp2 deficient mice 
Lupus  2013;22(9):870-872.
Methyl-CpG-binding protein 2 (MeCP2) is a key transcriptional regulator that can induce either silencing or activation of target genes. Genetic polymorphisms in the MECP2/IRAK1 locus have been associated with increased susceptibility to multiple autoimmune diseases such as lupus, primary Sjogren's syndrome, and more recently rheumatoid arthritis. Data from our group suggest that the disease risk variant in this locus is associated with gain of MeCP2 function. Recent findings indicate that MECP2 duplication in human results in defective T helper cell type 1 (TH1) response and IFN-γ production. Herein, we discuss the data from children with MECP2 duplication, human lupus, and from the human MECP2 transgenic and Mecp2 deficient mice to support a link between MECP2 overexpression and autoimmunity. We also provide findings from an Mecp2 deficient mouse that independently support a role for MeCP2 in the immune response and specifically in IFN-γ expression.
doi:10.1177/0961203313497119
PMCID: PMC3790641  PMID: 23861028
MeCP2; MECP2 duplication; lupus; autoimmunity; TH1 response
6.  Genome-wide DNA methylation study suggests epigenetic accessibility and transcriptional poising of interferon-regulated genes in naïve CD4+ T cells from lupus patients 
Journal of autoimmunity  2013;43:78-84.
Systemic lupus erythematosus is an autoimmune disease characterized by multi-system involvement and autoantibody production. Abnormal T cell DNA methylation and type-I interferon play an important role in the pathogenesis of lupus. We performed a genome-wide DNA methylation study in two independent sets of lupus patients and matched healthy controls to characterize the DNA methylome in naïve CD4+ T cells in lupus. DNA methylation was quantified for over 485,000 methylation sites across the genome, and differentially methylated sites between lupus patients and controls were identified and then independently replicated. Gene expression analysis was also performed from the same cells to investigate the relationship between the DNA methylation changes observed and mRNA expression levels. We identified and replicated 86 differentially methylated CG sites between patients and controls in 47 genes, with the majority being hypomethylated. We observed significant hypomethylation in interferon-regulated genes in naïve T cells from lupus patients, including IFIT1, IFIT3, MX1, STAT1, IFI44L, USP18, TRIM22 and BST2, suggesting epigenetic transcriptional accessibility in these genetic loci. Indeed, the majority of the hypomethylated genes (21 out of 35 hypomethylated genes) are regulated by type I interferon. The hypomethylation in interferon-regulated genes was not related to lupus disease activity. Gene expression analysis showed overexpression of these genes in total but not naïve CD4+ T cells from lupus patients. Our data suggest epigenetic “poising” of interferon-regulated genes in lupus naïve CD4+ T cells, argue for a novel pathogenic implication for abnormal T cell DNA methylation in lupus, and suggest a mechanism for type-I interferon hyper-responsiveness in lupus T cells.
doi:10.1016/j.jaut.2013.04.003
PMCID: PMC3790645  PMID: 23623029
Lupus; naïve CD4+ T cells; methylome; DNA methylation
7.  Functional characterization of the MECP2/IRAK1 lupus risk haplotype in human T cells and a human MECP2 transgenic mouse 
Journal of autoimmunity  2013;41:168-174.
Genetic polymorphism in MECP2/IRAK1 on chromosome Xq28 is a confirmed and replicated susceptibility locus for lupus. High linkage disequilibrium in this locus suggests that both MECP2 and IRAK1 are candidate genes for the disease. DNA methylation changes in lupus T cells play a central role in the pathogenesis of lupus, and MeCp-2 (encoded by MECP2) is a master regulator of gene expression and is also known to recruit DNA methyltransferase 1 (DNMT1) during DNA synthesis. Using human T cells from normal individuals with either the lupus risk or the lupus protective haplotype in MECP2/IRAK1, we demonstrate that polymorphism in this locus increases MECP2 isoform 2 mRNA expression in stimulated but not unstimulated T cells. By assessing DNA methylation levels across over 485,000 methylation sites across the entire genome, we further demonstrate that the lupus risk variant in this locus is associated with significant DNA methylation changes, including in the HLA-DR and HLA-DQ loci, as well as interferon-related genes such as IFI6, IRF6, and BST2. Further, using a human MECP2 transgenic mouse, we show that overexpression of MECP2 alters gene expression in stimulated T cells. This includes overexpression of Eif2c2 that regulates the expression of multiple microRNAs (such as miR-21), and the histone demethylase Jhdm1d. In addition, we show that MECP2 transgenic mice develop antinuclear antibodies. Our data suggest that the lupus associated variant in the MECP2/IRAK1 locus has the potential to affect all 3 epigenetic mechanisms: DNA methylation, microRNA expression, and histone modification. Importantly, these data support the notion that variants within the MECP2 gene can alter DNA methylation in other genetic loci including the HLA and interferon-regulated genes, thereby providing evidence for genetic-epigenetic interaction in lupus.
doi:10.1016/j.jaut.2012.12.012
PMCID: PMC3622940  PMID: 23428850
MECP2; IRAK1; lupus; epigenetics; polymorphism; DNA methylation; T cells; transgenic mouse
8.  Sex-specific differences in the relationship between genetic susceptibility, T cell DNA demethylation and lupus flare severity 
Journal of Autoimmunity  2012;38(2-3):J216-J222.
Lupus is less common in men than women, and the reason is incompletely understood. Current evidence indicates that lupus flares when genetically predisposed individuals encounter environmental agents that trigger the disease, and that the environmental contribution is mediated at least in part by T cell DNA demethylation. We hypothesized that lupus disease activity is directly related to total genetic risk and inversely related to T cell DNA methylation levels in each patient. Since women are predisposed to lupus in part because of their second X chromosome, we also hypothesized that men would require a greater genetic risk, a greater degree of autosomal T cell DNA demethylation, or both, to achieve a lupus flare equal in severity to women. Genetic risk was determined by genotyping men and women with lupus across 32 confirmed lupus susceptibility loci. The methylation status of two T cell autosomal genes known to demethylate in proportion to disease activity, KIR2DL4 (KIR) and PRF1, was measured by bisulfite sequencing. Lupus disease activity was determined by the SLEDAI. Interactions between genetic score, T cell DNA demethylation, and the SLEDAI score were compared between the men and women by regression analysis. Combining the degree of DNA demethylation with the genetic risk score for each patient demonstrated that the (genetic risk)/(DNA methylation) ratio increased directly with disease activity in both men and women with lupus. Importantly, men required a greater (genetic risk)/(DNA methylation) ratio to achieve a SLEDAI score equivalent to women (p=0.010 for KIR and p=0.0054 for PRF1). This difference was not explained by a difference in the genetic risk or T cell DNA demethylation alone, suggesting a genetic-epigenetic interaction. These results suggest that genetic risk and T cell DNA demethylation interact in lupus patients to influence the severity of lupus flares, and that men require a higher genetic risk and/or greater degree of T cell DNA demethylation to achieve a lupus flare equal in severity to women.
doi:10.1016/j.jaut.2011.11.008
PMCID: PMC3313010  PMID: 22305513
Genetic risk; epigenetics; DNA methylation; lupus; genetic-epigenetic interaction; sex-disparity
9.  Analysis of autosomal genes reveals gene–sex interactions and higher total genetic risk in men with systemic lupus erythematosus 
Annals of the Rheumatic Diseases  2011;71(5):694-699.
Objectives
Systemic lupus erythematosus (SLE) is a sexually dimorphic autoimmune disease which is more common in women, but affected men often experience a more severe disease. The genetic basis of sexual dimorphism in SLE is not clearly defined. A study was undertaken to examine sex-specific genetic effects among SLE susceptibility loci.
Methods
A total of 18 autosomal genetic susceptibility loci for SLE were genotyped in a large set of patients with SLE and controls of European descent, consisting of 5932 female and 1495 male samples. Sex-specific genetic association analyses were performed. The sex–gene interaction was further validated using parametric and nonparametric methods. Aggregate differences in sex-specific genetic risk were examined by calculating a cumulative genetic risk score for SLE in each individual and comparing the average genetic risk between male and female patients.
Results
A significantly higher cumulative genetic risk for SLE was observed in men than in women. (P = 4.52×10−8) A significant sex–gene interaction was seen primarily in the human leucocyte antigen (HLA) region but also in IRF5, whereby men with SLE possess a significantly higher frequency of risk alleles than women. The genetic effect observed in KIAA1542 is specific to women with SLE and does not seem to have a role in men.
Conclusions
The data indicate that men require a higher cumulative genetic load than women to develop SLE. These observations suggest that sex bias in autoimmunity could be influenced by autosomal genetic susceptibility loci.
doi:10.1136/annrheumdis-2011-200385
PMCID: PMC3324666  PMID: 22110124
10.  Evidence for gene-gene epistatic interactions among susceptibility loci for systemic lupus erythematosus 
Arthritis and Rheumatism  2012;64(2):485-492.
Objective
Several confirmed genetic susceptibility loci for lupus have been described. To date, no clear evidence for genetic epistasis is established in lupus. We test for gene-gene interactions in a number of known lupus susceptibility loci.
Methods
Eighteen SNPs tagging independent and confirmed lupus susceptibility loci were genotyped in a set of 4,248 lupus patients and 3,818 normal healthy controls of European descent. Epistasis was tested using a 2-step approach utilizing both parametric and non-parametric methods. The false discovery rate (FDR) method was used to correct for multiple testing.
Results
We detected and confirmed gene-gene interactions between the HLA region and CTLA4, IRF5, and ITGAM, and between PDCD1 and IL21 in lupus patients. The most significant interaction detected by parametric analysis was between rs3131379 in the HLA region and rs231775 in CTLA4 (Interaction odds ratio=1.19, z-score= 3.95, P= 7.8×10−5 (FDR≤0.05), PMDR= 5.9×10−45). Importantly, our data suggest that in lupus patients the presence of the HLA lupus-risk alleles in rs1270942 and rs3131379 increases the odds of also carrying the lupus-risk allele in IRF5 (rs2070197) by 17% and 16%, respectively (P= 0.0028 and 0.0047).
Conclusion
We provide evidence for gene-gene epistasis in systemic lupus erythematosus. These findings support a role for genetic interaction contributing to the complexity of lupus heritability.
doi:10.1002/art.33354
PMCID: PMC3268866  PMID: 21952918
11.  Identification of novel genetic susceptibility loci in African-American lupus patients using a candidate gene association study 
Arthritis and rheumatism  2011;63(11):3493-3501.
Objective
Candidate gene and genome-wide association studies have identified several disease susceptibility loci in lupus patients. These studies have been largely performed in European-derived and Asian lupus patients. In this study, we examine if some of these same susceptibility loci increase lupus risk in African-American individuals.
Methods
Single nucleotide polymorphisms tagging 15 independent lupus susceptibility loci were genotyped in a set of 1,724 lupus patients and 2,024 normal healthy controls of African-American descent. The loci examined included: PTPN22, FCGR2A, TNFSF4, STAT4, CTLA4, PDCD1, PXK, BANK1, MSH5 (HLA region), CFB (HLA region), C8orf13-BLK region, MBL2, KIAA1542, ITGAM, and MECP2/IRAK1.
Results
We provide the first evidence for genetic association between lupus and five susceptibility loci in African-American patients (C8orf13-BLK, BANK1, TNFSF4, KIAA1542 andCTLA4; P values= 8.0 × 10−6, 1.9 × 10−5, 5.7 × 10−5, 0.00099, 0.0045, respectively). Further, we confirm the genetic association between lupus and five additional lupus susceptibility loci (ITGAM, MSH5, CFB, STAT4, and FCGR2A; P values= 7.5 × 10−11, 5.2 × 10−8, 8.7 × 10−7, 0.0058, and 0.0070, respectively), and provide evidence for a genome-wide significance for the association between ITGAM and MSH5 (HLA region) for the first time in African-American lupus patients.
Conclusion
These findings provide evidence for novel genetic susceptibility loci for lupus in African-Americans and demonstrate that the majority of lupus susceptibility loci examined confer lupus risk across multiple ethnicities.
doi:10.1002/art.30563
PMCID: PMC3205224  PMID: 21792837
12.  A putative functional variant within the ubiquitin-associated domain-containing protein 2 gene (UBAC2) is associated with increased risk of Behçet’s disease 
Arthritis and rheumatism  2011;63(11):3607-3612.
Objectives
Using a genome-wide association scan and DNA pooling, we previously identified 5 novel genetic susceptibility loci for Behçet’s disease. Herein, we establish the genetic effect within the UBAC2 gene, replicate this genetic association, and identify a functional variant within this locus.
Methods
A total of 676 Behçet’s disease patients and 1,096 controls were studied. The discovery set included 156 patients and 167 controls from Turkey, and the replication sets included 376 patients and 369 controls, and 144 patients and 560 controls, from Turkey and Italy, respectively. Genotyping of 14 SNPs within and around UBAC2 was performed using TaqMan SNP genotyping assays.
Results
The genetic association between Behçet’s disease and UBAC2 was established, replicated and confirmed (Meta-analysis OR= 1.84, meta-analysis P= 1.69X10−7). Haplotype analysis identified both a disease risk and a protective haplotype (P= 0.00014 and 0.0075, respectively). Using conditional haplotype analysis we identified the SNP rs7999348 (A/G) within UBAC2 as the most likely SNP with a genetic effect independent of the haplotypic effect formed by the remaining associated SNPs in this locus. Indeed, we demonstrate that rs7999348 tags a functional variant associated with increased mRNA expression of a UBAC2 transcript variant in PBMCs of individuals homozygous for the Behçet’s disease-associated “G” allele. Further, our data suggest the possibility of multiple genetic effects that increase susceptibility to Behçet’s disease in the UBAC2 locus.
Conclusion
We established and confirmed the genetic association between UBAC2 and Behçet’s disease in three independent sets of patients and controls. We identified the minor allele in rs7999348 as a disease-risk allele that tags altered UBAC2 expression.
doi:10.1002/art.30604
PMCID: PMC3205238  PMID: 21918955
13.  Phenotypic associations of genetic susceptibility loci in systemic lupus erythematosus 
Annals of the rheumatic diseases  2011;70(10):1752-1757.
Objective
Systemic lupus erythematosus is a clinically heterogeneous autoimmune disease. A number of genetic loci that increase lupus susceptibility have been established. This study examines if these genetic loci also contribute to the clinical heterogeneity in lupus.
Materials and methods
4001 European-derived, 1547 Hispanic, 1590 African-American and 1191 Asian lupus patients were genotyped for 16 confirmed lupus susceptibility loci. Ancestry informative markers were genotyped to calculate and adjust for admixture. The association between the risk allele in each locus was determined and compared in patients with and without the various clinical manifestations included in the ACR criteria.
Results
Renal disorder was significantly correlated with the lupus risk allele in ITGAM (p=5.0×10−6, OR 1.25, 95% CI 1.12 to 1.35) and in TNFSF4 (p=0.0013, OR 1.14, 95% CI 1.07 to 1.25). Other significant findings include the association between risk alleles in FCGR2A and malar rash (p=0.0031, OR 1.11, 95% CI 1.17 to 1.33), ITGAM and discoid rash (p=0.0020, OR 1.20, 95% CI 1.06 to 1.33), STAT4 and protection from oral ulcers (p=0.0027, OR 0.89, 95% CI 0.83 to 0.96) and IL21 and haematological disorder (p=0.0027, OR 1.13, 95% CI 1.04 to 1.22). All these associations are significant with a false discovery rate of <0.05 and pass the significance threshold using Bonferroni correction for multiple testing.
Conclusion
Significant associations were found between lupus clinical manifestations and the FCGR2A, ITGAM, STAT4, TNSF4 and IL21 genes. The findings suggest that genetic profiling might be a useful tool to predict disease manifestations in lupus patients in the future.
doi:10.1136/ard.2011.154104
PMCID: PMC3232181  PMID: 21719445
14.  Fine mapping and trans-ethnic genotyping establish IL2/IL21 genetic association with lupus and localize this genetic effect to IL21 
Arthritis and rheumatism  2011;63(6):1689-1697.
Objective
Genetic association of the IL2/IL21 region at 4q27 has been previously reported in lupus and a number of autoimmune and inflammatory diseases. Herein, using a very large cohort of lupus patients and controls, we localize this genetic effect to the IL21 gene.
Methods
We genotyped 45 tag SNPs across the IL2/IL21 locus in two large independent lupus sample sets. We studied a European-derived set consisting of 4,248 lupus patients and 3,818 healthy controls, and an African-American set of 1,569 patients and 1,893 healthy controls. Imputation in 3,004 WTCCC additional control individuals was also performed. Genetic association between the genotyped markers was determined, and pair-wise conditional analysis was performed to localize the independent genetic effect in the IL2/IL21 locus in lupus.
Results
We established and confirmed the genetic association between IL2/IL21 and lupus. Using conditional analysis and trans-ethnic mapping, we localized the genetic effect in this locus to two SNPs in high linkage disequilibrium; rs907715 located within IL21 (OR=1.16 (1.10–1.22), P= 2.17 ×10−8), and rs6835457 located in the 3’-UTR flanking region of IL21 (OR= 1.11 (1.05–1.17), P= 9.35×10−5).
Conclusion
We have established the genetic association between lupus and IL2/IL21 with a genome-wide level of significance. Further, we localized this genetic association within the IL2/IL21 linkage disequilibrium block to IL21. If other autoimmune IL2/IL21 genetic associations are similarly localized, then the IL21 risk alleles would be predicted to operate in a fundamental mechanism that influences the course of a number of autoimmune disease processes.
doi:10.1002/art.30320
PMCID: PMC3106139  PMID: 21425124
15.  Genome-wide DNA methylation patterns in CD4+ T cells from patients with systemic lupus erythematosus 
Epigenetics  2011;6(5):593-601.
Systemic lupus erythematosus is a chronic-relapsing autoimmune disease of incompletely understood etiology. Recent evidence strongly supports an epigenetic contribution to the pathogenesis of lupus. To understand the extent and nature of dysregulated DNA methylation in lupus T cells, we performed a genome-wide DNA methylation study in CD4+ T cells in lupus patients compared to normal healthy controls. Cytosine methylation was quantified in 27,578 CG sites located within the promoter regions of 14,495 genes. We identified 236 hypomethylated and 105 hypermethylated CG sites in lupus CD4+ T cells compared to normal controls, consistent with widespread DNA methylation changes in lupus T cells. Of interest, hypomethylated genes in lupus T cells include CD9, which is known to provide potent T-cell co-stimulation signals. Other genes with known involvement in autoimmunity such as MMP9 and PDGFRA were also hypomethylated. The BST2 gene, an interferon-inducible membrane-bound protein that helps restrict the release of retroviral particles was also hypomethylated in lupus patients. Genes involved in folate biosynthesis, which plays a role in DNA methylation, were overrepresented among hypermethylated genes. In addition, the transcription factor RUNX3 was hypermethylated in patients, suggesting an impact on T-cell maturation. Protein-protein interaction maps identified a transcription factor, HNF4a, as a regulatory hub affecting a number of differentially methylated genes. Apoptosis was also an overrepresented ontology in these interaction maps. Further, our data suggest that the methylation status of RAB22A, STX1B2, LGALS3BP, DNASE1L1 and PREX1 correlates with disease activity in lupus patients.
doi:10.4161/epi.6.5.15374
PMCID: PMC3121972  PMID: 21436623
lupus; T cells; CD4+ T cells; methylation; methylome
16.  Early disease onset is predicted by a higher genetic risk for lupus and is associated with a more severe phenotype in lupus patients 
Annals of the rheumatic diseases  2010;70(1):151-156.
Background
Systemic lupus erythematosus (SLE) is a chronic, multiorgan, autoimmune disease that affects people of all ages and ethnicities.
Objectives
To explore the relationship between age at disease onset and many of the diverse manifestations of SLE. Additionally, to determine the relationship between age of disease onset and genetic risk in patients with SLE.
Methods
The relationship between the age at disease onset and SLE manifestations were explored in a multiracial cohort of 1317 patients. Patients with SLE were genotyped across 19 confirmed genetic susceptibility loci for SLE. Logistic regression was used to determine the relationships between the number of risk alleles present and age of disease onset.
Results
Childhood-onset SLE had higher odds of proteinuria, malar rash, anti-dsDNA antibody, haemolytic anaemia, arthritis and leucopenia (OR=3.03, 2.13, 2.08, 2.50, 1.89, 1.53, respectively; p values <0.0001, 0.0004, 0.0005, 0.0024, 0.0114, 0.045, respectively). In female subjects, the odds of having cellular casts were 2.18 times higher in childhood-onset than in adult-onset SLE (p=0.0027). With age of onset ≥50, the odds of having proteinuria, cellular casts, anti-nRNP antibody, anti-Sm antibody, anti-dsDNA antibody and seizures were reduced. However, late adult-onset patients with SLE have higher odds of developing photosensitivity than early adult-onset patients. Each SLE-susceptibility risk allele carried within the genome of patients with SLE increased the odds of having a childhood-onset disease in a race-specific manner: by an average of 48% in Gullah and 25% in African-Americans, but this was not significant in Hispanic and European-American lupus patients.
Conclusions
The genetic contribution towards predicting early-onset disease in patients with SLE is quantified for the first time. A more severe SLE phenotype is found in patients with early-onset disease in a large multi-racial cohort, independent of gender, race and disease duration.
doi:10.1136/ard.2010.141697
PMCID: PMC3034281  PMID: 20881011
18.  An update on belimumab for the treatment of lupus 
B-lymphocyte stimulator (BLyS), a homeostatic factor for B-cell differentiation and survival, has a major role in B-cell expansion and autoreactivity that characterize systemic lupus erythematosus (SLE). Belimumab, a BLyS-specific inhibitor, has shown promising evidence of efficacy in several preclinical and clinical studies in SLE. Two recent large randomized controlled trials yielded a significant positive effect of the drug compared to placebo in patients with active disease. In this review, we discuss basic aspects of B-cell and BLyS biology in SLE and summarize the evidence supporting a role of belimumab in SLE, from animal studies to phase III clinical trials.
doi:10.2147/BTT.S13804
PMCID: PMC3044792  PMID: 21383914
B lymphocyte stimulator; lupus erythematosus; belimumab
19.  Confirmation of an Association Between rs6822844 at the IL2–IL21 Region and Multiple Autoimmune Diseases 
Arthritis and rheumatism  2010;62(2):323-329.
Objective
Autoimmune diseases often have susceptibility genes in common, indicating similar molecular mechanisms. Increasing evidence suggests that rs6822844 at the IL2–IL21 region is strongly associated with multiple autoimmune diseases in individuals of European descent. This study was undertaken to attempt to replicate the association between rs6822844 and 6 different immune-mediated diseases in non-European populations, and to perform disease-specific and overall meta-analyses using data from previously published studies.
Methods
We evaluated case–control associations between rs6822844 and celiac disease (CD) in subjects from Argentina; rheumatoid arthritis (RA), type 1 diabetes mellitus (DM), primary Sjögren's syndrome (SS), and systemic lupus erythematosus (SLE) in subjects from Colombia; and Behçet's disease (BD) in subjects from Turkey. Allele and gene distributions were compared between cases and controls. Meta-analyses were performed using data from the present study and previous studies.
Results
We detected significant associations of rs6822844 with SLE (P = 0.008), type 1 DM (P = 0.014), RA (P = 0.019), and primary SS (P = 0.033) but not with BD (P = 0.34) or CD (P = 0.98). We identified little evidence of population differentiation (FST = 0.01) within cases and controls from Argentina and Colombia, suggesting that association was not influenced by population substructure. Disease-specific meta-analysis indicated significant association for RA (Pmeta = 3.61 × 10–6), inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis) (Pmeta = 3.48 × 10–12), type 1 DM (Pmeta = 5.33 × 10–5), and CD (Pmeta = 5.30 × 10–3). Overall meta-analysis across all autoimmune diseases reinforced association with rs6822844 (23 data sets; Pmeta = 2.61 × 10–25, odds ratio 0.73 [95% confidence interval 0.69–0.78]).
Conclusion
Our results indicate that there is an association between rs6822844 and multiple auto-immune diseases in non-European populations. Meta-analysis results strongly reinforce this robust association across multiple autoimmune diseases in both European-derived and non-European populations.
doi:10.1002/art.27222
PMCID: PMC3028384  PMID: 20112382
20.  A polymorphism within interleukin-21 receptor (IL21R) confers risk for systemic lupus erythematosus 
Arthritis and rheumatism  2009;60(8):2402-2407.
Objective
Interleukin (IL) 21 is a member of the type I cytokine superfamily that exerts a variety of effects on the immune system including B cell activation, plasma cell differentiation, and immunoglobulin production. The expression of IL21R is reduced in B cells from lupus patients, while IL21 serum levels are increased in both lupus patients and some lupus-murine models. We recently reported that polymorphisms within the IL21 gene are associated with increased susceptibility to lupus. Herein, we examined the genetic association between SNPs within IL21R and lupus.
Methods
We genotyped 17 SNPs in the IL21R gene in two large cohorts of lupus patients and ethnically-matched healthy controls. Genotyping was performed with the Illumina BeadStation 500GX instrument using Illumina Infinum II genotyping assays.
Results
We identified and confirmed the association between rs3093301 within the IL21R gene and lupus in two independent European-derived and Hispanic cohorts (meta analysis odds ratio= 1.16, 95% CI= 1.08-1.25, meta analysis p=1.0×10-4).
Conclusion
We identified IL21R as a novel susceptibility gene for lupus.
doi:10.1002/art.24658
PMCID: PMC2782592  PMID: 19644854
21.  Variants within MECP2, a key transcriptional regulator, are associated with increased susceptibility to lupus and differential gene expression in lupus patients 
Arthritis and rheumatism  2009;60(4):1076-1084.
Objective
Both genetic and epigenetic factors play an important role in the pathogenesis of lupus. Herein, we study methyl-CpG-binding protein 2 (MECP2) polymorphism in a large cohort of lupus patients and controls, and determine functional consequences of the lupus-associated MECP2 haplotype.
Methods
We genotyped 18 SNPs within MECP2, located on chromosome Xq28, in a large cohort of European-derived lupus patients and controls. We studied the functional effects of the lupus-associated MECP2 haplotype by determining gene expression profiles in B cell lines from female lupus patients with and without the lupus-associated MECP2 risk haplotype.
Results
We confirm, replicate, and extend the genetic association between lupus and genetic markers within MECP2 in a large independent cohort of European-derived lupus patients and controls (OR= 1.35, p= 6.65×10−11). MECP2 is a dichotomous transcriptional regulator that either activates or represses gene expression. We identified 128 genes that are differentially expressed in lupus patients with the disease-associated MECP2 haplotype; most (~81%) are upregulated. Genes that were upregulated have significantly more CpG islands in their promoter regions compared to downregulated genes. Gene ontology analysis using the differentially expressed genes revealed significant association with epigenetic regulatory mechanisms suggesting that these genes are targets for MECP2 regulation in B cells. Further, at least 13 of the 104 upregulated genes are interferon-regulated genes. The disease-risk MECP2 haplotype is associated with increased expression of the MECP2 transcriptional co-activator CREB1, and decreased expression of the co-repressor HDAC1.
Conclusion
Polymorphism in the MECP2 locus is associated with lupus and, at least in part, contributes to the interferon signature observed in lupus patients.
doi:10.1002/art.24360
PMCID: PMC2734382  PMID: 19333917
22.  The DNA methylation signature of human TCRαβ+CD4−CD8− double negative T cells reveals CG demethylation and a unique epigenetic architecture permissive to a broad stimulatory immune response 
T cell receptor (TCR) αβ+CD4−CD8− double negative T cells represent a rare T cell subset implicated in the pathogenesis of several autoimmune diseases. We investigated the DNA methylation signature of double negative T cells to gain insight into the epigenetic architecture of peripheral blood primary human double negative T cells compared to autologous CD4+ and CD8+ T cells. We identified 2984 CG sites across the genome with unique loss of DNA methylation in double negative T cells, and showed significant reduction in mRNA expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. DNA methylation was increased in CD8A/CD8B and CD4 consistent with epigenetic repression of both the CD8 and CD4 genetic loci in double negative T cells. We show a consistent increase in non-CG methylation in double negative T cells, a finding suggestive of pluripotency. Network analyses indicate a strong relationship between double negative T cells and functions related to cell –cell interaction, cell adhesion, and cell activation pathways. Our data also suggest a robust pro-inflammatory epigenetic signature in double negative T cells, consistent with a transcriptional permissiveness in key inflammatory cytokines including IFNγ, IL-17F, IL-12B, IL-5, IL-18, TNFSF11 (RANKL), and TNFSF13B (BLYS or BAFF). These findings highlight an epigenetic basis for a role of double negative T cells in autoimmunity.
doi:10.1016/j.clim.2014.10.007
PMCID: PMC4278938  PMID: 25451162
Double negative; T cell; Epigenome; Methylome; DNA methylation
23.  Dehydroepiandrosterone in systemic lupus erythematosus 
Current rheumatology reports  2008;10(4):286-291.
Dehydroepiandrosterone (DHEA) is a weak androgen that exerts pleomorphic effects on the immune system. The hormone has no known receptor, and consequently, the mechanism of action of DHEA on immunocompetent cells remains poorly understood. Interestingly, serum levels of DHEA are decreased in patients with inflammatory disease including lupus, and these levels seem to inversely correlate with disease activity. Following encouraging studies demonstrating beneficial effects of DHEA supplementation in murine lupus models, a number of clinical studies have tested the effect of DHEA administration in lupus patients. DHEA treatment could improve patient’s overall quality of life assessment measures and glucocorticoid requirements in some lupus patients with mild to moderate disease, however, the effect of DHEA on disease activity in lupus patients remains controversial. Long term safety assessment studies are required in light of the reported effect of DHEA supplementation in lowering HDL cholesterol in lupus patients.
PMCID: PMC2701249  PMID: 18662508
24.  Ethnicity-specific epigenetic variation in naïve CD4+ T cells and the susceptibility to autoimmunity 
Background
Genetic and epigenetic variability contributes to the susceptibility and pathogenesis of autoimmune diseases. T cells play an important role in several autoimmune conditions, including lupus, which is more common and more severe in people of African descent. To investigate inherent epigenetic differences in T cells between ethnicities, we characterized genome-wide DNA methylation patterns in naïve CD4+ T cells in healthy African-Americans and European-Americans, and then confirmed our findings in lupus patients.
Results
Impressive ethnicity-specific clustering of DNA methylation profiling in naïve CD4+ T cells was revealed. Hypomethylated loci in healthy African-Americans were significantly enriched in pro-apoptotic and pro-inflammatory genes. We also found hypomethylated genes in African-Americans to be disproportionately related to autoimmune diseases including lupus. We then confirmed that these genes, such as IL32, CD226, CDKN1A, and PTPRN2 were similarly hypomethylated in lupus patients of African-American compared to European-American descent. Using patch DNA methylation and luciferase reporter constructs, we showed that methylation of the IL32 promoter region reduces gene expression in vitro. Importantly, bisulfite DNA sequencing demonstrated that cis-acting genetic variants within and directly disrupting CpG sites account for some ethnicity-specific variability in DNA methylation.
Conclusion
Ethnicity-specific inherited epigenetic susceptibility loci in CD4+ T cells provide clues to explain differences in the susceptibility to autoimmunity and possibly other T cell-related diseases between populations.
Electronic supplementary material
The online version of this article (doi:10.1186/s13072-015-0037-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s13072-015-0037-1
PMCID: PMC4659164  PMID: 26609326
Epigenetic; Autoimmunity; Lupus; Ethnicity specific; Genetic; T cell
25.  Systemic lupus erythematosus complicated by diffuse alveolar haemorrhage: risk factors, therapy and survival 
Lupus Science & Medicine  2015;2(1):e000117.
Objectives
While diffuse alveolar haemorrhage (DAH) is recognised as a life-threatening complication of systemic lupus erythematosus (SLE), little is known about its risk factors and response to treatment. We describe 22 cases of DAH in a US lupus cohort of approximately 1000 patients, and compare them to 66 controls from the same outpatient cohort.
Methods
We captured variables pertaining to diagnoses of SLE and secondary antiphospholipid syndrome (APS), and analysed them by univariate testing. Those variables with p values <0.05 were then further considered in a multivariate model. Kaplan-Meier curves were constructed for each group, and survival was analysed by Log-rank test.
Results
Of the 22 patients with DAH, 59% were diagnosed with DAH within 5 years of lupus diagnosis. By univariate testing, several manifestations of SLE and APS were more common in patients with DAH, including history of thrombocytopenia, cardiac valve disease, low C3, leucopenia, neuropsychiatric features, haemolysis, arterial thrombosis, lupus anticoagulant, secondary APS and low C4. On multivariate analysis, history of thrombocytopenia and low C3 were maintained as independent risk factors. Importantly, only two patients had platelet counts <50 000/µL at the time of the DAH episode, arguing that DAH was not simply a haemorrhagic complication of thrombocytopenia. All patients were treated with increased immunosuppression, including various combinations of corticosteroids, plasmapheresis, cyclophosphamide, rituximab and mycophenolate mofetil. Notably, all patients in the cohort survived their initial episode of DAH. While the patients with DAH did well in the short-term, their long-term survival was significantly worse than controls. Several of the deaths were attributable to thrombotic complications after recovering from DAH.
Conclusions
To the best of our knowledge, this is the largest case–control study of lupus DAH to date. History of thrombocytopenia was strongly predictive of DAH (OR ∼40). A number of APS manifestations correlated with DAH by univariate analysis, and deserve further consideration in larger studies.
doi:10.1136/lupus-2015-000117
PMCID: PMC4586940  PMID: 26430514
Diffuse alveolar hemorrhage; Thrombocytopenia; Antiphospholipid Syndrome

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