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1.  Dysregulation of the Microvasculature in Nonlesional Non-Sun-exposed Skin of Patients with Lupus Nephritis 
The Journal of rheumatology  2012;39(3):510-515.
Membrane endothelial protein C receptor (mEPCR) is highly expressed in peritubular capillaries of kidneys from patients with active and poorly responsive lupus nephritis (LN). We investigated the hypothesis that changes in the microvasculature are widespread with extension to the dermal vasculature.
Skin biopsies from uninvolved skin (buttocks) were performed in 27 patients with LN and 5 healthy controls. Sections were stained with specific antibodies reactive with mEPCR, adiponectin, intercellular adhesion molecule-1 (ICAM-1), and CD31; then assessed by enumeration of stained blood vessels (percentage positive blood vessels) blinded to knowledge of clinical information.
There was a significant increase in the prevalence of blood vessels that stained for mEPCR and ICAM-1 in patients compared to controls [94% vs 59% (p = 0.045) and 81% vs 67% (p = 0.037), respectively]. Adiponectin staining and CD31 staining were similar between the groups (45% vs 43% and 98% vs 92%). Dermal staining for mEPCR was greater in patients with proliferative glomerulonephritis than in those with membranous disease (96% vs 60%; p = 0.029). A composite of poor prognostic renal markers and death was significantly associated with greater expression of mEPCR staining.
These data are consistent with the notion that in patients with LN, activation of the microvasculature extends beyond the clinically targeted organ. The insidious expression of this widespread vasculopathy may be a contributor to longterm comorbidities.
PMCID: PMC4054860  PMID: 22298906
2.  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.
PMCID: PMC3790645  PMID: 23623029
Lupus; naïve CD4+ T cells; methylome; DNA methylation
3.  End-Stage Renal Disease in African Americans With Lupus Nephritis Is Associated With APOL1 
Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE) that exhibits familial aggregation and may progress to end-stage renal disease (ESRD). LN is more prevalent among African Americans than among European Americans. This study was undertaken to investigate the hypothesis that the apolipoprotein L1 gene (APOL1) nephropathy risk alleles G1/G2, common in African Americans and rare in European Americans, contribute to the ethnic disparity in risk.
APOL1 G1 and G2 nephropathy alleles were genotyped in 855 African American SLE patients with LN-ESRD (cases) and 534 African American SLE patients without nephropathy (controls) and tested for association under a recessive genetic model, by logistic regression.
Ninety percent of the SLE patients were female. The mean ± SD age at SLE diagnosis was significantly lower in LN-ESRD cases than in SLE non-nephropathy controls (27.3 ± 10.9 years versus 39.5 ± 12.2 years). The mean ± SD time from SLE diagnosis to development of LN-ESRD in cases was 7.3 ± 7.2 years. The G1/G2 risk alleles were strongly associated with SLE-ESRD, with 25% of cases and 12% of controls having 2 nephropathy alleles (odds ratio [OR] 2.57, recessive model P = 1.49 × 10−9), and after adjustment for age, sex, and ancestry admixture (OR 2.72, P = 6.23 × 10−6). The age-, sex-, and admixture-adjusted population attributable risk for ESRD among patients with G1/G2 polymorphisms was 0.26, compared to 0.003 among European American patients. The mean time from SLE diagnosis to ESRD development was ~2 years earlier among individuals with APOL1 risk genotypes (P = 0.01).
APOL1 G1/G2 alleles strongly impact the risk of LN-ESRD in African Americans, as well as the time to progression to ESRD. The high frequency of these alleles in African Americans with near absence in European Americans explains an important proportion of the increased risk of LN-ESRD in African Americans.
PMCID: PMC4002759  PMID: 24504811
4.  Circulating levels of soluble MER in lupus reflect M2c activation of monocytes/macrophages, autoantibody specificities and disease activity 
Arthritis Research & Therapy  2013;15(6):R212.
Systemic lupus erythematosus (SLE) is characterized by impaired efferocytosis and aberrant activation of innate immunity. We asked if shedding of MER receptor tyrosine kinase (MerTK) and AXL into soluble (s) ectodomains was related to immunological and clinical aspects of SLE.
Levels of sMER and sAXL in the plasma of 107 SLE patients and 45 matched controls were measured by ELISA. In 40 consecutive SLE patients, we examined potential correlations between either sMER or sAXL and plasma levels of sCD163, a marker of M2 activation. All three soluble receptors were measured in supernatants of monocytes/macrophages cultured in various immunological conditions. Membrane expression of MerTK, AXL and CD163 was assessed by flow cytometry.
Both sMER and sAXL were associated with anti-chromatin and anti-phospholipid autoantibodies, and with hematological and renal involvement. However, sMER and sAXL did not significantly correlate with each other; sAXL correlated with growth arrest-specific 6 (Gas6), whereas sMER correlated with reduced free protein S (PROS) levels. Only sMER showed significant associations with lupus-specific anti-dsDNA, anti-Sm, anti-ribonucleoprotein (anti-RNP) and anti-Ro60 autoantibodies. Strong correlations with disease activity indices (Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), complement reduction, titer of circulating anti-dsDNA) were found for sMER, not for sAXL. Patients with active SLEDAI, nephritis, anti-dsDNA and anti-Ro60 positivity showed higher levels of sMER compared to controls. Levels of sMER, not sAXL, correlated with sCD163 levels, and these correlated with SLEDAI. Production of sMER and sCD163 occurred under “M2c” polarizing conditions, whereas sAXL was released upon type-I IFN exposure.
Alterations in homeostasis of anti-inflammatory and efferocytic “M2c” monocytes/macrophages may have a role in immunopathogenesis of SLE.
PMCID: PMC3978923  PMID: 24325951
5.  Two Independent Functional Risk Haplotypes in TNIP1 are Associated with Systemic Lupus Erythematosus 
Arthritis and rheumatism  2012;64(11):3695-3705.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and altered type I interferon expression. Genetic surveys and genome-wide association studies have identified more than 30 SLE susceptibility genes. One of these genes, TNIP1, encodes the ABIN1 protein. ABIN1 functions in the immune system by restricting the NF-κB signaling. In order to better understand the genetic factors that influence association with SLE in genes that regulate the NF-κB pathway, we analyzed a dense set of genetic markers spanning TNIP1 and TAX1BP1, as well as the TNIP1 homolog, TNIP2, in case-control sets of diverse ethnic origins.
We fine-mapped TNIP1, TNIP2, and TAX1BP1 in a total of 8372 SLE cases and 7492 healthy controls from European-ancestry, African-American, Hispanic, East Asian, and African-American Gullah populations. Levels of TNIP1 mRNA and ABIN1 protein were analyzed using quantitative RT-PCR and Western blotting, respectively, in EBV-transformed human B cell lines.
We found significant associations between genetic variants within TNIP1 and SLE but not in TNIP2 or TAX1BP1. After resequencing and imputation, we identified two independent risk haplotypes within TNIP1 in individuals of European-ancestry that were also present in African-American and Hispanic populations. These risk haplotypes produced lower levels of TNIP1 mRNA and ABIN1 protein suggesting they harbor hypomorphic functional variants that influence susceptibility to SLE by restricting ABIN1 expression.
Our results confirmed the association signals between SLE and TNIP1 variants in multiple populations and provide new insight into the mechanism by which TNIP1 variants may contribute to SLE pathogenesis.
PMCID: PMC3485412  PMID: 22833143
6.  Impact of Genetic Ancestry and Socio-Demographic Status on the Clinical Expression of Systemic Lupus Erythematosus in Amerindian-European Populations 
Arthritis and rheumatism  2012;64(11):3687-3694.
Amerindian-Europeans, Asians and African-Americans have an excess morbidity from SLE and higher prevalence of lupus nephritis than Caucasians. The aim of this study was to analyze the relationship between genetic ancestry and socio-demographic characteristics and clinical features in a large cohort of Amerindian-European SLE patients.
A total of 2116 SLE patients of Amerindian-European origin and 4001 SLE patients of European descent with clinical data were used in the study. Genotyping of 253 continental ancestry informative markers was performed on the Illumina platform. The STRUCTURE and ADMIXTURE software were used to determine genetic ancestry of each individual. Correlation between ancestry and socio-demographic and clinical data were analyzed using logistic regression.
The average Amerindian genetic ancestry of 2116 SLE patients was 40.7%. There was an increased risk of having renal involvement (P<0.0001, OR= 3.50 95%CI 2.63-4.63) and an early age of onset with the presence of Amerindian genetic ancestry (P<0.0001). Amerindian ancestry protected against photosensitivity (P<0.0001, OR= 0.58 95%CI 0.44-0.76), oral ulcers (P<0.0001, OR= 0.55 95%CI 0.42-0.72), and serositis (P<0.0001, OR= 0.56 95%CI 0.41-0.75) after adjustment by age, gender and age of onset. However, gender and age of onset had stronger effects on malar rash, discoid rash, arthritis and neurological involvement than genetic ancestry.
In general, genetic Amerindian ancestry correlates with lower socio-demographic status and increases the risk for developing renal involvement and SLE at an earlier age of onset.
PMCID: PMC3485439  PMID: 22886787
7.  Preferential Binding to Elk-1 by SLE-Associated IL10 Risk Allele Upregulates IL10 Expression 
PLoS Genetics  2013;9(10):e1003870.
Immunoregulatory cytokine interleukin-10 (IL-10) is elevated in sera from patients with systemic lupus erythematosus (SLE) correlating with disease activity. The established association of IL10 with SLE and other autoimmune diseases led us to fine map causal variant(s) and to explore underlying mechanisms. We assessed 19 tag SNPs, covering the IL10 gene cluster including IL19, IL20 and IL24, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported IL10 variant, rs3024505 located at 1 kb downstream of IL10, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (P = 2.7×10−8, OR = 1.30), but not in non-EA ancestries. SNP imputation conducted in EA dataset identified three additional SLE-associated SNPs tagged by rs3024505 (rs3122605, rs3024493 and rs3024495 located at 9.2 kb upstream, intron 3 and 4 of IL10, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of IL10 mRNA in PBMCs and circulating IL-10 protein in SLE patients and controls. Using nuclear extracts of peripheral blood cells from SLE patients for electrophoretic mobility shift assays, we identified specific binding of transcription factor Elk-1 to oligodeoxynucleotides containing the risk (G) allele of rs3122605, suggesting rs3122605 as the most likely causal variant regulating IL10 expression. Elk-1 is known to be activated by phosphorylation and nuclear localization to induce transcription. Of interest, phosphorylated Elk-1 (p-Elk-1) detected only in nuclear extracts of SLE PBMCs appeared to increase with disease activity. Co-expression levels of p-Elk-1 and IL-10 were elevated in SLE T, B cells and monocytes, associated with increased disease activity in SLE B cells, and were best downregulated by ERK inhibitor. Taken together, our data suggest that preferential binding of activated Elk-1 to the IL10 rs3122605-G allele upregulates IL10 expression and confers increased risk for SLE in European Americans.
Author Summary
Systemic lupus erythematosus (SLE), a debilitating autoimmune disease characterized by the production of pathogenic autoantibodies, has a strong genetic basis. Variants of the IL10 gene, which encodes cytokine interleukin-10 (IL-10) with known function of promoting B cell hyperactivity and autoantibody production, are associated with SLE and other autoimmune diseases, and serum IL-10 levels are elevated in SLE patients correlating with increased disease activity. In this study, to discover SLE-predisposing causal variant(s), we assessed variants within the genomic region containing IL10 and its gene family member IL19, IL20 and IL24 for association with SLE in case and control subjects from diverse ancestries. We identified SLE-associated SNP rs3122605 located at 9.2 kb upstream of IL10 as the most likely causal variant in subjects of European ancestry. The SLE-risk allele of rs3122605 was dose-dependently associated with elevated IL10 expression at both mRNA and protein levels in peripheral blood samples from SLE patients and controls, which could be explained, at least in part, by its preferential binding to Elk-1, a transcription factor activated in B cells during active disease of SLE patients. Elk-1-mediated IL-10 overexpression could be downregulated by inhibiting activation of mitogen-activated protein kinases, suggesting a potential therapeutic target for SLE.
PMCID: PMC3794920  PMID: 24130510
8.  Plasma from systemic lupus patients compromises cholesterol homeostasis: a potential mechanism linking autoimmunity to atherosclerotic cardiovascular disease 
Rheumatology international  2009;30(5):591-598.
Atherosclerotic cardiovascular disease (ASCVD) contributes to morbidity and mortality in systemic lupus erythematosus (SLE). Immunologic derangements may disrupt cholesterol balance in vessel wall monocytes/macrophages and endothelium. We determined whether lupus plasma impacts expression of cholesterol 27-hydroxylase, an anti-atherogenic cholesterol-degrading enzyme that promotes cellular cholesterol efflux, in THP-1 human monocytes and primary human aortic endothelial cells (HAEC). THP-1 monocytes and HAEC were incubated in medium containing SLE patient plasma or apparently healthy control human plasma (CHP). SLE plasma decreased 27-hydroxylase message in THP-1 monocytes by 47 ± 8% (p < 0.008) and in HAEC by 51 ± 5.5% (n = 5, p < 0.001). THP-1 macrophages were incubated in 25% lupus plasma or CHP and cholesterol-loaded (50 µg ml−1 acetylated low density lipoprotein). Lupus plasma more than doubled macrophage foam cell transformation (74 ± 3% vs.35 § 3% for CHP, n = 3, p < 0.001). Impaired cholesterol homeostasis in SLE provides further evidence of immune involvement in atherogenesis. Strategies to inhibit or reverse arterial cholesterol accumulation may benefit SLE patients.
PMCID: PMC3736583  PMID: 19547978
Lupus erythematosus; Systemic; Atherosclerosis; Cholesterol; Macrophage scavenger receptor; Foam cells
9.  PTPN22 Association in Systemic Lupus Erythematosus (SLE) with Respect to Individual Ancestry and Clinical Sub-Phenotypes 
PLoS ONE  2013;8(8):e69404.
Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a negative regulator of T-cell activation associated with several autoimmune diseases, including systemic lupus erythematosus (SLE). Missense rs2476601 is associated with SLE in individuals with European ancestry. Since the rs2476601 risk allele frequency differs dramatically across ethnicities, we assessed robustness of PTPN22 association with SLE and its clinical sub-phenotypes across four ethnically diverse populations. Ten SNPs were genotyped in 8220 SLE cases and 7369 controls from in European-Americans (EA), African-Americans (AA), Asians (AS), and Hispanics (HS). We performed imputation-based association followed by conditional analysis to identify independent associations. Significantly associated SNPs were tested for association with SLE clinical sub-phenotypes, including autoantibody profiles. Multiple testing was accounted for by using false discovery rate. We successfully imputed and tested allelic association for 107 SNPs within the PTPN22 region and detected evidence of ethnic-specific associations from EA and HS. In EA, the strongest association was at rs2476601 (P = 4.7×10−9, OR = 1.40 (95% CI = 1.25–1.56)). Independent association with rs1217414 was also observed in EA, and both SNPs are correlated with increased European ancestry. For HS imputed intronic SNP, rs3765598, predicted to be a cis-eQTL, was associated (P = 0.007, OR = 0.79 and 95% CI = 0.67–0.94). No significant associations were observed in AA or AS. Case-only analysis using lupus-related clinical criteria revealed differences between EA SLE patients positive for moderate to high titers of IgG anti-cardiolipin (aCL IgG >20) versus negative aCL IgG at rs2476601 (P = 0.012, OR = 1.65). Association was reinforced when these cases were compared to controls (P = 2.7×10−5, OR = 2.11). Our results validate that rs2476601 is the most significantly associated SNP in individuals with European ancestry. Additionally, rs1217414 and rs3765598 may be associated with SLE. Further studies are required to confirm the involvement of rs2476601 with aCL IgG.
PMCID: PMC3737240  PMID: 23950893
10.  Variable association of reactive intermediate genes with systemic lupus erythematosus (SLE) in populations with different African ancestry 
The Journal of rheumatology  2013;40(6):842-849.
Little is known about the genetic etiology of systemic lupus erythematosus (SLE) in individuals of African ancestry, despite its higher prevalence and greater disease severity. Overproduction of nitric oxide (NO) and reactive oxygen species are implicated in the pathogenesis and severity of SLE, making NO synthases and other reactive intermediate related genes biological candidates for disease susceptibility. This study analyzed variation in reactive intermediate genes for association with SLE in two populations with African ancestry.
A total of 244 SNPs from 53 regions were analyzed in non-Gullah African Americans (AA; 1432 cases and 1687 controls) and the genetically more homogeneous Gullah of the Sea Islands of South Carolina (133 cases and 112 controls) and. Single-marker, haplotype, and two-locus interaction tests were computed for these populations.
The glutathione reductase gene GSR (rs2253409, P=0.0014, OR [95% CI]=1.26 [1.09–1.44]) was the most significant single-SNP association in AA. In the Gullah, the NADH dehydrogenase NDUFS4 (rs381575, P=0.0065, OR [95%CI]=2.10 [1.23–3.59]) and nitric oxide synthase gene NOS1 (rs561712, P=0.0072, OR [95%CI]=0.62 [0.44–0.88]) were most strongly associated with SLE. When both populations were analyzed together, GSR remained the most significant effect (rs2253409, P=0.00072, OR [95%CI]=1.26 [1.10–1.44]). Haplotype and two-locus interaction analyses also uncovered different loci in each population.
These results suggest distinct patterns of association with SLE in African-derived populations; specific loci may be more strongly associated within select population groups.
PMCID: PMC3735344  PMID: 23637325
systemic lupus erythematosus; African Americans; genetic association studies; oxygen compounds; single nucleotide polymorphism
11.  Derivation and Validation of Systemic Lupus International Collaborating Clinics Classification Criteria for Systemic Lupus Erythematosus 
Arthritis and rheumatism  2012;64(8):2677-2686.
The Systemic Lupus Collaborating Clinics (SLICC) revised and validated the American College of Rheumatology (ACR) SLE classification criteria in order to improve clinical relevance, meet stringent methodology requirements and incorporate new knowledge in SLE immunology.
The classification criteria were derived from a set of 702 expert-rated patient scenarios. Recursive partitioning was used to derive an initial rule that was simplified and refined based on SLICC physician consensus. SLICC validated the classification criteria in a new validation sample of 690 SLE patients and controls.
Seventeen criteria were identified. The SLICC criteria for SLE classification requires: 1) Fulfillment of at least four criteria, with at least one clinical criterion AND one immunologic criterion OR 2) Lupus nephritis as the sole clinical criterion in the presence of ANA or anti-dsDNA antibodies. In the derivation set, the SLICC classification criteria resulted in fewer misclassifications than the current ACR classification criteria (49 versus 70, p=0.0082), had greater sensitivity (94% versus 86%, p<0.0001) and equal specificity (92% versus 93%, p=0.39). In the validation set, the SLICC Classification criteria resulted in fewer misclassifications (62 versus 74, p=0.24), had greater sensitivity (97% versus 83%, p<0.0001) but less specificity (84% versus 96%, p<0.0001).
The new SLICC classification criteria performed well on a large set of patient scenarios rated by experts. They require that at least one clinical criterion and one immunologic criterion be present for a classification of SLE. Biopsy confirmed nephritis compatible with lupus (in the presence of SLE autoantibodies) is sufficient for classification.
PMCID: PMC3409311  PMID: 22553077
12.  Trans-Ancestral Studies Fine Map the SLE-Susceptibility Locus TNFSF4 
PLoS Genetics  2013;9(7):e1003554.
We previously established an 80 kb haplotype upstream of TNFSF4 as a susceptibility locus in the autoimmune disease SLE. SLE-associated alleles at this locus are associated with inflammatory disorders, including atherosclerosis and ischaemic stroke. In Europeans, the TNFSF4 causal variants have remained elusive due to strong linkage disequilibrium exhibited by alleles spanning the region. Using a trans-ancestral approach to fine-map the locus, utilising 17,900 SLE and control subjects including Amerindian/Hispanics (1348 cases, 717 controls), African-Americans (AA) (1529, 2048) and better powered cohorts of Europeans and East Asians, we find strong association of risk alleles in all ethnicities; the AA association replicates in African-American Gullah (152,122). The best evidence of association comes from two adjacent markers: rs2205960-T (P = 1.71×10−34, OR = 1.43[1.26–1.60]) and rs1234317-T (P = 1.16×10−28, OR = 1.38[1.24–1.54]). Inference of fine-scale recombination rates for all populations tested finds the 80 kb risk and non-risk haplotypes in all except African-Americans. In this population the decay of recombination equates to an 11 kb risk haplotype, anchored in the 5′ region proximal to TNFSF4 and tagged by rs2205960-T after 1000 Genomes phase 1 (v3) imputation. Conditional regression analyses delineate the 5′ risk signal to rs2205960-T and the independent non-risk signal to rs1234314-C. Our case-only and SLE-control cohorts demonstrate robust association of rs2205960-T with autoantibody production. The rs2205960-T is predicted to form part of a decameric motif which binds NF-κBp65 with increased affinity compared to rs2205960-G. ChIP-seq data also indicate NF-κB interaction with the DNA sequence at this position in LCL cells. Our research suggests association of rs2205960-T with SLE across multiple groups and an independent non-risk signal at rs1234314-C. rs2205960-T is associated with autoantibody production and lymphopenia. Our data confirm a global signal at TNFSF4 and a role for the expressed product at multiple stages of lymphocyte dysregulation during SLE pathogenesis. We confirm the validity of trans-ancestral mapping in a complex trait.
Author Summary
Systemic lupus erythematosus (SLE/lupus) is a complex disease in which the body's immune cells cause inflammation in one or more systems to cause the associated morbidity. Hormones, the environment and genes are all causal contributors to SLE and over the past several years the genetic component of SLE has been firmly established. Several genes which are regulators of the immune system are associated with disease risk. We have established one of these, the tumour-necrosis family superfamily member 4 (TNFSF4) gene, as a lupus susceptibility gene in Northern Europeans. A major obstacle in pinpointing the marker(s) at TNFSF4 which best explain the risk of SLE has been the strong correlation (linkage disequilibrium, LD) between adjacent markers across the TNFSF4 region in this population. To address this, we have typed polymorphisms in several populations in addition to the European groups. The mixed ancestry of these populations gives a different LD pattern than that found in Europeans, presenting a method of pinpointing the section of the TNFSF4 region which results in SLE susceptibility. The Non-European populations have allowed identification of a polymorphism likely to regulate expression of TNFSF4 to increase susceptibility to SLE.
PMCID: PMC3715547  PMID: 23874208
13.  Genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 and other loci 
Nature genetics  2008;40(2):204-210.
Systemic lupus erythematosus (SLE) is a common systemic autoimmune disease with complex etiology but strong clustering in families (λS = ~30). We performed a genome-wide association scan using 317,501 SNPs in 720 women of European ancestry with SLE and in 2,337 controls, and we genotyped consistently associated SNPs in two additional independent sample sets totaling 1,846 affected women and 1,825 controls. Aside from the expected strong association between SLE and the HLA region on chromosome 6p21 and the previously confirmed non-HLA locus IRF5 on chromosome 7q32, we found evidence of association with replication (1.1 × 10−7 < Poverall < 1.6 × 10−23; odds ratio 0.82–1.62)in four regions: 16p11.2 (ITGAM), 11p15.5 (KIAA1542), 3p14.3 (PXK) and 1q25.1 (rs10798269). We also found evidence for association (P < 1 × 10−5) at FCGR2A, PTPN22 and STAT4, regions previously associated with SLE and other autoimmune diseases, as well as at ≥9 other loci (P < 2 × 10−7). Our results show that numerous genes, some with known immune-related functions, predispose to SLE.
PMCID: PMC3712260  PMID: 18204446
Arthritis and Rheumatism  2012;64(7):2311-2318.
Which serologic and clinical findings predict adverse pregnancy outcome (APO) in patients with antiphospholipid antibody (aPL) is controversial.
PROMISSE is a multicenter, prospective observational study of risk factors for APO in patients with aPL (lupus anticoagulant [LAC], anticardiolipin antibody [aCL] and/or antibody to β2 glycoprotein I [anti-β2-GP-I]). We tested the hypothesis that a pattern of clinical and serological variables can identify women at highest risk for APO.
Between 2003 and 2011 we enrolled 144 pregnant patients, of whom 28 had APO. Thirty-nine percent of patients with LAC had APO, compared to 3% who did not have LAC (p < 0.0001). Only 8% of women with IgG aCL ≥40 u/mL but not LAC suffered APO, compared to 43% of those with LAC (p = 0.002). IgM aCL or IgG or IgM anti-β2-GP-I did not predict APO. In bivariate analysis, APO occurred in 52% of patients with and 13% of patients without prior thrombosis (p = 0.00005), and in 23% with SLE compared to 17% without SLE (not significant); SLE was a predictor in multivariate analysis. Prior pregnancy loss did not predict APO, nor did maternal race. Simultaneous aCL, anti-β2-GP-I, and LAC did not predict APO better than did LAC alone.
LAC is the primary predictor of APO after 12 weeks gestation in aPL-associated pregnancies. ACL and anti-β2-GP-I, if LAC is not also present, do not predict APO.
PMCID: PMC3357451  PMID: 22275304
15.  Evaluation of TRAF6 in a Large Multi-Ancestral Lupus Cohort 
Arthritis and Rheumatism  2012;64(6):1960-1969.
Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with significant immune system aberrations resulting from complex heritable genetics as well as environmental factors. TRAF6 is a candidate gene for SLE, which has a major role in several signaling pathways that are important for immunity and organ development.
Fifteen single-nucleotide polymorphisms (SNPs), across TRAF6 were evaluated in 7,490 SLE and 6,780 control subjects from different ancestries. Population-based case-control association analyses and meta-analyses were performed. P values, false discovery rate q values, and odds ratios with 95% confidence intervals were calculated.
Evidence of associations in multiple SNPs was detected. The best overall p values were obtained for SNPs rs5030437 and rs4755453 (p=7.85×10−5 and p=4.73×10−5, respectively) without significant heterogeneity among populations (p=0.67 and p=0.50 in Q-statistic). In addition, rs540386 previously reported to be associated with RA was found to be in LD with these two SNPs (r2= 0.95) and demonstrated evidence of association with SLE in the same direction (meta-analysis p=9.15×10−4, OR=0.89, 95%CI=0.83–0.95). Thrombocytopenia improved the overall results in different populations (meta-analysis p=1.99×10−6, OR=0.57, 95%CI=0.45–0.72, for rs5030470). Finally evidence of family based association in 34 African-American pedigrees with the presence of thrombocytopenia were detected in one available SNP rs5030437 with Z score magnitude of 2.28 (p=0.02) under a dominant model.
Our data indicate the presence of association of TRAF6 with SLE in agreement with the previous report of association with RA. These data provide further support for the involvement of TRAF6 in the pathogenesis of autoimmunity.
PMCID: PMC3380425  PMID: 22231568
TRAF6; polymorphism; systemic lupus erythematosus
17.  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.
PMCID: PMC3313010  PMID: 22305513
Genetic risk; epigenetics; DNA methylation; lupus; genetic-epigenetic interaction; sex-disparity
18.  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.
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.
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.
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.
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.
PMCID: PMC3324666  PMID: 22110124
19.  Monitoring cartilage loss in the hands and wrists in rheumatoid arthritis with magnetic resonance imaging in a multi-center clinical trial: IMPRESS (NCT00425932) 
Magnetic resonance imaging (MRI) is increasingly being used in clinical trials of rheumatoid arthritis (RA) because of its superiority over x-ray radiography (XR) in detecting and monitoring change in bone erosion, osteitis and synovitis. However, in contrast to XR, the MRI scoring method that was used in most clinical trials did not include cartilage loss. This limitation has been an obstacle to accepting MRI as a potential alternative to XR in clinical trials. Cross-sectional studies have shown MRI to be sensitive for cartilage loss in the hands and wrist; although, longitudinal sensitivity to change has not yet been confirmed. In this study we examined the ability of MRI to monitor change in cartilage loss in patients with RA in a multi-site clinical trial setting.
Thirty-one active RA patients from a clinical trial (IMPRESS) who were randomized equally into treatment with either rituximab + methotrexate or placebo + methotrexate had MRI of the dominant hand/wrist at baseline, 12 weeks and 24 weeks at 3 clinical sites in the US. Twenty-seven of these patients also had XR of both hands/wrists and both feet at baseline and 24 weeks. One radiologist scored all XR images using the van der Heijde-modified Sharp method blinded to visit order. The same radiologist scored MR images for cartilage loss using a previously validated 9-point scale, and bone erosion using the Outcome Measures in Rheumatology Clinical Trials (OMERACT) RA MRI Score (RAMRIS) blinded to visit order and XR scores. Data from the two treatment arms were pooled for this analysis.
Mean MRI cartilage score increased at 12 and 24 weeks, and reached statistical significance at 24 weeks. XR total Sharp score, XR erosion score and XR joint-space narrowing (JSN) score all increased at 24 weeks, but only XR total Sharp score increased significantly.
To our knowledge, this is the first publication of a study demonstrating MRI's ability to monitor cartilage loss in a multi-site clinical trial. Combined with MRI's established performance in monitoring bone erosions in RA, these findings suggest that MRI may offer a superior alternative to XR in multi-site clinical trials of RA.
PMCID: PMC4060230  PMID: 23514433
20.  IRF5 haplotypes demonstrate diverse serological associations which predict serum interferon alpha activity and explain the majority of the genetic association with systemic lupus erythematosus 
Annals of the Rheumatic Diseases  2011;71(3):463-468.
High serum interferon α (IFNα) activity is a heritable risk factor for systemic lupus erythematosus (SLE). Auto-antibodies found in SLE form immune complexes which can stimulate IFNα production by activating endosomal Toll-like receptors and interferon regulatory factors (IRFs), including IRF5. Genetic variation in IRF5 is associated with SLE susceptibility; however, it is unclear how IRF5 functional genetic elements contribute to human disease.
1034 patients with SLE and 989 controls of European ancestry, 555 patients with SLE and 679 controls of African–American ancestry, and 73 patients with SLE of South African ancestry were genotyped at IRF5 polymorphisms, which define major haplotypes. Serum IFNα activity was measured using a functional assay.
In European ancestry subjects, anti-double-stranded DNA (dsDNA) and anti-Ro antibodies were each associated with different haplotypes characterised by a different combination of functional genetic elements (OR > 2.56, p >003C; 1.9×10−14 for both). These IRF5 haplotype-auto-antibody associations strongly predicted higher serum IFNα in patients with SLE and explained > 70% of the genetic risk of SLE due to IRF5. In African–American patients with SLE a similar relationship between serology and IFNα was observed, although the previously described European ancestry-risk haplotype was present at admixture proportions in African–American subjects and absent in African patients with SLE.
The authors define a novel risk haplotype of IRF5 that is associated with anti-dsDNA antibodies and show that risk of SLE due to IRF5 genotype is largely dependent upon particular auto-antibodies. This suggests that auto-antibodies are directly pathogenic in human SLE, resulting in increased IFNα in cooperation with particular combinations of IRF5 functional genetic elements.
SLE is a systemic autoimmune disorder affecting multiple organ systems including the skin, musculoskeletal, renal and haematopoietic systems. Humoral autoimmunity is a hallmark of SLE, and patients frequently have circulating auto-antibodies directed against dsDNA, as well as RNA binding proteins (RBP). Anti-RBP autoantibodies include antibodies which recognize Ro, La, Smith (anti-Sm), and ribonucleoprotein (anti-nRNP), collectively referred to as anti-retinol-binding protein). Anti-retinol-binding protein and anti-dsDNA auto-antibodies are rare in the healthy population.1 These auto-antibodies can be present in sera for years preceding the onset of clinical SLE illness2 and are likely pathogenic in SLE.34
PMCID: PMC3307526  PMID: 22088620
21.  MicroRNA-3148 Modulates Allelic Expression of Toll-Like Receptor 7 Variant Associated with Systemic Lupus Erythematosus 
PLoS Genetics  2013;9(2):e1003336.
We previously reported that the G allele of rs3853839 at 3′untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10−10, odds ratio (OR) (95%CI) = 1.27 (1.17–1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10−11, OR = 1.24 [1.18–1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3′UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R2 = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3′UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta = 2.0×10−19, OR = 1.25 [1.20–1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.
Author Summary
Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease contributed to by excessive innate immune activation involving toll-like receptors (TLRs, particularly TLR7/8/9) and type I interferon (IFN) signaling pathways. TLR7 responds against RNA–containing nuclear antigens and activates IFN-α pathway, playing a pivotal role in the development of SLE. While a genomic duplication of Tlr7 promotes lupus-like disease in the Y-linked autoimmune accelerator (Yaa) murine model, the lack of common copy number variations at TLR7 in humans led us to identify a functional single nucleotide polymorphism (SNP), rs3853839 at 3′ UTR of the TLR7 gene, associated with SLE susceptibility in Eastern Asians. In this study, we fine-mapped the TLR7-TLR8 region and confirmed rs3853839 exhibiting the strongest association with SLE in European Americans, African Americans, and Amerindian/Hispanics. Individuals carrying the risk G allele of rs3853839 exhibited increased TLR7 expression at the both mRNA and protein level and decreased transcript degradation. MicroRNA-3148 (miR-3148) downregulated the expression of non-risk allele (C) containing transcripts preferentially, suggesting a likely mechanism for increased TLR7 levels in risk-allele carriers. This trans-ancestral mapping provides evidence for the global association with SLE risk at rs3853839, which resides in a microRNA–gene regulatory site affecting TLR7 expression.
PMCID: PMC3585142  PMID: 23468661
22.  Admixture Mapping in Lupus Identifies Multiple Functional Variants within IFIH1 Associated with Apoptosis, Inflammation, and Autoantibody Production 
PLoS Genetics  2013;9(2):e1003222.
Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with a strong genetic component. African-Americans (AA) are at increased risk of SLE, but the genetic basis of this risk is largely unknown. To identify causal variants in SLE loci in AA, we performed admixture mapping followed by fine mapping in AA and European-Americans (EA). Through genome-wide admixture mapping in AA, we identified a strong SLE susceptibility locus at 2q22–24 (LOD = 6.28), and the admixture signal is associated with the European ancestry (ancestry risk ratio ∼1.5). Large-scale genotypic analysis on 19,726 individuals of African and European ancestry revealed three independently associated variants in the IFIH1 gene: an intronic variant, rs13023380 [Pmeta = 5.20×10−14; odds ratio, 95% confidence interval = 0.82 (0.78–0.87)], and two missense variants, rs1990760 (Ala946Thr) [Pmeta = 3.08×10−7; 0.88 (0.84–0.93)] and rs10930046 (Arg460His) [Pdom = 1.16×10−8; 0.70 (0.62–0.79)]. Both missense variants produced dramatic phenotypic changes in apoptosis and inflammation-related gene expression. We experimentally validated function of the intronic SNP by DNA electrophoresis, protein identification, and in vitro protein binding assays. DNA carrying the intronic risk allele rs13023380 showed reduced binding efficiency to a cellular protein complex including nucleolin and lupus autoantigen Ku70/80, and showed reduced transcriptional activity in vivo. Thus, in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates nucleolin, Ku70/80, or other nucleic acid regulatory proteins. This could promote antibody hypermutation and auto-antibody generation, further destabilizing the cellular network. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis.
Author Summary
African-Americans (AA) are at increased risk of systemic lupus erythematosus (SLE), but the genetic basis of this risk increase is largely unknown. We used admixture mapping to localize disease-causing genetic variants that differ in frequency across populations. This approach is advantageous for localizing susceptibility genes in recently admixed populations like AA. Our genome-wide admixture scan identified seven admixture signals, and we followed the best signal at 2q22–24 with fine-mapping, imputation-based association analysis and experimental validation. We identified two independent coding variants and a non-coding variant within the IFIH1 gene associated with SLE. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis.
PMCID: PMC3575474  PMID: 23441136
23.  Evidence for gene-gene epistatic interactions among susceptibility loci for systemic lupus erythematosus 
Arthritis and Rheumatism  2012;64(2):485-492.
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.
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.
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).
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.
PMCID: PMC3268866  PMID: 21952918
24.  Genetic and Physical Interaction of the B-Cell SLE-Associated Genes BANK1 and BLK 
Annals of the Rheumatic Diseases  2011;71(1):136-142.
Altered signaling in B-cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signaling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterize the role of BANK1 and BLK in SLE, we performed a genetic interaction analysis hypothesizing that genetic interactions could reveal functional pathways relevant to disease pathogenesis.
We Used the method GPAT16 to analyze the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localization, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK.
Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from Northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK.
As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, we tested the possibility that BANK1 and BLK could also show a protein-protein interaction. We demonstrated co-immunoprecipitation and co-localization of BLK and BANK1. In a Daudi cell line and primary naïve B-cells the endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies.
Here, we show a genetic interaction between BANK1 and BLK, and demonstrate that these molecules interact physically. Our results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signaling pathway.
PMCID: PMC3268679  PMID: 21978998
systemic lupus erythematosus; genetics; polymorphism; B-cells; autoantibodies
25.  A functional haplotype of UBE2L3 confers risk for Systemic Lupus Erythematosus 
Genes and immunity  2012;13(5):380-387.
Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical manifestations characterized by the development of pathogenic autoantibodies manifesting in inflammation of target organs such as the kidneys, skin and joints. Genome-wide association studies have identified genetic variants in the UBE2L3 region that are associated with SLE in subjects of European and Asian ancestry. UBE2L3 encodes an ubiquitin-conjugating enzyme, UBCH7, involved in cell proliferation and immune function. In this study, we sought to further characterize the genetic association in the region of UBE2L3 and use molecular methods to determine the functional effect of the risk haplotype. We identified significant associations between variants in the region of UBE2L3 and SLE in individuals of European and Asian ancestry that exceeded a Bonferroni corrected threshold (P < 1 × 10−4). A single risk haplotype was observed in all associated populations. Individuals harboring the risk haplotype display a significant increase in both UBE2L3 mRNA expression (P = 0.0004) and UBCH7 protein expression (P = 0.0068). The results suggest that variants carried on the SLE associated UBE2L3 risk haplotype influence autoimmunity by modulating UBCH7 expression.
PMCID: PMC3411915  PMID: 22476155
Systemic Lupus Erythematosus; UBE2L3; Multi Ethnic Association Study; UBCH7 Expression

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