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26.  Fine Mapping of Xq28: Both MECP2 and IRAK1 Contribute to Risk for Systemic Lupus Erythematosus in Multiple Ancestral Groups 
Annals of the rheumatic diseases  2012;72(3):437-444.
Objectives
The Xq28 region containing IRAK1 and MECP2 has been identified as a risk locus for systemic lupus erythematosus (SLE) in previous genetic association studies. However, due to the strong linkage disequilibrium between IRAK1 and MECP2, it remains unclear which gene is affected by the underlying causal variant(s) conferring risk of SLE.
Methods
We fine-mapped ≥136 SNPs in a ~227kb region on Xq28, containing IRAK1, MECP2 and 7 adjacent genes (L1CAM, AVPR2, ARHGAP4, NAA10, RENBP, HCFC1 and TMEM187), for association with SLE in 15,783 case-control subjects derived from 4 different ancestral groups.
Results
Multiple SNPs showed strong association with SLE in European Americans, Asians and Hispanics at P<5×10−8 with consistent association in subjects with African ancestry. Of these, 6 SNPs located in the TMEM187-IRAK1-MECP2 region captured the underlying causal variant(s) residing in a common risk haplotype shared by all 4 ancestral groups. Among them, rs1059702 best explained the Xq28 association signals in conditional testings and exhibited the strongest P value in trans-ancestral meta-analysis (Pmeta=1.3×10−27, OR=1.43), and thus was considered to be the most-likely causal variant. The risk allele of rs1059702 results in the amino acid substitution S196F in IRAK1 and had previously been shown to increase NF-κB activity in vitro. We also found that the homozygous risk genotype of rs1059702 was associated with lower mRNA levels of MECP2, but not IRAK1, in SLE patients (P=0.0012) and healthy controls (P=0.0064).
Conclusion
These data suggest contributions of both IRAK1 and MECP2 to SLE susceptibility.
doi:10.1136/annrheumdis-2012-201851
PMCID: PMC3567234  PMID: 22904263
Systemic Lupus Erythematosus; Gene Polymorphism; Xq28; IRAK1; MECP2
27.  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.
doi:10.1371/journal.pgen.1003336
PMCID: PMC3585142  PMID: 23468661
28.  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.
doi:10.1371/journal.pgen.1003222
PMCID: PMC3575474  PMID: 23441136
29.  Influenza vaccination can induce new onset anticardiolipins but not β2-glycoprotein-I antibodies among patients with systemic lupus erythematosus 
Lupus  2012;21(2):168-174.
Summary
Background
Antiphospholipid syndrome is characterized by autoantibodies against cardiolipins (aCL), lupus anticoagulant, and independent β2-glycoprotein (β2GPI). Controversy exists as to whether vaccination triggers the development of anti-phospholipid antibodies (aPL) in systemic lupus erythematosus (SLE) patients.
Methods
SLE patients (101) and matched controls (101) were enrolled from 2005 to 2009 and received seasonal influenza vaccinations. Sera were tested by ELISA for aCL at baseline, 2, 6, and 12 weeks after vaccination. Vaccine responses were ranked according to an overall anti-influenza antibody response index. Individuals with positive aCL were further tested for β2GPI antibodies.
Results
SLE patients and healthy controls developed new onset aCL post-vaccination (12/101 cases and 7/101 controls, OR 1.81, p=0.34). New onset moderate aCL are slightly enriched in African American SLE patients (5/36 cases; p=0.094). The optical density (OD) measurements for aCL reactivity in patients were significantly higher than baseline at 2 weeks (p<0.05), 6 weeks (p<0.05), and 12 weeks (p<0.05) post vaccination. No new β2GPI antibodies were detected among patients with new aCL reactivity. Vaccine response was not different between patients with and without new onset aCL reactivity (p=0.43).
Conclusions
This study shows transient increases in aCL, but not anti-β2GPI responses, after influenza vaccination.
doi:10.1177/0961203311429554
PMCID: PMC3268677  PMID: 22235049
Influenza; vaccine; antiphospholipid antibodies; systemic lupus erythematosus
30.  Evaluation of genetic association between an ITGAM non-synonymous SNP (rs1143679) and multiple autoimmune diseases 
Autoimmunity Reviews  2011;11(4):276-280.
Many autoimmune diseases (ADs) share similar underlying pathology and have a tendency to cluster within families, supporting the involvement of shared susceptibility genes. To date, most of the genetic variants associated with systemic lupus erythematosus (SLE) susceptibility also show association with others ADs. ITGAM and its associated ‘predisposing’ variant (rs1143679, Arg77His), predicted to alter the tertiary structures of the ligand-binding domain of ITGAM, may play a key role for SLE pathogenesis. The aim of this study is to examine whether the ITGAM variant is also associated with other ADs. We evaluated case-control association between rs1143679 and ADs (N=18,457) including primary Sjögren’s syndrome, systemic sclerosis, multiple sclerosis, rheumatoid arthritis, juvenile idiopathic arthritis, celiac disease, and type-1 diabetes. We also performed meta-analyses using our data in addition to available published data. Although the risk allele ‘A’ is relatively more frequent among cases for each disease, it was not significantly associated with any other ADs tested in this study. However, the meta-analysis for systemic sclerosis was associated with rs1143679 (pmeta=0.008). In summary, this study explored the role of ITGAM in general autoimmunity in seven non-lupus ADs, and only found association for systemic sclerosis when our results were combined with published results. Thus ITGAM may not be a general autoimmunity gene but this variant may be specifically associated with SLE and systemic sclerosis.
doi:10.1016/j.autrev.2011.07.007
PMCID: PMC3224188  PMID: 21840425
ITGAM; autoimmune diseases; genetic susceptibility
31.  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
32.  Genetic and Physical Interaction of the B-Cell SLE-Associated Genes BANK1 and BLK 
Annals of the Rheumatic Diseases  2011;71(1):136-142.
Objectives
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1136/annrheumdis-2011-200085
PMCID: PMC3268679  PMID: 21978998
systemic lupus erythematosus; genetics; polymorphism; B-cells; autoantibodies
33.  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.
doi:10.1038/gene.2012.6
PMCID: PMC3411915  PMID: 22476155
Systemic Lupus Erythematosus; UBE2L3; Multi Ethnic Association Study; UBCH7 Expression
34.  B Lymphocyte Stimulator Levels in Systemic Lupus Erythematosus: Higher Circulating Levels in African American Patients and Increased Production after Influenza Vaccination in Patients with Low Baseline Levels 
Arthritis and rheumatism  2011;63(12):3931-3941.
Objective
Examine the relationship between circulating B lymphocyte stimulator (BLyS) levels and humoral responses to influenza vaccination in systemic lupus erythematosus (SLE) patients, as well as the effect of vaccination on BLyS levels. Clinical and serologic features of SLE that are associated with elevated BLyS levels will also be investigated.
Methods
Clinical history, disease activity measurements and blood specimens were collected from sixty SLE patients at baseline and after influenza vaccination. Sera were tested for BLyS levels, lupus-associated autoantibodies, serum IFN-α activity, 25-hydroxyvitamin D, and humoral responses to influenza vaccination.
Results
Thirty percent of SLE patients had elevated BLyS levels, with African American patients having higher BLyS levels than European American patients (p=0.006). Baseline BLyS levels in patients were not correlated with humoral responses to influenza vaccination (p=0.863), and BLyS levels increased post-vaccination only in the subset of patients in the lowest quartile of BLyS levels (p=0.0003). Elevated BLyS levels were associated with increased disease activity as measured by SLEDAI, PGA, and SLAM in European Americans (p=0.035, p=0.016, p=0.018, respectively), but not in African Americans. Elevated BLyS levels were also associated with anti-nRNP (p=0.0003) and decreased 25(OH)D (p=0.018). Serum IFN-α activity was a significant predictor of elevated BLyS in a multivariate analysis (p=0.002).
Conclusion
African American SLE patients have higher BLyS levels regardless of disease activity. Humoral response to influenza vaccination is not correlated with baseline BLyS levels in SLE patients and only those patients with low baseline BLyS levels demonstrate an increased BLyS response after vaccination.
doi:10.1002/art.30598
PMCID: PMC3234134  PMID: 22127709
Systemic lupus erythematosus (SLE); Cytokines
35.  MHC Class II and Non-MHC Class II Genes Differentially Influence Humoral Immunity to Bacillus anthracis Lethal Factor and Protective Antigen 
Toxins  2012;4(12):1451-1467.
Anthrax Lethal Toxin consists of Protective Antigen (PA) and Lethal Factor (LF), and current vaccination strategies focus on eliciting antibodies to PA. In human vaccination, the response to PA can vary greatly, and the response is often directed toward non-neutralizing epitopes. Variable vaccine responses have been shown to be due in part to genetic differences in individuals, with both MHC class II and other genes playing roles. Here, we investigated the relative contribution of MHC class II versus non-MHC class II genes in the humoral response to PA and LF immunization using three immunized strains of inbred mice: A/J (H-2k at the MHC class II locus), B6 (H-2b), and B6.H2k (H-2k). IgG antibody titers to LF were controlled primarily by the MHC class II locus, whereas IgG titers to PA were strongly influenced by the non-MHC class II genetic background. Conversely, the humoral fine specificity of reactivity to LF appeared to be controlled primarily through non-MHC class II genes, while the specificity of reactivity to PA was more dependent on MHC class II. Common epitopes, reactive in all strains, occurred in both LF and PA responses. These results demonstrate that MHC class II differentially influences humoral immune responses to LF and PA.
doi:10.3390/toxins4121451
PMCID: PMC3528256  PMID: 23342680
Bacillus anthracis; protective antigen; lethal factor; vaccine; antibody response; MHC class II; mouse; genetic background
36.  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
37.  Individual antibody and T cell responses to vaccination and infection with the 2009 pandemic swine-origin H1N1 influenza virus 
Journal of clinical immunology  2011;31(5):900-912.
Introduction
The 2009 swine origin H1N1 influenza virus (swH1N1) provided an opportunity to study immune responses to a new influenza strain in the context of seasonal influenza vaccination. Our goals were: to assess whether analyzing multiple parameters of immune responsiveness to influenza has an advantage over evaluating hemagglutination inhibition (HAI) titer alone, to determine whether vaccination with the seasonal vaccine induced cross-reactive immunity to swH1N1 in some individuals, and to determine whether the immune response against swH1N1 is higher after infection than vaccination.
Methods
Antibody and T cell responses were studied in ten subjects who were first immunized with the 2009-10 seasonal influenza subunit vaccine, then six weeks later with the swH1N1 monovalent subunit vaccine. The amount of antibody against native virus glycoproteins, overall avidity of these antibodies, and HAI titer were measured. T cells were evaluated for proliferation and IFNγ secretion in response to the vaccine in vitro. Individuals with influenza-like illness were also evaluated, adding a microplate neuraminidase-inhibition (NAI) test.
Results
The immune response to influenza was highly variable and immune parameters did not increase in parallel. The seasonal vaccine induced antibodies recognizing the pandemic virus in 50% of subjects. Antibody affinity and NAI activity to swH1N1 were higher after natural infection than vaccination.
Conclusions
Evaluation of several immune parameters gives a more complete measure of immune responsiveness to influenza infection or vaccination than the HAI test alone.
doi:10.1007/s10875-011-9563-1
PMCID: PMC3197711  PMID: 21732013
pandemic 2009 H1N1 influenza; vaccine response; antibodies and T cells after infection
38.  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
39.  Multiple Autoantibodies Display Association with Lymphopenia, Proteinuria, and Cellular Casts in a Large, Ethnically Diverse SLE Patient Cohort 
Autoimmune Diseases  2012;2012:819634.
Purpose. This study evaluates high-throughput autoantibody screening and determines associated systemic lupus erythematosus (SLE) clinical features in a large lupus cohort. Methods. Clinical and demographic information, along with serum samples, were obtained from each SLE study participant after appropriate informed consent. Serum samples were screened for 10 distinct SLE autoantibody specificities and examined for association with SLE ACR criteria and subcriteria using conditional logistic regression analysis. Results. In European-American SLE patients, autoantibodies against 52 kD Ro and RNP 68 are independently enriched in patients with lymphopenia, anti-La, and anti-ribosomal P are increased in patients with malar rash, and anti-dsDNA and anti-Sm are enriched in patients with proteinuria. In African-American SLE patients, cellular casts associate with autoantibodies against dsDNA, Sm, and Sm/nRNP. Conclusion. Using a high-throughput, bead-based method of autoantibody detection, anti-dsDNA is significantly enriched in patienets with SLE ACR renal criteria as has been previously described. However, lymphopenia is associated with several distinct autoantibody specificities. These findings offer meaningful information to allow clinicians and clinical investigators to understand which autoantibodies correlate with select SLE clinical manifestations across common racial groups using this novel methodology which is expanding in clinical use.
doi:10.1155/2012/819634
PMCID: PMC3439936  PMID: 22988489
40.  Vitamin D deficiency is associated with an increased autoimmune response in healthy individuals and in patients with systemic lupus erythematosus 
Annals of the rheumatic diseases  2011;70(9):1569-1574.
Objectives
Vitamin D deficiency is widespread and has been associated with many chronic diseases, including autoimmune disorders. A study was undertaken to explore the impact of low vitamin D levels on autoantibody production in healthy individuals, as well as B cell hyperactivity and interferon α (IFNα) activity in patients with systemic lupus erythematosus (SLE).
Methods
Serum samples from 32 European American female patients with SLE and 32 matched controls were tested for 25-hydroxyvitamin D (25(OH)D) levels, lupus-associated autoantibodies and serum IFNα activity. Isolated peripheral blood mononuclear cells were tested for intracellular phospho-ERK 1/2 as a measure of B cell activation status.
Results
Vitamin D deficiency (25(OH)D <20 ng/ml) was significantly more frequent among patients with SLE (n=32, 69%) and antinuclear antibody (ANA)-positive controls (n=14, 71%) compared with ANA-negative controls (n=18, 22%) (OR 7.7, 95% CI 2.0 to 29.4, p=0.003 and OR 8.8, 95% CI 1.8 to 43.6, p=0.011, respectively). Patients with high B cell activation had lower mean (SD) 25(OH)D levels than patients with low B cell activation (17.2 (5.1) vs 24.2 (3.9) ng/ml; p=0.009). Patients with vitamin D deficiency also had higher mean (SD) serum IFNα activity than patients without vitamin D deficiency (3.5 (6.6) vs 0.3 (0.3); p=0.02).
Conclusions
The observation that ANA-positive healthy controls are significantly more likely to be deficient in vitamin D than ANA-negative healthy controls, together with the finding that vitamin D deficiency is associated with certain immune abnormalities in SLE, suggests that vitamin D plays an important role in autoantibody production and SLE pathogenesis.
doi:10.1136/ard.2010.148494
PMCID: PMC3149865  PMID: 21586442
41.  Anti-Peptidoglycan Antibodies and Fcγ Receptors Are the Key Mediators of Inflammation in Gram-Positive Sepsis 
Gram-positive bacteria are an important public health problem, but it is unclear how they cause systemic inflammation in sepsis. Our previous work showed that peptidoglycan (PGN) induced proinflammatory cytokines in human cells by binding to an unknown extracellular receptor, followed by phagocytosis leading to the generation of NOD ligands. In this study, we used flow cytometry to identify host factors that supported PGN binding to immune cells. PGN binding required plasma, and plasma from all tested healthy donors contained IgG recognizing PGN. Plasma depleted of IgG or of anti-PGN Abs did not support PGN binding or PGN-triggered cytokine production. Adding back intact but not F(ab′)2 IgG restored binding and cytokine production. Transfection of HEK293 cells with FcγRIIA enabled PGN binding and phagocytosis. These data establish a key role for anti-PGN IgG and FcγRs in supporting inflammation to a major structural element of Gram-positive bacteria and suggest that anti-PGN IgG contributes to human pathology in Gram-positive sepsis.
doi:10.4049/jimmunol.1201302
PMCID: PMC3424298  PMID: 22815288
42.  Influenza vaccination responses in human systemic lupus erythematosus: impact of clinical and demographic features 
Arthritis and rheumatism  2011;63(8):2396-2406.
Objective
Vaccination against common pathogens, such as influenza, is recommended for SLE patients to decrease infections and improve health. However, most vaccination response reports are limited to evaluation of SLE patients with quiescent disease. This study focuses on understanding the clinical, serological, therapeutic, and demographic factors which influence the response to influenza vaccination in patients with a range of disease activities.
Methods
Blood specimens and disease activity information were collected from seventy-two SLE patients at baseline and 2, 6 and 12 weeks after influenza vaccination. Influenza-specific antibody responses were assessed for antibody concentration (Bmax), relative affinity (Ka), and hemagglutination inhibition (HAI). Using a cumulative score, the subjects were evenly divided into high and low responders. Autoantibody levels were evaluated at each time-point by immunofluorescence and standard ELISAs.
Results
Low responders to the vaccine were more likely to have hematologic criteria (p=0.009), exhibit more ACR criteria (p=0.05), and be on concurrent prednisone treatment (p=0.04). Interestingly, European American patients were more likely to be low responders than African Americans (p = 0.03). Following vaccination, low responders were more likely to experience disease flares (p=0.01) and to have increased ANA titers (p = 0.04). Baseline serum interferon alpha activity was significantly higher in patients that experienced a flare after vaccination compared to a matched group of patients that did not flare (p= 0.04).
Conclusions
Ancestral background, prednisone treatment, hematological criteria and evidence of increased disease flares were associated with low antibody responses to influenza vaccination in SLE patients.
doi:10.1002/art.30388
PMCID: PMC3149742  PMID: 21598235
43.  Immunodominance of Antigenic Site B over Site A of Hemagglutinin of Recent H3N2 Influenza Viruses 
PLoS ONE  2012;7(7):e41895.
H3N2 influenza viruses have now circulated in the human population for 43 years since the pandemic of 1968, accumulating sequence changes in the hemagglutinin (HA) and neuraminidase (NA) that are believed to be predominantly due to selection for escape from antibodies. Examination of mutations that persist and accumulate led to identification of antigenically significant mutations that are contained in five antigenic sites (A–E) mapped on to the H3 HA. In early H3N2 isolates, antigenic site A appeared to be dominant while in the 1990s site B seemed more important. To obtain experimental evidence for dominance of antigenic sites on modern H3 HAs, we have measured antibodies in plasma of human subjects who received the 2006–07 trivalent subunit influenza vaccine (H3 component A/Wisconsin/67/05) or the 2008–09 formulation (H3 component A/Uruguay/716/07). Plasmas were tested against expressed HA of Wisconsin-like influenza A/Oklahoma/309/06 and site-directed mutants in antigenic site A (NNES121-124ITEG, N126T, N133D, TSSS135-138GSNA, K140I, RSNNS142-146PGSG), and antigenic site B (HL156-157KS, KFK158-160GST, NDQI189-192QEQT, A196V). “Native ELISA” analysis and escape mutant selection with two human monoclonal antibodies demonstrated that antibody E05 binds to antigenic site A and 1_C02 binds to site B. We find that most individuals, after vaccination in seasons 2006–07 and/or 2008–09, showed dominance of antigenic site B recognition over antigenic site A. A minority showed dominance of site A in 2006 but these were reduced in 2008 when the vaccine virus had a site A mutation. A better understanding of immunodominance may allow prediction of future antigenic drift and assist in vaccine strain selection.
doi:10.1371/journal.pone.0041895
PMCID: PMC3405050  PMID: 22848649
44.  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
45.  Anthrax Vaccination Induced Anti-Lethal Factor IgG: Fine Specificity and Neutralizing Capacity 
Vaccine  2011;29(20):3670-3678.
The efficacy biomarker of the currently licensed anthrax vaccine (AVA) is based on quantity and neutralizing capacity of anti-Protective Antigen (anti-PA) antibodies. However, animal studies have demonstrated that antibodies to Lethal Factor (LF) can provide protection against in vivo bacterial spore challenges. Improved understanding of the fine specificities of humoral immune responses that provide optimum neutralization capacity may enhance the efficacy of future passive immune globulin preparations to treat and prevent inhalation anthrax morbidity and mortality. This study (n = 1000) was designed to identify AVA vaccinated individuals who generate neutralizing antibodies and to determine what specificities correlate with protection. The number of vaccine doses, years post vaccination, and PA titer were associated with in vitro neutralization, reinforcing previous reports. In addition, African American individuals had lower serologic neutralizing activity than European Americans, suggesting a genetic role in the generation of these neutralizing antibodies. Of the vaccinated individuals, only 69 (6.9%) had moderate levels of anti-LF IgG compared to 244 (24.4%) with low and 687 (68.7%) with extremely low levels of IgG antibodies to LF. Using overlapping decapeptide analysis, we identified six common LF antigenic regions targeted by those individuals with moderate levels of antibodies to LF and high in vitro toxin neutralizing activity. Affinity purified antibodies directed against antigenic epitopes within the PA binding and ADP-ribotransferase-like domains of LF were able to protect mice against lethal toxin challenge. Findings from these studies have important implications for vaccine design and immunotherapeutic development.
doi:10.1016/j.vaccine.2011.03.011
PMCID: PMC3233230  PMID: 21420416
Bacillus anthracis; Anthrax; Anthrax Vaccine Adsorbed; Lethal Factor; Protective Antigen; correlate of protection
46.  Role of MYH9 and APOL1 in African and non-African populations with Lupus Nephritis 
Genes and Immunity  2011;13(3):232-238.
Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by autoantibody production and organ damage. Lupus nephritis (LN) is one of the most severe manifestations of SLE. Multiple studies reported associations between renal diseases and variants in the non-muscle myosin heavy chain 9 (MYH9) and the neighboring apolipoprotein L 1 (APOL1) genes. We evaluated 167 variants spanning MYH9 for association with LN in a multiethnic sample. The two previously identified risk variants in APOL1 were also tested for association with LN in European-Americans (EAs) (N = 579) and African-Americans (AAs) (N = 407). Multiple peaks of association exceeding a Bonferroni corrected p-value of p < 2.03 × 10−3 were observed between LN and MYH9 in EAs (N=4620), with the most pronounced association at rs2157257 (p = 4.7 × 10−4; odds ratio [OR]=1.205). A modest effect with MYH9 was also detected in Gullah (rs8136069, p = 0.0019, OR = 2.304). No association between LN and MYH9 was found in AAs, Asians, Amerindians or Hispanics. This study provides the first investigation of MYH9 in LN in non-Africans and of APOL1 in LN in any population and presents novel insight into the potential role of MYH9 in LN in EAs.
doi:10.1038/gene.2011.82
PMCID: PMC3330160  PMID: 22189356
MYH9; APOL1; lupus nephritis; systemic lupus erythematosus; multiethnic association study
47.  Network Analysis of Associations between Serum Interferon Alpha Activity, Autoantibodies, and Clinical Features in Systemic Lupus Erythematosus 
Arthritis and rheumatism  2011;63(4):1044-1053.
Background
Interferon-alpha (IFN-α) is a primary pathogenic factor in systemic lupus erythematosus (SLE), and high IFN-α levels may be associated with particular clinical manifestations. The prevalence of individual clinical and serologic features differs significantly by ancestry. We used multivariate and network analyses to detect associations between clinical and serologic disease manifestations and serum IFN-α activity in a large diverse SLE cohort.
Methods
1089 SLE patients were studied (387 African-American, 186 Hispanic-American, and 516 European-American). Presence or absence of ACR clinical criteria for SLE, autoantibodies, and serum IFN-α activity data were analyzed in univariate and multivariate models. Iterative multivariate logistic regression was performed in each background separately to establish the network of associations between variables that were independently significant following Bonferroni correction.
Results
In all ancestral backgrounds, high IFN-α activity was associated with anti-Ro and anti-dsDNA antibodies (p-values 4.6×10−18 and 2.9 × 10−16 respectively). Younger age, non-European ancestry, and anti-RNP were also independently associated with increased serum IFN-α activity (p≤6.7×10−4). We found 14 unique associations between variables in network analysis, and only 7 of these associations were shared by more than one ancestral background. Associations between clinical criteria were different in different ancestral backgrounds, while autoantibody-IFN-α relationships were similar across backgrounds. IFN-α activity and autoantibodies were not associated with ACR clinical features in multivariate models.
Conclusions
Serum IFN-α activity was strongly and consistently associated with autoantibodies, and not independently associated with clinical features in SLE. IFN-α may be more relevant to humoral tolerance and initial pathogenesis than later clinical disease manifestations.
doi:10.1002/art.30187
PMCID: PMC3068224  PMID: 21162028
systemic lupus erythematosus; interferon alpha; autoantibodies; ancestry
48.  Effects of IRF5 Lupus Risk Haplotype on Pathways Predicted to Influence B Cell Functions 
Both genetic and environmental interactions affect systemic lupus erythematosus (SLE) development and pathogenesis. One known genetic factor associated with lupus is a haplotype of the interferon regulatory factor 5 (IRF5) gene. Analysis of global gene expression microarray data using gene set enrichment analysis identified multiple interferon- and inflammation-related gene sets significantly overrepresented in cells with the risk haplotype. Pathway analysis using expressed genes from the significant gene sets impacted by the IRF5 risk haplotype confirmed significant correlation with the interferon pathway, Toll-like receptor pathway, and the B-cell receptor pathway. SLE patients with the IRF5 risk haplotype have a heightened interferon signature, even in an unstimulated state (P = 0.011), while patients with the IRF5 protective haplotype have a B cell interferon signature similar to that of controls. These results identify multiple genes in functionally significant pathways which are affected by IRF5 genotype. They also establish the IRF5 risk haplotype as a key determinant of not only the interferon response, but also other B-cell pathways involved in SLE.
doi:10.1155/2012/594056
PMCID: PMC3304673  PMID: 22500098
49.  The Lupus Family Registry and Repository 
Rheumatology (Oxford, England)  2010;50(1):47-59.
The Lupus Family Registry and Repository (LFRR) was established with the goal of assembling and distributing materials and data from families with one or more living members diagnosed with SLE, in order to address SLE genetics. In the present article, we describe the problems and solutions of the registry design and biometric data gathering; the protocols implemented to guarantee data quality and protection of participant privacy and consent; and the establishment of a local and international network of collaborators. At the same time, we illustrate how the LFRR has enabled progress in lupus genetics research, answering old scientific questions while laying out new challenges in the elucidation of the biologic mechanisms that underlie disease pathogenesis. Trained staff ascertain SLE cases, unaffected family members and population-based controls, proceeding in compliance with the relevant laws and standards; participant consent and privacy are central to the LFRR’s effort. Data, DNA, serum, plasma, peripheral blood and transformed B-cell lines are collected and stored, and subject to strict quality control and safety measures. Coded data and materials derived from the registry are available for approved scientific users. The LFRR has contributed to the discovery of most of the 37 genetic associations now known to contribute to lupus through 104 publications. The LFRR contains 2618 lupus cases from 1954 pedigrees that are being studied by 76 approved users and their collaborators. The registry includes difficult to obtain populations, such as multiplex pedigrees, minority patients and affected males, and constitutes the largest collection of lupus pedigrees in the world. The LFRR is a useful resource for the discovery and characterization of genetic associations in SLE.
doi:10.1093/rheumatology/keq302
PMCID: PMC3307518  PMID: 20864496
Systemic lupus erythematosus; Registry; Repository; Autoimmune diseases; Genetics; Heritability; Genome-wide association studies; Linkage analysis; Minorities; Women
50.  Anthrax Lethal Toxin-Induced Gene Expression Changes in Mouse Lung 
Toxins  2011;3(9):1111-1130.
A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain. Expression of genes known to regulate vascular permeability was modulated by LeTx in the lungs of the more susceptible A/J strain. Unexpectedly, the largest set of genes with altered expression was immune specific, characterized by the up-regulation of lymphoid genes and the down-regulation of myeloid genes. Transcripts encoding neutrophil chemoattractants, modulators of tumor regulation and angiogenesis were also differentially expressed in both mouse strains. These studies provide new directions for the investigation of vascular leakage and pulmonary edema induced by anthrax LeTx.
doi:10.3390/toxins3091111
PMCID: PMC3202878  PMID: 22039574
Lethal Toxin; lung; gene expression

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