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1.  Analysis of autosomal genes reveals gene–sex interactions and higher total genetic risk in men with systemic lupus erythematosus 
Annals of the Rheumatic Diseases  2011;71(5):694-699.
Objectives
Systemic lupus erythematosus (SLE) is a sexually dimorphic autoimmune disease which is more common in women, but affected men often experience a more severe disease. The genetic basis of sexual dimorphism in SLE is not clearly defined. A study was undertaken to examine sex-specific genetic effects among SLE susceptibility loci.
Methods
A total of 18 autosomal genetic susceptibility loci for SLE were genotyped in a large set of patients with SLE and controls of European descent, consisting of 5932 female and 1495 male samples. Sex-specific genetic association analyses were performed. The sex–gene interaction was further validated using parametric and nonparametric methods. Aggregate differences in sex-specific genetic risk were examined by calculating a cumulative genetic risk score for SLE in each individual and comparing the average genetic risk between male and female patients.
Results
A significantly higher cumulative genetic risk for SLE was observed in men than in women. (P = 4.52×10−8) A significant sex–gene interaction was seen primarily in the human leucocyte antigen (HLA) region but also in IRF5, whereby men with SLE possess a significantly higher frequency of risk alleles than women. The genetic effect observed in KIAA1542 is specific to women with SLE and does not seem to have a role in men.
Conclusions
The data indicate that men require a higher cumulative genetic load than women to develop SLE. These observations suggest that sex bias in autoimmunity could be influenced by autosomal genetic susceptibility loci.
doi:10.1136/annrheumdis-2011-200385
PMCID: PMC3324666  PMID: 22110124
2.  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.
Objective
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.
Methods
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.
Results
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.
Conclusions
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
doi:10.1136/annrheumdis-2011-200463
PMCID: PMC3307526  PMID: 22088620
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  Association of PPP2CA polymorphisms with SLE susceptibility in multiple ethnic groups 
Arthritis and rheumatism  2011;63(9):2755-2763.
Objective
T cells from patients with SLE express increased amounts of PP2Ac which contribute to decreased production of IL-2. Because IL-2 is important in the regulation of several aspects of the immune response, it has been proposed that PP2Ac contributes to the expression of SLE. This study was designed to determine whether genetic variants of PPP2AC are linked to the expression of SLE and specific clinical manifestations and account for the increased expression of PP2Ac.
Methods
We conducted a trans-ethnic study consisting of 8,695 SLE cases and 7,308 controls from four different ancestries. Eighteen single-nucleotide polymorphisms (SNPs) across the PPP2CA were genotyped using an Illumina custom array. PPP2CA expression in SLE and control T cells was analyzed by real-time PCR.
Results
A 32-kb haplotype comprised of multiple SNPs of PPP2CA showed significant association with SLE in Hispanic Americans (HA), European Americans (EA) and Asians but not in African-Americans (AA). Conditional analyses revealed that SNP rs7704116 in intron 1 showed consistently strong association with SLE across Asian, EA and HA populations (pmeta=3.8×10−7, OR=1.3[1.14–1.31]). In EA, the largest ethnic dataset, the risk A allele of rs7704116 was associated with the presence of renal disease, anti-dsDNA and anti-RNP antibodies. PPP2CA expression was approximately 2-fold higher in SLE patients carrying the rs7704116 AG genotype than those carrying GG genotype (p = 0.008).
Conclusion
Our data provide the first evidence for an association between PPP2CA polymorphisms and elevated PP2Ac transcript levels in T cells, which implicates a new molecular pathway for SLE susceptibility in EA, HA and Asians.
doi:10.1002/art.30452
PMCID: PMC3163110  PMID: 21590681
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies 
The Journal of Clinical Investigation  2011;121(8):3109-3119.
During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies.
doi:10.1172/JCI57834
PMCID: PMC3148747  PMID: 21785218
19.  Evaluation of the TREX1 gene in a large multi-ancestral lupus cohort 
Genes and immunity  2011;12(4):270-279.
Systemic Lupus Erythematosus (SLE) is a prototypic autoimmune disorder with a complex pathogenesis in which genetic, hormonal and environmental factors play a role. Rare mutations in the TREX1 gene, the major mammalian 3′-5′ exonuclease, have been reported in sporadic SLE cases. Some of these mutations have also been identified in a rare pediatric neurologic condition featuring an inflammatory encephalopathy known as Aicardi-Goutières syndrome (AGS). We sought to investigate the frequency of these mutations in a large multi-ancestral cohort of SLE cases and controls.
Methods
Forty single-nucleotide polymorphisms (SNPs), including both common and rare variants, across the TREX1 gene were evaluated in ∼8370 patients with SLE and ∼7490 control subjects. Stringent quality control procedures were applied and principal components and admixture proportions were calculated to identify outliers for removal from analysis. Population-based case-control association analyses were performed. P values, false discovery rate q values, and odds ratios with 95% confidence intervals were calculated.
Results
The estimated frequency of TREX1 mutations in our lupus cohort was 0.5%. Five heterozygous mutations were detected at the Y305C polymorphism in European lupus cases but none were observed in European controls. Five African cases incurred heterozygous mutations at the E266G polymorphism and, again, none were observed in the African controls. A rare homozygous R114H mutation was identified in one Asian SLE patient whereas all genotypes at this mutation in previous reports for SLE were heterozygous. Analysis of common TREX1 SNPs (MAF >10%) revealed a relatively common risk haplotype in European SLE patients with neurologic manifestations, especially seizures, with a frequency of 58% in lupus cases compared to 45% in normal controls (p=0.0008, OR=1.73, 95% CI=1.25-2.39). Finally, the presence or absence of specific autoantibodies in certain populations produced significant genetic associations. For example, a strong association with anti-nRNP was observed in the European cohort at a coding synonymous variant rs56203834 (p=2.99E-13, OR=5.2, 95% CI=3.18-8.56).
Conclusion
Our data confirm and expand previous reports and provide additional support for the involvement of TREX1 in lupus pathogenesis.
doi:10.1038/gene.2010.73
PMCID: PMC3107387  PMID: 21270825
20.  Association Between a Functional Variant Downstream of TNFAIP3 and Systemic Lupus Erythematosus 
Nature genetics  2011;43(3):253-258.
Systemic Lupus Erythematosus (SLE, OMIM 152700) is an autoimmune disease characterized by self-reactive antibodies resulting in systemic inflammation and organ failure. TNFAIP3, encoding the ubiquitin-modifying enzyme A20, is an established susceptibility locus for SLE. By fine mapping and genomic resequencing in ethnically diverse populations we fully characterized the TNFAIP3 risk haplotype and isolated a novel TT>A polymorphic dinucleotide associated with SLE in subjects of European (P = 1.58 × 10−8; odds ratio (OR) = 1.70) and Korean (P = 8.33 × 10−10; OR = 2.54) ancestry. This variant, located in a region of high conservation and regulatory potential, bound a nuclear protein complex comprised of NF-κB subunits with reduced avidity. Furthermore, compared with the non-risk haplotype, the haplotype carrying this variant resulted in reduced TNFAIP3 mRNA and A20 protein expression. These results establish this TT>A variant as the most likely functional polymorphism responsible for the association between TNFAIP3 and SLE.
doi:10.1038/ng.766
PMCID: PMC3103780  PMID: 21336280
21.  Association of Genetic Variants in Complement Factor H and Factor H-Related Genes with Systemic Lupus Erythematosus Susceptibility 
PLoS Genetics  2011;7(5):e1002079.
Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, Pmeta = 6.6×10−8, OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, Pmeta = 2.9×10−7, OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ∼146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (Pmeta = 3.2×10−7, OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (Pmeta = 3.5×10−4, OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE.
Author Summary
Systemic lupus erythematosus (SLE) is a complex autoimmune disease, associated with increased complement activation. Previous studies have provided evidence for the presence of SLE susceptibility gene(s) in the chromosome 1q31-32 locus. Within 1q32, genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) may contribute to the development of SLE, because genetic variants of these genes impair complement regulation and predispose to various human diseases. In this study, we tested association of genetic variants in the region containing CFH and CFHRs with SLE. We identified genetic variants predisposing to SLE in European American, African American, and Asian populations, which might be attributed to the deletion of CFHR3 and CFHR1 genes but not previously identified disease-associated exonic variants of CFH. This study provides the first evidence for consistent association between CFH/CFHRs and SLE across multi-ancestral SLE datasets, providing new insights into the role of complement regulators in the pathogenesis of SLE.
doi:10.1371/journal.pgen.1002079
PMCID: PMC3102741  PMID: 21637784

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