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1.  A Meta-Analysis Identifies New Loci Associated with Body Mass index in Individuals of African Ancestry 
Monda, Keri L. | Chen, Gary K. | Taylor, Kira C. | Palmer, Cameron | Edwards, Todd L. | Lange, Leslie A. | Ng, Maggie C.Y. | Adeyemo, Adebowale A. | Allison, Matthew A. | Bielak, Lawrence F. | Chen, Guanji | Graff, Mariaelisa | Irvin, Marguerite R. | Rhie, Suhn K. | Li, Guo | Liu, Yongmei | Liu, Youfang | Lu, Yingchang | Nalls, Michael A. | Sun, Yan V. | Wojczynski, Mary K. | Yanek, Lisa R. | Aldrich, Melinda C. | Ademola, Adeyinka | Amos, Christopher I. | Bandera, Elisa V. | Bock, Cathryn H. | Britton, Angela | Broeckel, Ulrich | Cai, Quiyin | Caporaso, Neil E. | Carlson, Chris | Carpten, John | Casey, Graham | Chen, Wei-Min | Chen, Fang | Chen, Yii-Der I. | Chiang, Charleston W.K. | Coetzee, Gerhard A. | Demerath, Ellen | Deming-Halverson, Sandra L. | Driver, Ryan W. | Dubbert, Patricia | Feitosa, Mary F. | Freedman, Barry I. | Gillanders, Elizabeth M. | Gottesman, Omri | Guo, Xiuqing | Haritunians, Talin | Harris, Tamara | Harris, Curtis C. | Hennis, Anselm JM | Hernandez, Dena G. | McNeill, Lorna H. | Howard, Timothy D. | Howard, Barbara V. | Howard, Virginia J. | Johnson, Karen C. | Kang, Sun J. | Keating, Brendan J. | Kolb, Suzanne | Kuller, Lewis H. | Kutlar, Abdullah | Langefeld, Carl D. | Lettre, Guillaume | Lohman, Kurt | Lotay, Vaneet | Lyon, Helen | Manson, JoAnn E. | Maixner, William | Meng, Yan A. | Monroe, Kristine R. | Morhason-Bello, Imran | Murphy, Adam B. | Mychaleckyj, Josyf C. | Nadukuru, Rajiv | Nathanson, Katherine L. | Nayak, Uma | N’Diaye, Amidou | Nemesure, Barbara | Wu, Suh-Yuh | Leske, M. Cristina | Neslund-Dudas, Christine | Neuhouser, Marian | Nyante, Sarah | Ochs-Balcom, Heather | Ogunniyi, Adesola | Ogundiran, Temidayo O. | Ojengbede, Oladosu | Olopade, Olufunmilayo I. | Palmer, Julie R. | Ruiz-Narvaez, Edward A. | Palmer, Nicholette D. | Press, Michael F. | Rampersaud, Evandine | Rasmussen-Torvik, Laura J. | Rodriguez-Gil, Jorge L. | Salako, Babatunde | Schadt, Eric E. | Schwartz, Ann G. | Shriner, Daniel A. | Siscovick, David | Smith, Shad B. | Wassertheil-Smoller, Sylvia | Speliotes, Elizabeth K. | Spitz, Margaret R. | Sucheston, Lara | Taylor, Herman | Tayo, Bamidele O. | Tucker, Margaret A. | Van Den Berg, David J. | Velez Edwards, Digna R. | Wang, Zhaoming | Wiencke, John K. | Winkler, Thomas W. | Witte, John S. | Wrensch, Margaret | Wu, Xifeng | Yang, James J. | Levin, Albert M. | Young, Taylor R. | Zakai, Neil A. | Cushman, Mary | Zanetti, Krista A. | Zhao, Jing Hua | Zhao, Wei | Zheng, Yonglan | Zhou, Jie | Ziegler, Regina G. | Zmuda, Joseph M. | Fernandes, Jyotika K. | Gilkeson, Gary S. | Kamen, Diane L. | Hunt, Kelly J. | Spruill, Ida J. | Ambrosone, Christine B. | Ambs, Stefan | Arnett, Donna K. | Atwood, Larry | Becker, Diane M. | Berndt, Sonja I. | Bernstein, Leslie | Blot, William J. | Borecki, Ingrid B. | Bottinger, Erwin P. | Bowden, Donald W. | Burke, Gregory | Chanock, Stephen J. | Cooper, Richard S. | Ding, Jingzhong | Duggan, David | Evans, Michele K. | Fox, Caroline | Garvey, W. Timothy | Bradfield, Jonathan P. | Hakonarson, Hakon | Grant, Struan F.A. | Hsing, Ann | Chu, Lisa | Hu, Jennifer J. | Huo, Dezheng | Ingles, Sue A. | John, Esther M. | Jordan, Joanne M. | Kabagambe, Edmond K. | Kardia, Sharon L.R. | Kittles, Rick A. | Goodman, Phyllis J. | Klein, Eric A. | Kolonel, Laurence N. | Le Marchand, Loic | Liu, Simin | McKnight, Barbara | Millikan, Robert C. | Mosley, Thomas H. | Padhukasahasram, Badri | Williams, L. Keoki | Patel, Sanjay R. | Peters, Ulrike | Pettaway, Curtis A. | Peyser, Patricia A. | Psaty, Bruce M. | Redline, Susan | Rotimi, Charles N. | Rybicki, Benjamin A. | Sale, Michèle M. | Schreiner, Pamela J. | Signorello, Lisa B. | Singleton, Andrew B. | Stanford, Janet L. | Strom, Sara S. | Thun, Michael J. | Vitolins, Mara | Zheng, Wei | Moore, Jason H. | Williams, Scott M. | Zhu, Xiaofeng | Zonderman, Alan B. | Kooperberg, Charles | Papanicolaou, George | Henderson, Brian E. | Reiner, Alex P. | Hirschhorn, Joel N. | Loos, Ruth JF | North, Kari E. | Haiman, Christopher A.
Nature genetics  2013;45(6):690-696.
Genome-wide association studies (GWAS) have identified 36 loci associated with body mass index (BMI), predominantly in populations of European ancestry. We conducted a meta-analysis to examine the association of >3.2 million SNPs with BMI in 39,144 men and women of African ancestry, and followed up the most significant associations in an additional 32,268 individuals of African ancestry. We identified one novel locus at 5q33 (GALNT10, rs7708584, p=3.4×10−11) and another at 7p15 when combined with data from the Giant consortium (MIR148A/NFE2L3, rs10261878, p=1.2×10−10). We also found suggestive evidence of an association at a third locus at 6q16 in the African ancestry sample (KLHL32, rs974417, p=6.9×10−8). Thirty-two of the 36 previously established BMI variants displayed directionally consistent effect estimates in our GWAS (binomial p=9.7×10−7), of which five reached genome-wide significance. These findings provide strong support for shared BMI loci across populations as well as for the utility of studying ancestrally diverse populations.
doi:10.1038/ng.2608
PMCID: PMC3694490  PMID: 23583978
2.  Fli-1 transcription Factor Affects Glomerulonephritis Development by Regulating Expression of Monocyte chemoattractant protein-1 in Endothelial Cells in the Kidney 
Clinical immunology (Orlando, Fla.)  2012;145(3):201-208.
Expression of transcription factor Fli-1 is implicated in the development of glomerulonephritis. Fli-1 heterozygous knockout (Fli1+/−) NZM2410 mice, a murine model of lupus, had significantly improved survival and reduced glomerulonephritis. In this study, we found that infiltrated inflammatory cells were significantly decreased in the kidneys from Fli-1+/− NZM2410 mice. The expression of Monocyte chemoattractant protein-1 (MCP-1) was significantly decreased in kidneys from Fli-1+/− NZM2410 mice. The primary endothelial cells isolated from the kidneys of Fli-1+/− NZM2410 mice produced significantly less MCP-1. In endothelial cells transfected with specific Fli-1 siRNA the production of MCP-1 was significantly reduced compared to cells transfected with negative control siRNA. By Chromatin Immunoprecipitation (ChIP) assay, we further demonstrated that Fli-1 directly binds to the promoter of the MCP-1 gene. Our data indicate that Fli-1 impacts glomerulonephritis development by regulating expression of inflammatory chemokine MCP-1 and inflammatory cell infiltration in the kidneys in the NZM2410 mice.
doi:10.1016/j.clim.2012.09.006
PMCID: PMC3501541  PMID: 23108091
Glomerulonephritis; inflammatory chemokines; infiltrations; transcription factors
3.  Two Independent Functional Risk Haplotypes in TNIP1 are Associated with Systemic Lupus Erythematosus 
Arthritis and rheumatism  2012;64(11):3695-3705.
Objective
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1002/art.34642
PMCID: PMC3485412  PMID: 22833143
4.  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.
Objective
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1002/art.34650
PMCID: PMC3485439  PMID: 22886787
5.  Comparison of autoantibody specificities between traditional and bead-based assays in a large, diverse collection of SLE patients and family members 
Arthritis and rheumatism  2012;64(11):3677-3686.
Objective
The replacement of standard immunofluorescence anti-nuclear antibody (ANA) methods with bead-based assays is a new clinical option. A large, multi-racial cohort of SLE patients, blood relatives and unaffected control individuals was evaluated for familial aggregation and subset clustering of autoantibodies by high-throughput serum screening technology and traditional methods.
Methods
Serum samples (1,540 SLE patients, 1,127 unaffected relatives, and 906 healthy, population-based controls) were analyzed for SLE autoantibodies using a bead-based assay, immunofluorescence, and immunodiffusion. Autoantibody prevalence, disease sensitivity, clustering, and association with standard immunodiffusion results were evaluated.
Results
ANA frequency in SLE patient sera were 89%, 73%, and 67% by BioPlex 2200 and 94%, 84%, and 86% by immunofluorescence in African-American, Hispanic, and European-American patients respectively. 60kD Ro, La, Sm, nRNP A, and ribosomal P prevalence were compared across assays, with sensitivities ranging from 0.92 to 0.83 and specificities ranging from 0.90 to 0.79. Cluster autoantibody analysis showed association of three subsets: 1) 60kD Ro, 52kD Ro and La, 2) spliceosomal proteins, and 3) dsDNA, chromatin, and ribosomal P. Familial aggregation of Sm/RNP, ribosomal P, and 60kD Ro in SLE patient sibling pairs was observed (p ≤ 0.004). Simplex pedigree patients had a greater prevalence for dsDNA (p=0.0003) and chromatin (p=0.005) autoantibodies than multiplex patients.
Conclusion
ANA frequencies detected by a bead-based assay are lower in European-American SLE patients compared to immunofluorescence. These assays have strong positive predictive values across racial groups, provide useful information for clinical care, and provide unique insights to familial aggregation and autoantibody clustering.
doi:10.1002/art.34651
PMCID: PMC3490432  PMID: 23112091
systemic lupus erythematosus; autoantibodies; ancestry
6.  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.
doi:10.1371/journal.pgen.1003870
PMCID: PMC3794920  PMID: 24130510
7.  Estrogen receptor alpha modulates toll-like receptor signaling in murine lupus 
Systemic lupus erythematosus (SLE) is a disease that disproportionately affects females. Despite significant research effort, the mechanisms underlying the female predominance in this disease are largely unknown. Previously, we showed that estrogen receptor alpha knockout (ERαKO) lupus prone female mice had significantly less pathologic renal disease and proteinuria, and significantly prolonged survival. Since autoantibody levels and number and percentage of B/T cells were not significantly impacted by ERα genotype, we hypothesized that the primary benefit of ERα deficiency in lupus nephritis was via modulation of the innate immune response. Using BMDCs and spleen cells/B cells from female wild-type or ERαKO mice, we found that ERαKO-derived cells have a significantly reduced inflammatory response after stimulation with TLR agonists. Our results indicate that the inflammatory response to TLR ligands is significantly impacted by the presence of ERα despite the absence of estradiol, and may partially explain the protective effect of ERα deficiency in lupus-prone animals.
doi:10.1016/j.clim.2012.04.001
PMCID: PMC3737583  PMID: 22659029
Estrogen receptor alpha; Systemic lupus; erythematosus; Dendritic cells; Toll-like receptors
8.  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.
doi:10.1371/journal.pone.0069404
PMCID: PMC3737240  PMID: 23950893
9.  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.
Objective
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.
Methods
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.
Results
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.
Conclusion
These results suggest distinct patterns of association with SLE in African-derived populations; specific loci may be more strongly associated within select population groups.
doi:10.3899/jrheum.120989
PMCID: PMC3735344  PMID: 23637325
systemic lupus erythematosus; African Americans; genetic association studies; oxygen compounds; single nucleotide polymorphism
10.  The HLA–DRB1 Shared Epitope Is Associated With Susceptibility to Rheumatoid Arthritis in African Americans Through European Genetic Admixture 
Arthritis and rheumatism  2008;58(2):349-358.
Objective
To determine whether shared epitope (SE)–containing HLA–DRB1 alleles are associated with rheumatoid arthritis (RA) in African Americans and whether their presence is associated with higher degrees of global (genome-wide) genetic admixture from the European population.
Methods
In this multicenter cohort study, African Americans with early RA and matched control subjects were analyzed. In addition to measurement of serum anti–cyclic citrullinated peptide (anti-CCP) antibodies and HLA–DRB1 genotyping, a panel of >1,200 ancestry-informative markers was analyzed in patients with RA and control subjects, to estimate the proportion of European ancestry.
Results
The frequency of SE-containing HLA–DRB1 alleles was 25.2% in African American patients with RA versus 13.6% in control subjects (P = 0.00005). Of 321 patients with RA, 42.1% had at least 1 SE-containing allele, compared with 25.3% of 166 control subjects (P = 0.0004). The mean estimated percent European ancestry was associated with SE-containing HLA–DRB1 alleles in African Americans, regardless of disease status (RA or control). As reported in RA patients of European ancestry, there was a significant association of the SE with the presence of the anti-CCP antibody: 86 (48.9%) of 176 patients with anti-CCP antibody–positive RA had at least 1 SE allele, compared with 36 (32.7%) of 110 patients with anti-CCP antibody–negative RA (P = 0.01, by chi-square test).
Conclusion
HLA–DRB1 alleles containing the SE are strongly associated with susceptibility to RA in African Americans. The absolute contribution is less than that reported in RA among populations of European ancestry, in which ~50–70% of patients have at least 1 SE allele. As in Europeans with RA, the SE association was strongest in the subset of African American patients with anti-CCP antibodies. The finding of a higher degree of European ancestry among African Americans with SE alleles suggests that a genetic risk factor for RA was introduced into the African American population through admixture, thus making these individuals more susceptible to subsequent environmental or unknown factors that trigger the disease.
doi:10.1002/art.23166
PMCID: PMC3726059  PMID: 18240241
11.  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.
doi:10.1371/journal.pgen.1003554
PMCID: PMC3715547  PMID: 23874208
12.  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.
doi:10.1038/ng.81
PMCID: PMC3712260  PMID: 18204446
13.  A role for estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) in collagen biosynthesis in mouse skin 
Hormonal regulation of the dermal collagenous extracellular matrix plays a key role in maintaining proper tissue homeostasis, however the factors and pathways involved in this process are not fully defined. This study investigated the role of estrogen receptors (ERs) in the regulation of collagen biosynthesis in mice lacking ERα or ERβ. Collagen content was significantly increased in the skin of ΕRα-/- mice as measured by acetic acid extraction and the hydroxyproline assay and correlated with the decreased levels of MMP-15 and elevated collagen production by ΕRα-/- fibroblasts. In contrast, collagen content was decreased in the skin of ERβ-/- mice despite markedly increased collagen production by ERβ-/- fibroblasts. However, expression of several matrix metalloproteinases (MMPs), including MMP-8 and -15 was significantly elevated suggesting increased degradation of dermal collagen. Furthermore, ERβ-/- mice were characterized by significantly reduced levels of small leucine proteoglycans (SLRPs), lumican and decorin, leading to the defects in collagen fibrillogenesis and possibly less stable collagen fibrils. ERα-/- mice also exhibited fibrils with irregular structure and size, which correlated with increased levels of lumican and decorin. Together, these results demonstrate distinct functions of estrogen receptors in the regulation of collagen biosynthesis in mouse skin in vivo.
doi:10.1038/jid.2012.264
PMCID: PMC3502697  PMID: 22895361
14.  Evaluation of TRAF6 in a Large Multi-Ancestral Lupus Cohort 
Arthritis and Rheumatism  2012;64(6):1960-1969.
Objective
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1002/art.34361
PMCID: PMC3380425  PMID: 22231568
TRAF6; polymorphism; systemic lupus erythematosus
15.  Endothelial Nitric Oxide Synthase Reduces Crescentic and Necrotic Glomerular Lesions, Reactive Oxygen Production, and MCP1 Production in Murine Lupus Nephritis 
PLoS ONE  2013;8(5):e64650.
Systemic lupus erythematosus, in both animal models and in humans, is characterized by autoantibody production followed by immune complex deposition in target tissues. Ensuing target organ damage is modulated by reactive intermediates, including reactive nitrogen and oxygen species, through as of now incompletely understood mechanisms. Endothelial nitric oxide synthase is known to impact vascular reactivity; however its impact on reactive intermediate production and inflammatory renal disease is less well defined. In this study, we assessed the impact of endothelial nitric oxide synthase (eNOS) on disease in lupus prone MRL/lpr mice. Mice lacking eNOS developed earlier more severe disease with decreased survival. eNOS deficient mice died sooner and developed significantly more glomerular crescents, necrosis, inflammatory infiltrates and vasculitis, indicating a role for eNOS in modulating these renal lesions. Immune complex deposition was similar between groups, indicating the impact of eNOS is distal to antibody/complement glomerular deposition. Urinary nitric oxide production was decreased in the eNOS deficient mice, while proteinuria was increased. Urinary monocyte chemotactic protein-1 was also increased in the knockout mice. CD4+ T cells from MRL/lpr mice demonstrated mitochondrial hyperpolarization, increased nitric oxide and superoxide production and increased calcium flux compared to B6 control mice. Deficiency of eNOS resulted in decreased nitric oxide and mitochondrial calcium levels but had no effect on mitochondrial hyperpolarization. Renal cortices from MRL/lpr mice that are eNOS deficient demonstrated increased superoxide production, which was blocked by both nitric oxide synthase and NADPH oxidase inhibitors. These studies thus demonstrate a key role for eNOS in modulating renal disease in lupus prone MRL/lpr mice. The impact appears to be mediated by effects on superoxide production in the kidney, impacting downstream mediators such as monocyte chemotactic protein-1. These results suggest that modulation of eNOS may be a novel therapeutic approach to treating lupus nephritis.
doi:10.1371/journal.pone.0064650
PMCID: PMC3669382  PMID: 23741359
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  Variation in the ICAM1–ICAM4–ICAM5 locus is associated with systemic lupus erythematosus susceptibility in multiple ancestries 
Annals of the rheumatic diseases  2012;71(11):1809-1814.
Objective
Systemic lupus erythematosus (SLE; OMIM 152700) is a chronic autoimmune disease for which the aetiology includes genetic and environmental factors. ITGAM, integrin αΜ (complement component 3 receptor 3 subunit) encoding a ligand for intracellular adhesion molecule (ICAM) proteins, is an established SLE susceptibility locus. This study aimed to evaluate the independent and joint effects of genetic variations in the genes that encode ITGAM and ICAM.
Methods
The authors examined several markers in the ICAM1–ICAM4–ICAM5 locus on chromosome 19p13 and the single ITGAM polymorphism (rs1143679) using a large-scale case–control study of 17 481 unrelated participants from four ancestry populations. The single marker association and gene–gene interaction were analysed for each ancestry, and a meta-analysis across the four ancestries was performed.
Results
The A-allele of ICAM1–ICAM4–ICAM5 rs3093030, associated with elevated plasma levels of soluble ICAM1, and the A-allele of ITGAM rs1143679 showed the strongest association with increased SLE susceptibility in each of the ancestry populations and the trans-ancestry meta-analysis (ORmeta=1.16, 95% CI 1.11 to 1.22; p=4.88×10−10 and ORmeta=1.67, 95% CI 1.55 to 1.79; p=3.32×10−46, respectively). The effect of the ICAM single-nucleotide polymorphisms (SNPs) was independent of the effect of the ITGAM SNP rs1143679, and carriers of both ICAM rs3093030-AA and ITGAM rs1143679-AA had an OR of 4.08 compared with those with no risk allele in either SNP (95% CI 2.09 to 7.98; p=3.91×10−5).
Conclusion
These findings are the first to suggest that an ICAM–integrin-mediated pathway contributes to susceptibility to SLE.
doi:10.1136/annrheumdis-2011-201110
PMCID: PMC3466387  PMID: 22523428

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