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1.  An Enhancer Element Harboring Variants Associated with Systemic Lupus Erythematosus Engages the TNFAIP3 Promoter to Influence A20 Expression 
PLoS Genetics  2013;9(9):e1003750.
Functional characterization of causal variants present on risk haplotypes identified through genome-wide association studies (GWAS) is a primary objective of human genetics. In this report, we evaluate the function of a pair of tandem polymorphic dinucleotides, 42 kb downstream of the promoter of TNFAIP3, (rs148314165, rs200820567, collectively referred to as TT>A) recently nominated as causal variants responsible for genetic association of systemic lupus erythematosus (SLE) with tumor necrosis factor alpha inducible protein 3 (TNFAIP3). TNFAIP3 encodes the ubiquitin-editing enzyme, A20, a key negative regulator of NF-κB signaling. A20 expression is reduced in subjects carrying the TT>A risk alleles; however, the underlying functional mechanism by which this occurs is unclear. We used a combination of electrophoretic mobility shift assays (EMSA), mass spectrometry (MS), reporter assays, chromatin immunoprecipitation-PCR (ChIP-PCR) and chromosome conformation capture (3C) EBV transformed lymphoblastoid cell lines (LCL) from individuals carrying risk and non-risk TNFAIP3 haplotypes to characterize the effect of TT>A on A20 expression. Our results demonstrate that the TT>A variants reside in an enhancer element that binds NF-κB and SATB1 enabling physical interaction of the enhancer with the TNFAIP3 promoter through long-range DNA looping. Impaired binding of NF-κB to the TT>A risk alleles or knockdown of SATB1 expression by shRNA, inhibits the looping interaction resulting in reduced A20 expression. Together, these data reveal a novel mechanism of TNFAIP3 transcriptional regulation and establish the functional basis by which the TT>A risk variants attenuate A20 expression through inefficient delivery of NF-κB to the TNFAIP3 promoter. These results provide critical functional evidence supporting a direct causal role for TT>A in the genetic predisposition to SLE.
Author Summary
A key objective of human genetics is the identification and characterization of variants responsible for association with complex diseases. A pair of single nucleotide polymorphisms (rs148314165, rs200820567) 42 kb downstream from the promoter of TNFAIP3, have been proposed as the variants responsible for association with systemic lupus erythematosus based on comprehensive genetic and bioinformatic analyses. TNFAIP3 encodes for the ubiquitin-editing enzyme, A20, which plays a central role in maintaining immune system homeostasis through restriction of NF-κB signaling. Cells that carry this risk haplotype express low levels of TNFAIP3 compared to cells carrying the nonrisk haplotype. How the risk alleles of rs148314165 and rs200820567 might influence low TNFAIP3 expression is unknown. In this paper, we demonstrate that these variants reside in an enhancer element that binds NF-κB and SATB1 enabling the interaction of the enhancer with the TNFAIP3 promoter through long-range DNA looping. Impaired binding of NF-κB directly to the risk alleles or shRNA-mediated knockdown of SATB1 inhibits interaction of the enhancer with the TNFAIP3 promoter resulting in reduced A20 expression. These results clarify the functional mechanism by which rs148314165 and rs200820567 attenuate A20 expression and support a causal role for these variants in the predisposition to autoimmune disease.
doi:10.1371/journal.pgen.1003750
PMCID: PMC3764111  PMID: 24039598
2.  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
3.  CSK regulatory polymorphism is associated with systemic lupus erythematosus and influences B cell signaling and activation 
Nature genetics  2012;44(11):1227-1230.
C-src tyrosine kinase, Csk, physically interacts with the intracellular phosphatase Lyp (PTPN22) and can modify the activation state of downstream Src kinases, such as Lyn, in lymphocytes. We identified an association of Csk with systemic lupus erythematosus (SLE) and refined its location to an intronic polymorphism rs34933034 (OR 1.32, p = 1.04 × 10−9). The risk allele is associated with increased CSK expression and augments inhibitory phosphorylation of Lyn. In carriers of the risk allele, B cell receptor (BCR)-mediated activation of mature B cells, as well as plasma IgM, are increased. Moreover, the fraction of transitional B cells is doubled in the cord blood of carriers of the risk allele compared to non-risk haplotypes due to an expansion of the late transitional cells, a stage targeted by selection mechanisms. This suggests that the Lyp-Csk complex increases susceptibility to lupus at multiple maturation and activation points of B cells.
doi:10.1038/ng.2439
PMCID: PMC3715052  PMID: 23042117
4.  Polymorphism at the TNF superfamily gene TNFSF4 confers susceptibility to systemic lupus erythematosus 
Nature genetics  2007;40(1):83-89.
Systemic lupus erythematosus (SLE) is a multisystem complex autoimmune disease of uncertain etiology (OMIM 152700). Over recent years a genetic component to SLE susceptibility has been established1–3. Recent successes with association studies in SLE have identified genes including IRF5 (refs. 4,5) and FCGR3B6. Two tumor necrosis factor (TNF) superfamily members located within intervals showing genetic linkage with SLE are TNFSF4 (also known as OX40L; 1q25), which is expressed on activated antigen-presenting cells (APCs)7,8 and vascular endothelial cells9, and also its unique receptor, TNFRSF4 (also known as OX40; 1p36), which is primarily expressed on activated CD4+ T cells10. TNFSF4 produces a potent co-stimulatory signal for activated CD4+ T cells after engagement of TNFRSF4 (ref. 11). Using both a family-based and a case-control study design, we show that the upstream region of TNFSF4 contains a single risk haplotype for SLE, which is correlated with increased expression of both cell-surface TNFSF4 and the TNFSF4 transcript. We hypothesize that increased expression of TNFSF4 predisposes to SLE either by quantitatively augmenting T cell–APC interaction or by influencing the functional consequences of T cell activation via TNFRSF4.
doi:10.1038/ng.2007.47
PMCID: PMC3705866  PMID: 18059267
5.  Sex chromosome Aneuploides among Men with Systemic Lupus Erythematosus 
Journal of Autoimmunity  2011;38(2-3):J129-J134.
About 90% of patients with systemic lupus erythematosus (SLE) are female. We hypothesize that the number of X chromosomes, not sex, is a determinate of risk of SLE. Number of X chromosomes was determined by single nucleotide typing and then confirmed by karyotype or fluorescent in situ hybridization in a large group of men with SLE. Presence of an sry gene was assessed by rtPCR. We calculated 96% confidence intervals using the Adjusted Wald method, and used Bayes’ theorem to estimate the prevalence of SLE among 47,XXY and 46,XX men. Among 316 men with SLE, 7 had 47,XXY and 1 had 46,XX. The rate of Klinefelter’s syndrome (47,XXY) was statistically different from that found in control men and from the known prevalence in the population. The 46,XX man had an sry gene, which encodes the testes determining factor, on an X chromosome as a result of an abnormal crossover during meiosis. In the case of 46,XX, 1 of 316 was statistically different from the known population prevalence of 1 in 20,000 live male births. A previously reported 46,XX man with SLE had a different molecular mechanism in which there were no common gene copy number abnormalities with our patient. Thus, men with SLE are enriched for conditions with additional X chromosomes. Especially since 46,XX men are generally normal males, except for infertility, these data suggest the number of X chromosomes, not phenotypic sex, is responsible for the sex bias of SLE.
doi:10.1016/j.jaut.2011.10.004
PMCID: PMC3309073  PMID: 22154021
Systemic lupus erythematosus; Klinefelter’s syndrome; male 46; XX; female bias; X chromosome
6.  The Genomics of Autoimmune Disease in the Era of Genome-Wide Association Studies and Beyond 
Autoimmunity Reviews  2011;11(4):267-275.
Recent advances in the field of genetics have dramatically changed our understanding of autoimmune disease. Candidate gene and, more recently, genome-wide association (GWA) studies have led to an explosion in the number of loci and pathways known to contribute to autoimmune phenotypes. Since the 1970s, researchers have known that several alleles in the MHC region play a role in the pathogenesis of many autoimmune diseases. More recent work has identified numerous risk loci involving both the innate and adaptive immune responses. However, much remains to be learned about the heritability of autoimmune conditions. Most regions found through GWA scans have yet to isolate the association to the causal allele(s) responsible for conferring disease risk. A role for rare variants (allele frequencies of <1%) has begun to emerge. Future research will use next generation sequencing (NGS) technology to comprehensively evaluate the human genome for risk variants. Whole transcriptome sequencing is now possible, which will provide much more detailed gene expression data. The dramatic drop in the cost and time required to sequence the entire human genome will ultimately make it possible for this technology to be used as a clinical diagnostic tool.
doi:10.1016/j.autrev.2011.10.003
PMCID: PMC3288956  PMID: 22001415
Genetics; Genomics; Genome-wide association study; Autoimmune disease
7.  Identification of novel coding mutation in C1qA gene in an African-American pedigree with lupus and C1q deficiency 
Lupus  2012;21(10):1113-1118.
Objectives
Homozygous C1q deficiency is an extremely rare condition and strongly associated with systemic lupus erythematosus. To assess and characterize C1q deficiency in an African-American lupus pedigree, C1q genomic region was evaluated in the lupus cases and family members.
Methods
Genomic DNA from patient was obtained and C1q A, B and C gene cluster was sequenced using next generation sequencing method. The identified mutation was further confirmed by direct Sanger sequencing method in the patient and all blood relatives. C1q levels in serum were measured using sandwich ELISA method.
Results
In an African-American patient with lupus and C1q deficiency, we identified and confirmed a novel homozygote start codon mutation in C1qA gene that changes amino acid Methionine to Arginine at position 1. The Met1Arg mutation prevents protein translation (Met1Arg). Mutation analyses of the patient’s family members also revealed the Met1Arg homozygote mutation in her deceased brother who also had lupus with absence of total complement activity consistent with a recessive pattern of inheritance.
Conclusion
The identification of new mutation in C1qA gene that disrupts the start codon (ATG to AGG (Met1Arg)), has not been reported previously and it expands the knowledge and importance of the C1q gene in the pathogenesis of lupus especially in high risk African-American population.
doi:10.1177/0961203312443993
PMCID: PMC3508769  PMID: 22472776
8.  Contributions of mass spectrometry-based proteomics to defining cellular mechanisms and diagnostic markers for systemic lupus erythematosus 
Systematic lupus erythematosus (SLE) is a complex disease for which molecular diagnostics are limited and pathogenesis is not clearly understood. Important information is provided in this regard by identification and characterization of more specific molecular and cellular targets in SLE immune cells and target tissue and markers of early-onset and effective response to treatment of SLE complications. In recent years, advances in proteomic technologies and applications have facilitated such discoveries. Here we provide a review of insights into SLE pathogenesis, diagnosis and treatment that have been provided by mass spectrometry-based proteomic approaches.
doi:10.1186/ar3701
PMCID: PMC3392812  PMID: 22364570
9.  Genetic Analyses of Interferon Pathway-Related Genes Reveals Multiple New Loci Associated with Systemic Lupus Erythematosus (SLE) 
Arthritis and rheumatism  2011;63(7):2049-2057.
Objective
The overexpression of interferon (IFN)-inducible genes is a prominent feature of SLE, serves as a marker for active and more severe disease, and is also observed in other autoimmune and inflammatory conditions. The genetic variations responsible for sustained activation of IFN responsive genes are unknown.
Methods
We systematically evaluated association of SLE with a total of 1,754 IFN-pathway related genes, including IFN-inducible genes known to be differentially expressed in SLE patients and their direct regulators. We performed a three-stage design where two cohorts (total n=939 SLE cases, 3,398 controls) were analyzed independently and jointly for association with SLE, and the results were adjusted for the number of comparisons.
Results
A total of 16,137 SNPs passed all quality control filters of which 316 demonstrated replicated association with SLE in both cohorts. Nine variants were further genotyped for confirmation in an average of 1,316 independent SLE cases and 3,215 independent controls. Association with SLE was confirmed for several genes, including the transmembrane receptor CD44 (rs507230, P = 3.98×10−12), cytokine pleiotrophin (PTN) (rs919581, P = 5.38×10−04), the heat-shock DNAJA1 (rs10971259, P = 6.31×10−03), and the nuclear import protein karyopherin alpha 1 (KPNA1) (rs6810306, P = 4.91×10−02).
Conclusion
This study expands the number of candidate genes associated with SLE and highlights the potential of pathway-based approaches for gene discovery. Identification of the causal alleles will help elucidate the molecular mechanisms responsible for activation of the IFN system in SLE.
doi:10.1002/art.30356
PMCID: PMC3128183  PMID: 21437871
10.  Effects of IRF5 Lupus Risk Haplotype on Pathways Predicted to Influence B Cell Functions 
Both genetic and environmental interactions affect systemic lupus erythematosus (SLE) development and pathogenesis. One known genetic factor associated with lupus is a haplotype of the interferon regulatory factor 5 (IRF5) gene. Analysis of global gene expression microarray data using gene set enrichment analysis identified multiple interferon- and inflammation-related gene sets significantly overrepresented in cells with the risk haplotype. Pathway analysis using expressed genes from the significant gene sets impacted by the IRF5 risk haplotype confirmed significant correlation with the interferon pathway, Toll-like receptor pathway, and the B-cell receptor pathway. SLE patients with the IRF5 risk haplotype have a heightened interferon signature, even in an unstimulated state (P = 0.011), while patients with the IRF5 protective haplotype have a B cell interferon signature similar to that of controls. These results identify multiple genes in functionally significant pathways which are affected by IRF5 genotype. They also establish the IRF5 risk haplotype as a key determinant of not only the interferon response, but also other B-cell pathways involved in SLE.
doi:10.1155/2012/594056
PMCID: PMC3304673  PMID: 22500098
11.  Peripheral blood gene expression profiling in Sjögren’s syndrome 
Genes and Immunity  2009;10(4):285-296.
Sjögren’s syndrome (SS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of exocrine glands. Affected cases commonly present with oral and ocular dryness, thought to be the result of inflammatory cell-mediated gland dysfunction. To identify important molecular pathways involved in SS, we used high-density microarrays to define global gene expression profiles in peripheral blood. We first analyzed 21 SS cases and 23 controls and identified a prominent pattern of overexpressed genes that are inducible by interferons (IFNs). These results were confirmed by evaluation of a second independent dataset of 17 SS cases and 22 controls. Additional inflammatory and immune-related pathways with altered expression patterns in SS cases included B and T cell receptor, IGF-1, GM-CSF, PPARα/RXRα, and PI3/AKT signaling. Exploration of these data for relationships to clinical features of disease revealed that expression levels for most IFN-inducible genes were positively correlated with titers of anti-Ro/SSA (P<0.001) and anti-La/SSB (P<0.001) autoantibodies. Diagnostic and therapeutic approaches targeting IFN signaling pathway may prove most effective in the subset of SS cases who produce anti-Ro/SSA and anti-La/SSB autoantibodies. Our results strongly support innate and adaptive immune processes in the pathogenesis of SS and provide numerous candidate disease markers for further study.
doi:10.1038/gene.2009.20
PMCID: PMC3273959  PMID: 19404300
12.  Exon 6 variants carried on systemic lupus erythematosus (SLE) risk haplotypes modulate IRF5 function 
Autoimmunity  2010;44(2):82-89.
Interferon response factor 5 (IRF5) regulates innate immune responses to viral infection. IRF5 genetic variants have been shown to be strongly associated with risk for systemic lupus erythematosus (SLE). Functional roles of IRF5 exon 6 structural variants that occur as part of a SLE risk-associated haplotype, including a 30-bp in/del (in/del-10) and a 48-bp splice-site variant (SV-16), have not been established. In this study, we used IRF5 deficient cells overexpressing human IRF5 variants to investigate the roles of exon 6 in/del-10 and SV-16 in regulation of the apoptosis response, nuclear translocation, and ability to transactivate IRF5 responsive cytokines. We found that expression of IRF5 isoforms including either SV-16 or in/del-10 confers ability of IRF5 to impair the apoptotic response and correlates with reduced capacity for IRF5 nuclear translocation in MEFs after a DNA-damaging stimulus treatment. Interestingly, the presence or absence of both SV-16 and in/del-10 results in abrogation of both the anti-apoptotic and enhanced nuclear translocation effects of IRF5 expression. Only cells expressing IRF5 bearing SV-16 show increased IL-6 production upon LPS stimulation. MEFs expressing hIRF5 variants containing in/del-10 showed no significant difference from the control; however, cells carrying hIRF5 lacking both SV-16 and in/del-10 showed reduced IL-6 production. Our overall findings suggest that exon 6 SV-16 is more potent than in/del-10 for IRF5-driven resistance to apoptosis and promotion of cytokine production; however, in/del-10 co-expression can neutralize these effects of SV-16.
doi:10.3109/08916934.2010.491842
PMCID: PMC3104271  PMID: 20695768
SLE; IRF5 variants; exon 6; apoptosis; nuclear translocation
13.  Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes 
BMC Genomics  2010;11:485.
Background
Discontinuous genes have been observed in bacteria, archaea, and eukaryotic nuclei, mitochondria and chloroplasts. Gene discontinuity occurs in multiple forms: the two most frequent forms result from introns that are spliced out of the RNA and the resulting exons are spliced together to form a single transcript, and fragmented gene transcripts that are not covalently attached post-transcriptionally. Within the past few years, fragmented ribosomal RNA (rRNA) genes have been discovered in bilateral metazoan mitochondria, all within a group of related oysters.
Results
In this study, we have characterized this fragmentation with comparative analysis and experimentation. We present secondary structures, modeled using comparative sequence analysis of the discontinuous mitochondrial large subunit rRNA genes of the cupped oysters C. virginica, C. gigas, and C. hongkongensis. Comparative structure models for the large subunit rRNA in each of the three oyster species are generally similar to those for other bilateral metazoans. We also used RT-PCR and analyzed ESTs to determine if the two fragmented LSU rRNAs are spliced together. The two segments are transcribed separately, and not spliced together although they still form functional rRNAs and ribosomes.
Conclusions
Although many examples of discontinuous ribosomal genes have been documented in bacteria and archaea, as well as the nuclei, chloroplasts, and mitochondria of eukaryotes, oysters are some of the first characterized examples of fragmented bilateral animal mitochondrial rRNA genes. The secondary structures of the oyster LSU rRNA fragments have been predicted on the basis of previous comparative metazoan mitochondrial LSU rRNA structure models.
doi:10.1186/1471-2164-11-485
PMCID: PMC2996981  PMID: 20813041
14.  Variation in the ATP-binding cassette transporter 2 gene is a separate risk factor for Systemic Lupus Erythematosus within the MHC 
Genes and immunity  2009;10(4):350-355.
The ATP-binding cassette transporter (TAP) proteins are functionally relevant candidates for predisposition to Systemic Lupus Erythematosus (SLE) by virtue of their role in autoantigen presentation and location in the MHC. We tested if variation in the TAP genes (TAP1 and TAP2) is associated with SLE. We genotyped tag single nucleotide polymorphisms (SNPs) and performed family-based association analysis on 390 Caucasian pedigrees. We found significant evidence of association between TAP2 and SLE (rs241453, P = 1.33 × 10-6). Conditional logistic regression analysis suggests that this TAP2 effect is separate from the HLA-DRB1 alleles. Our analyses show that both rs241453 (P = 1.6 × 10-4) and HLA-DRB1*03xx (P = 2.3 × 10-4) have significant autonomous effects not due to linkage disequilibrium. Moreover, these loci exhibit a significant statistical interaction (P < 1.0 × 10-6), demonstrated by an increase in the odds ratio for the TAP2 association from OR = 2.00 (CI=1.17-3.42) in HLA-DRB1*03xx-negative subjects to OR = 4.29 (CI=1.88-9.76) in the subjects with at least one HLA-DRB1*03xx allele group. We report the largest association study of the TAP genes with SLE to date, and the first to test for its separate effect and interaction with the HLA alleles consistently associated with SLE.
doi:10.1038/gene.2009.21
PMCID: PMC2927958  PMID: 19387463
Systemic Lupus Erythematosus; TAP2; HLA-DRB1; family-based association analysis; conditional logistic regression analysis; interaction analysis
15.  Genetic Associations of LYN with Systemic Lupus Erythematosus 
Genes and immunity  2009;10(5):397-403.
We targeted LYN, a src-tyosine kinase involved in B cell activation, in case-control association studies using populations of European American, African American and Korean subjects. Our combined European-derived population, consisting of 2463 independent cases and 3131 unrelated controls, demonstrates significant association with rs6983130 in a female-only analysis with 2254 cases and 2228 controls (p=1.1 × 10−4, OR=0.81 (95% CI: 0.73 – 0.90)). This SNP is located in the 5′ UTR within the first intron near the transcription initiation site of LYN. Additional SNPs upstream of the first exon also show weak and sporadic association in subsets of the total European American population. Multivariate logistic regression analysis implicates rs6983130 as a protective factor for SLE susceptibility when anti-dsDNA, anti-chromatin, anti-52 kDa Ro or anti-Sm autoantibody status were used as covariates. Subset analysis of the European American female cases by ACR classification criteria reveals a reduction in the risk of hematologic disorder with rs6983130 compared to cases without hematologic disorders (p=1.5 × 10−3, OR=0.75 (95% C.I.=0.62-0.89)). None of the 90 SNPs tested demonstrate significant association with SLE in the African American or Korean populations. These results support an association of LYN with European-derived individuals with SLE, especially within autoantibody or clinical subsets.
doi:10.1038/gene.2009.19
PMCID: PMC2750001  PMID: 19369946
systemic lupus erythematosus; association; LYN; SNP
16.  Replication of the BANK1 genetic association with systemic lupus erythematosus in a European-Derived Population 
Genes and immunity  2009;10(5):531-538.
Systemic lupus erythematosus (SLE) is an autoimmune disease with highly variable clinical presentation. Patients suffer from immunological abnormalities that target T cell, B cell and accessory cell functions. B cells are hyperactive in SLE patients. An adaptor protein expressed in B cells called BANK1 (B-cell scaffold protein with ankyrin repeats) was reported in a previous study to be associated with SLE in a European population. The objective of this study is to assess the BANK1 genotype-phenotype association in an independent replication sample. We genotyped 38 single nucleotide polymorphisms (SNPs) in BANK1 on 1892 European-derived SLE patients and 2652 European-derived controls. The strongest associations with SLE and BANK1 were at rs17266594 (corrected p-value=1.97 × 10−5, OR=1.22, 95% C.I.(1.12–1.34)) and rs10516487 (corrected p-value=2.59 × 10−5, OR=1.22, 95% C.I.(1.11–1.34)). Our findings suggest that the association is explained by these two SNPs, confirming previous reports that these polymorphisms contribute to the risk of developing lupus. Analysis of patient subsets enriched for hematological, immunological and renal ACR criteria or the levels of autoantibodies, such as anti-RNP A and anti-SmRNP, uncovers additional BANK1 associations. Our results suggest that BANK1 polymorphisms alter immune system development and function to increase the risk for developing lupus.
doi:10.1038/gene.2009.18
PMCID: PMC2736873  PMID: 19339986
systemic lupus erythematosus; replication; association; European; BANK1
17.  Meta-analysis and Imputation Identifies a 109 kb Risk Haplotype Spanning TNFAIP3 Associated with Lupus Nephritis and Hematologic Manifestations 
Genes and immunity  2009;10(5):470-477.
TNFAIP3 encodes the ubiquitin modifying enzyme, A20, a key regulator of inflammatory signaling pathways. We previously reported association between TNFAIP3 variants and systemic lupus erythematosus (SLE). In order to further localize the risk variant(s), we performed a meta-analysis using genetic data available from two Caucasian case/control datasets (1453 total cases, 3381 total controls) and 713 SLE trio families. The best result was found at rs5029939 (P = 1.67 × 10−14, OR = 2.09, 95% CI 1.68–2.60). We then imputed SNPs from the CEU Phase II HapMap using genotypes from 431 SLE cases and 2155 controls. Imputation identified eleven SNPs in addition to three observed SNPs, which together, defined a 109 kb SLE risk segment surrounding TNFAIP3. When evaluating whether the rs5029939 risk allele was associated with SLE clinical manifestations, we observed that heterozygous carriers of the TNFAIP3 risk allele at rs5029939 have a two-fold increased risk of developing renal or hematologic manifestations compared to homozygous non-risk subjects. In summary, our study strengthens the genetic evidence that variants in the region of TNFAIP3 influence risk for SLE, particularly in patients with renal and hematologic manifestations, and narrows the risk effect to a 109 kb DNA segment that spans the TNFAIP3 gene.
doi:10.1038/gene.2009.31
PMCID: PMC2714405  PMID: 19387456
systemic lupus erythematosus; TNFAIP3; imputation; meta-analysis
18.  Genetic linkage and transmission disequilibrium of marker haplotypes at chromosome 1q41 in human systemic lupus erythematosus 
Arthritis Research  2001;3(5):299-305.
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies to a wide range of self-antigens. Recent genome screens have implicated numerous chromosomal regions as potential SLE susceptibility loci. Among these, the 1q41 locus is of particular interest, because evidence for linkage has been found in several independent SLE family collections. Additionally, the 1q41 locus appears to be syntenic with a susceptibility interval identified in the NZM2410 mouse model for SLE. Here, we report the results of genotyping of 11 microsatellite markers within the 1q41 region in 210 SLE sibpair and 122 SLE trio families. These data confirm the modest evidence for linkage at 1q41 in our family collection (LOD = 1.21 at marker D1S2616). Evidence for significant linkage disequilibrium in this interval was also found. Multiple markers in the region exhibit transmission disequilibrium, with the peak single marker multiallelic linkage disequilibrium noted at D1S490 (pedigree disequilibrium test [PDT] global P value = 0.0091). Two- and three-marker haplotypes from the 1q41 region similarly showed strong transmission distortion in the collection of 332 SLE families. The finding of linkage together with significant transmission disequilibrium provides strong evidence for a susceptibility locus at 1q41 in human SLE.
PMCID: PMC64842  PMID: 11549371
1q41; autoimmunity; linkage; systemic lupus erythematosus; transmission disequilibrium
19.  Dense genotyping of immune-related disease regions identifies 14 new susceptibility loci for juvenile idiopathic arthritis 
Nature genetics  2013;45(6):664-669.
Analysis of the ImmunoChip single nucleotide polymorphism (SNP) array in 2816 individuals, comprising the most common subtypes (oligoarticular and RF negative polyarticular) of juvenile idiopathic arthritis (JIA) and 13056 controls strengthens the evidence for association to three known JIA-risk loci (HLA, PTPN22 and PTPN2) and has identified fourteen risk loci reaching genome-wide significance (p < 5 × 10-8) for the first time. Eleven additional novel regions showed suggestive evidence for association with JIA (p < 1 × 10-6). Dense-mapping of loci along with bioinformatic analysis has refined the association to one gene for eight regions, highlighting crucial pathways, including the IL-2 pathway, in JIA disease pathogenesis. The entire ImmunoChip loci, HLA region and the top 27 loci (p < 1 × 10-6) explain an estimated 18%, 13% and 6% risk of JIA, respectively. Analysis of the ImmunoChip dataset, the largest cohort of JIA cases investigated to date, provides new insight in understanding the genetic basis for this childhood autoimmune disease.
doi:10.1038/ng.2614
PMCID: PMC3673707  PMID: 23603761
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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