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1.  BANK1 Regulates IgG Production in a Lupus Model by Controlling TLR7-Dependent STAT1 Activation 
PLoS ONE  2016;11(5):e0156302.
The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.Sle1.yaa mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.Sle1.yaa with Bank1-/- mice maintained several B and myeloid cell phenotypes close to normal wild-type levels. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. Bank1 deficiency did modify numbers of MZ B cells and total B cell numbers, as well as expression of CXCR4 by follicular helper T cells. Other T cell changes were not observed. Bank1 deficiency did not modify numbers of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from Bank1 deficient mice had strongly reduced Ifnb, Ifna4, Irf7, Aicda and Stat1 gene expression following TLR7 agonist stimulation. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from B6.Sle1.yaa.Bank1-/- mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, Bank1 deficiency in B6.Sle1.yaa mice reduced the production of IgG2c after in vitro TLR7 agonist stimulation. Our results demonstrate that Bank1 controls TLR7-mediated type I interferon production. Combined with the control of the nuclear translocation of IRF7, the modulation of STAT1 transcription and phosphorylation, Bank1 contributes to IgG production during development of autoimmune disease.
PMCID: PMC4882053  PMID: 27228057
2.  GWAS in an Amerindian ancestry population reveals novel systemic lupus erythematosus risk loci and the role of European admixture 
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a strong genetic component. Our aim was to perform the first genome-wide association study on individuals from the Americas enriched for Native American heritage.
We analyzed 3,710 individuals from four countries of Latin America and the Unites States diagnosed with SLE and healthy controls. Samples were genotyped with the HumanOmni1 BeadChip. Data of out-of-study controls was obtained for the HumanOmni2.5. Statistical analyses were performed using SNPTEST and SNPGWA. Data was adjusted for genomic control and FDR. Imputation was done using IMPUTE2, and HiBAG for classical HLA alleles.
The IRF5-TNPO3 region showed the strongest association and largest odds ratio (OR) (rs10488631, Pgcadj = 2.61×10−29, OR = 2.12, 95% CI: 1.88–2.39) followed by the HLA class II on the DQA2-DQB1 loci (rs9275572, Pgcadj = 1.11 × 10−16, OR = 1.62, 95% CI: 1.46–1.80; rs9271366, Pgcadj=6.46 × 10−12, OR = 2.06, 95% CI: 1.71–2.50). Other known SLE loci associated were ITGAM, STAT4, TNIP1, NCF2 and IRAK1. We identified a novel locus on 10q24.33 (rs4917385, Pgcadj =1.4×10−8) with a eQTL effect (Peqtl=8.0 × 10−37 at USMG5/miR1307), and describe novel loci. We corroborate SLE-risk loci previously identified in European and Asians. Local ancestry estimation showed that HLA allele risk contribution is of European ancestral origin. Imputation of HLA alleles suggested that autochthonous Native American haplotypes provide protection.
Our results show the insight gained by studying admixed populations to delineate the genetic architecture that underlies autoimmune and complex diseases.
PMCID: PMC4829354  PMID: 26606652
3.  Concordance of Increased B1 Cell Subset and Lupus Phenotypes in Mouse and Human Dependent on BLK Expression Levels 
Polymorphisms in the BLK gene have been associated with autoimmune diseases, including systemic lupus erythematosus (SLE), with risk correlating with reduced expression of BLK. How reduced expression of BLK causes autoimmunity is unknown. Using Blk+/+, Blk+/−, and Blk−/− mice, we show that aged female Blk+/− and Blk−/− mice produced higher anti-dsDNA IgG antibodies and developed immune complex-mediated glomerulonephritis, compared to Blk+/+ mice. Starting at young age, Blk+/− and Blk−/− mice accumulated increased numbers of splenic B1a cells, which differentiated into class-switched CD138+IgG-secreting B1a cells. Increased infiltration of B1a-like cells into the kidneys was also observed in aged Blk+/− and Blk−/− mice. In human, we found that healthy individuals had BLK genotype-dependent levels of anti-dsDNA IgG antibodies as well as increased numbers of a B1-like cell population, CD19+CD3−CD20+CD43+CD27+, in peripheral blood. Furthermore, we describe the presence of B1-like cells in the tubulointerstitial space of human lupus kidney biopsies. Taken together, our study reveals a previously unappreciated role of reduced BLK expression on extraperitoneal accumulation of B1a cells in mice, and the presence of IgG autoantibodies and B1-like cells in human.
PMCID: PMC4458437  PMID: 25972485
4.  Genetic associations of leptin-related polymorphisms with systemic lupus erythematosus 
Clinical immunology (Orlando, Fla.)  2015;161(2):157-162.
Leptin is abnormally elevated in the plasma of patients with systemic lupus erythematosus (SLE), where it is thought to promote and/or sustain pro-inflammatory responses. Whether this association could reflect an increased genetic susceptibility to develop SLE is not known, and studies of genetic associations with leptin-related polymorphisms in SLE patients have been so far inconclusive. Here we genotyped DNA samples from 15,706 SLE patients and healthy matched controls from four different ancestral groups, to correlate polymorphisms of genes of the leptin pathway to risk for SLE. It was found that although several SNPs showed weak associations, those associations did not remain significant after correction for multiple testing. These data do not support associations between defined leptin-related polymorphisms and increased susceptibility to develop SLE.
PMCID: PMC4658308  PMID: 26385092
systemic lupus erythematosus; leptin pathway; gene polymorphisms
5.  Genetic Association of CD247 (CD3ζ) with SLE in a Large-Scale Multiethnic Study 
Genes and immunity  2015;16(2):142-150.
A classic T-cell phenotype in Systemic lupus erythematosus (SLE) is the downregulation and replacement of the CD3ζ chain that alters TCR signaling. However, genetic associations with SLE in the human CD247 locus that encodes CD3ζ are not well established and require replication in independent cohorts. Our aim was therefore to examine, localize and validate CD247-SLE association in a large multi-ethnic population. We typed 44 contiguous CD247 SNPs in 8 922 SLE patients and 8 077 controls from four ethnically distinct populations. The strongest associations were found in the Asian population (11 SNPs in intron 1, 4.99×10−4
PMCID: PMC4371129  PMID: 25569266
Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder whose etiology is incompletely understood, but likely involves environmental triggers in genetically susceptible individuals. We sought to identify the genetic loci associated with SLE in a Korean population by performing an unbiased genome-wide association scan.
A total of 1,174 Korean SLE cases and 4,248 population controls were genotyped with strict quality control measures and analyzed for association. For select variants, replication was tested in an independent set of 1,412 SLE cases and 1,163 population controls of Korean and Chinese ancestries.
Eleven regions outside the HLA exceeded genome-wide significance (P<5×10−8). A novel SNP-SLE association was identified between FCHSD2 and P2RY2 peaking at rs11235667 (P = 1.0×10−8, odds ratio (OR) = 0.59) on a 33kb haplotype upstream to ATG16L2. Replication for rs11235667 resulted in Pmeta-rep=0.001 and Pmeta-overall=6.67×10−11 (OR=0.63). Within the HLA region, association peaked in the Class II region at rs116727542 with multiple independent effects. Classical HLA allele imputation identified HLA-DRB1*1501 and HLA-DQB1*0602, both highly correlated, as most strongly associated with SLE. We replicated ten previously established SLE risk loci: STAT1-STAT4, TNFSF4, TNFAIP3, IKZF1, HIP1, IRF5, BLK, WDFY4, ETS1 and IRAK1-MECP2. Of these loci, we identified previously unreported independent second effects in TNFAIP3 and TNFSF4 as well as differences in the association for a putative causal variant in the WDFY4 region.
Further studies are needed to identify true SLE risk effects in other suggestive loci and to identify the causal variant(s) in the regions of ATG16L2, FCHSD2, and P2RY2.
PMCID: PMC4981330  PMID: 26663301
PLoS ONE  2016;11(8):e0160270.
Systemic Autoimmune Diseases, a group of chronic inflammatory conditions, have variable symptoms and difficult diagnosis. In order to reclassify them based on genetic markers rather than clinical criteria, we performed clustering of Single Nucleotide Polymorphisms. However naive approaches tend to group patients primarily by their geographic origin. To reduce this “ancestry signal”, we developed SNPClust, a method to select large sources of ancestry-independent genetic variations from all variations detected by Principal Component Analysis. Applied to a Systemic Lupus Erythematosus case control dataset, SNPClust successfully reduced the ancestry signal. Results were compared with association studies between the cases and controls without or with reference population stratification correction methods. SNPClust amplified the disease discriminating signal and the ratio of significant associations outside the HLA locus was greater compared to population stratification correction methods. SNPClust will enable the use of ancestry-independent genetic information in the reclassification of Systemic Autoimmune Diseases. SNPClust is available as an R package and demonstrated on the public Human Genome Diversity Project dataset at
PMCID: PMC4973908  PMID: 27490238
Journal of immunology (Baltimore, Md. : 1950)  2013;191(12):10.4049/jimmunol.1301203.
BANK1, an adaptor protein expressed in B-cells, plays a little understood role in B-cell signaling. Because BANK1 contains an N-terminal putative Toll-interleukin 1 receptor (TIR) domain, we used mouse Bank1−/− splenic B cells to test if BANK1 affects signaling induced by the TLR9 agonist CpG. Following CpG stimulation BANK1 deficiency reduced p38 phosphorylation without affecting that of ERK or JNK and reduced IL-6 secretion. Bank1−/− B cells showed reduced phosphorylation of MNK1/2 and eIF4E, suggesting an effect on translation initiation, while Bank1−/− had no effect on IL6 mRNA stability, thus suggesting that BANK1 has no effect on MK2 signaling. IL-6 secretion observed when CpG stimulation was combined with anti-CD40, was reduced in the absence of BANK1. While in the presence of anti-CD40 stimulation CpG induced a stronger phosphorylation of AKT, mTOR and 4E-BP1, Bank1−/− had no effect on phosphorylation of mTOR and 4E-BP1, and weakly on AKT, implying that BANK1 does not affect the release of eIF4E by phospho-4E-BP1. Together these data establish a previously unrecognized role for BANK1 in CpG-induced responses by splenic B cells on p38 signaling and control of translation initiation of IL-6 via MNK1/2 and eIF4E.
PMCID: PMC3858538  PMID: 24227780
endosomal toll-like receptors; mitogen activated protein kinases; p38; IL-6; CD40; autoimmunity; systemic lupus erythematosus; B lymphocytes
Nature genetics  2015;47(12):1457-1464.
Systemic lupus erythematosus (SLE; OMIM 152700) is a genetically complex autoimmune disease characterized by loss of immune tolerance to nuclear and cell surface antigens. Previous genome-wide association studies (GWAS) had modest sample sizes, reducing their scope and reliability. Our study comprised 7,219 cases and 15,991 controls of European ancestry: a new GWAS, meta-analysis with a published GWAS and a replication study. We have mapped 43 susceptibility loci, including 10 novel associations. Assisted by dense genome coverage, imputation provided evidence for missense variants underpinning associations in eight genes. Other likely causal genes were established by examining associated alleles for cis-acting eQTL effects in a range of ex vivo immune cells. We found an over-representation (n=16) of transcription factors among SLE susceptibility genes. This supports the view that aberrantly regulated gene expression networks in multiple cell types in both the innate and adaptive immune response contribute to the risk of developing SLE.
PMCID: PMC4668589  PMID: 26502338
Human Molecular Genetics  2013;22(19):4021-4029.
Systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are two archetypal systemic autoimmune diseases which have been shown to share multiple genetic susceptibility loci. In order to gain insight into the genetic basis of these diseases, we performed a pan-meta-analysis of two genome-wide association studies (GWASs) together with a replication stage including additional SSc and SLE cohorts. This increased the sample size to a total of 21 109 (6835 cases and 14 274 controls). We selected for replication 19 SNPs from the GWAS data. We were able to validate KIAA0319L (P = 3.31 × 10−11, OR = 1.49) as novel susceptibility loci for SSc and SLE. Furthermore, we also determined that the previously described SLE susceptibility loci PXK (P = 3.27 × 10−11, OR = 1.20) and JAZF1 (P = 1.11 × 10−8, OR = 1.13) are shared with SSc. Supporting these new discoveries, we observed that KIAA0319L was overexpressed in peripheral blood cells of SSc and SLE patients compared with healthy controls. With these, we add three (KIAA0319L, PXK and JAZF1) and one (KIAA0319L) new susceptibility loci for SSc and SLE, respectively, increasing significantly the knowledge of the genetic basis of autoimmunity.
PMCID: PMC3766185  PMID: 23740937
Annals of the rheumatic diseases  2008;68(11):10.1136/ard.2008.097642.
To confirm and define the genetic association of STAT4 and systemic lupus erythematosus, investigate the possibility of correlations with differential splicing and/or expression levels, and genetic interaction with IRF5.
30 tag SNPs were genotyped in an independent set of Spanish cases and controls. SNPs surviving correction for multiple tests were genotyped in 5 new sets of cases and controls for replication. STAT4 cDNA was analyzed by 5’-RACE PCR and sequencing. Expression levels were measured by quantitative PCR.
In the fine-mapping, four SNPs were significant after correction for multiple testing, with rs3821236 and rs3024866 as the strongest signals, followed by the previously associated rs7574865, and by rs1467199. Association was replicated in all cohorts. After conditional regression analyses, two major independent signals represented by SNPs rs3821236 and rs7574865, remained significant across the sets. These SNPs belong to separate haplotype blocks. High levels of STAT4 expression correlated with SNPs rs3821236, rs3024866 (both in the same haplotype block) and rs7574865 but not with other SNPs. We also detected transcription of alternative tissue-specific exons 1, indicating presence of tissue-specific promoters of potential importance in the expression of STAT4. No interaction with associated SNPs of IRF5 was observed using regression analysis.
These data confirm STAT4 as a susceptibility gene for SLE and suggest the presence of at least two functional variants affecting levels of STAT4. Our results also indicate that both genes STAT4 and IRF5 act additively to increase risk for SLE.
PMCID: PMC3878433  PMID: 19019891
Association studies; systemic lupus erythematosus; STAT4 transcription factor; Interferon regulatory factor; genetic predisposition to disease
Arthritis and rheumatism  2012;64(11):3687-3694.
Amerindian-Europeans, Asians and African-Americans have an excess morbidity from SLE and higher prevalence of lupus nephritis than Caucasians. The aim of this study was to analyze the relationship between genetic ancestry and socio-demographic characteristics and clinical features in a large cohort of Amerindian-European SLE patients.
A total of 2116 SLE patients of Amerindian-European origin and 4001 SLE patients of European descent with clinical data were used in the study. Genotyping of 253 continental ancestry informative markers was performed on the Illumina platform. The STRUCTURE and ADMIXTURE software were used to determine genetic ancestry of each individual. Correlation between ancestry and socio-demographic and clinical data were analyzed using logistic regression.
The average Amerindian genetic ancestry of 2116 SLE patients was 40.7%. There was an increased risk of having renal involvement (P<0.0001, OR= 3.50 95%CI 2.63-4.63) and an early age of onset with the presence of Amerindian genetic ancestry (P<0.0001). Amerindian ancestry protected against photosensitivity (P<0.0001, OR= 0.58 95%CI 0.44-0.76), oral ulcers (P<0.0001, OR= 0.55 95%CI 0.42-0.72), and serositis (P<0.0001, OR= 0.56 95%CI 0.41-0.75) after adjustment by age, gender and age of onset. However, gender and age of onset had stronger effects on malar rash, discoid rash, arthritis and neurological involvement than genetic ancestry.
In general, genetic Amerindian ancestry correlates with lower socio-demographic status and increases the risk for developing renal involvement and SLE at an earlier age of onset.
PMCID: PMC3485439  PMID: 22886787
Annals of the Rheumatic Diseases  2014;75(1):242-252.
Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association.
Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR.
The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10−4, OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10−7, OR 0.71; case-only pmeta=1.9×10−4, OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR.
These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications.
PMCID: PMC4717392  PMID: 25180293
Systemic Lupus Erythematosus; Autoantibodies; Gene Polymorphism; B cells
Annals of the Rheumatic Diseases  2011;71(1):136-142.
Altered signaling in B-cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signaling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterize the role of BANK1 and BLK in SLE, we performed a genetic interaction analysis hypothesizing that genetic interactions could reveal functional pathways relevant to disease pathogenesis.
We Used the method GPAT16 to analyze the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localization, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK.
Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from Northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK.
As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, we tested the possibility that BANK1 and BLK could also show a protein-protein interaction. We demonstrated co-immunoprecipitation and co-localization of BLK and BANK1. In a Daudi cell line and primary naïve B-cells the endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies.
Here, we show a genetic interaction between BANK1 and BLK, and demonstrate that these molecules interact physically. Our results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signaling pathway.
PMCID: PMC3268679  PMID: 21978998
systemic lupus erythematosus; genetics; polymorphism; B-cells; autoantibodies
PLoS Genetics  2015;11(12):e1005602.
South America has a complex demographic history shaped by multiple migration and admixture events in pre- and post-colonial times. Settled over 14,000 years ago by Native Americans, South America has experienced migrations of European and African individuals, similar to other regions in the Americas. However, the timing and magnitude of these events resulted in markedly different patterns of admixture throughout Latin America. We use genome-wide SNP data for 437 admixed individuals from 5 countries (Colombia, Ecuador, Peru, Chile, and Argentina) to explore the population structure and demographic history of South American Latinos. We combined these data with population reference panels from Africa, Asia, Europe and the Americas to perform global ancestry analysis and infer the subcontinental origin of the European and Native American ancestry components of the admixed individuals. By applying ancestry-specific PCA analyses we find that most of the European ancestry in South American Latinos is from the Iberian Peninsula; however, many individuals trace their ancestry back to Italy, especially within Argentina. We find a strong gradient in the Native American ancestry component of South American Latinos associated with country of origin and the geography of local indigenous populations. For example, Native American genomic segments in Peruvians show greater affinities with Andean indigenous peoples like Quechua and Aymara, whereas Native American haplotypes from Colombians tend to cluster with Amazonian and coastal tribes from northern South America. Using ancestry tract length analysis we modeled post-colonial South American migration history as the youngest in Latin America during European colonization (9–14 generations ago), with an additional strong pulse of European migration occurring between 3 and 9 generations ago. These genetic footprints can impact our understanding of population-level differences in biomedical traits and, thus, inform future medical genetic studies in the region.
Author Summary
South America is home to over 400 million people who share a rich demographic history, including settlement by Native Americans, European colonization, and the African slave trade. We use genomic data to infer which populations from Europe and the Americas contributed to these admixture events. We provide evidence for multiple origins of the Native American ancestry of admixed South American Latinos. The Native American ancestral component correlates strongly with geography, indicating that admixture occurred between European colonists and local Native American populations throughout South America. We also show that the European ancestry of South American Latinos comes mainly from the Iberian peninsula, however, a significant number of Argentinians have European ancestry from other Southern European regions. The genetic signal of European admixture in South American populations is younger than the signal observed in Mexico and the Caribbean. We find evidence for a second pulse of European migration to many regions of South America subsequent to the original colonization. These results demonstrate the heterogeneous nature of the Latino population in South America and help elucidate the complex genetic and admixture events that shaped the population structure of the region.
PMCID: PMC4670080  PMID: 26636962
Annals of the Rheumatic Diseases  2012;71(7):1219-1226.
To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in SLE.
Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 SNPs. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL’s test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild-type form were made to analyse their effect on protein half-life using a protein stability assay, cycloheximide and Western blot. CHiP-qPCR for NFkB binding.
Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR = 2.31 (95% c.i. 1.38–3.86). The 71Thr decreased BLK protein half-life.
Our results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently lead to reduced levels of BLK protein.
PMCID: PMC3375585  PMID: 22696686
systemic lupus erythematosus; autoimmunity; genetics; polymorphism; B-cells; autoantibodies; B-lymphocyte tyrosine kinase
Current opinion in immunology  2010;22(6):698-705.
Association studies of over 1 million SNPs capturing most of the human genome common variation became possible thanks to the information provided by the HapMap International project and the development of high-throughput genotyping technologies at accessible prices. Genome-wide scans analyzing thousands of individuals have now identified most if not all of the major genes involved in susceptibility for several systemic autoimmune diseases. In particular, results for rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) are reviewed here. While most genes are shared between diseases, few seem to be unique reflecting that we still are long before knowing all genes, their interactions with other genes and the environment and their impact on biological functions.
PMCID: PMC2997852  PMID: 20933377
Genes and immunity  2014;16(1):15-23.
Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disorder characterized by inflammation of multiple organ systems and dysregulated interferon responses. SLE is both genetically and phenotypically heterogeneous, greatly reducing the power of case-control studies in SLE. Elevated circulating interferon alpha (IFN-α) is a stable, heritable trait in SLE, which has been implicated in primary disease pathogenesis. 40–50% of patients have high IFN-α, and high levels correspond with clinical differences. To study genetic heterogeneity in SLE, we performed a case-case study comparing patients with high vs. low IFN-α in over 1550 SLE cases, including GWAS and replication cohorts. In meta-analysis, the top associations in European ancestry were PRKG1 rs7897633 (PMeta=2.75 × 10−8) and PNP rs1049564 (PMeta=1.24 × 10−7). We also found evidence for cross-ancestral background associations with the ANKRD44 and PLEKHF2 loci. These loci have not been previously identified in case-control SLE genetic studies. Bioinformatic analyses implicated these loci functionally in dendritic cells and natural killer cells, both of which are involved in IFN-α production in SLE. As case-control studies of heterogeneous diseases reach a limit of feasibility with respect to subject number and detectable effect size, the study of informative pathogenic subphenotypes becomes an attractive strategy for genetic discovery in complex disease.
PMCID: PMC4305028  PMID: 25338677
Genes and immunity  2014;15(6):347-354.
In a Genome Wide Association Study (GWAS) of individuals of European ancestry afflicted with Systemic Lupus Erythematosus (SLE) the extensive utilization of imputation, stepwise multiple regression, lasso regularization, and increasing study power by utilizing False Discovery Rate (FDR) instead of a Bonferroni multiple test correction enabled us to identify 13 novel non-human leukocyte antigen (HLA) genes and confirmed the association of 4 genes previously reported to be associated. Novel genes associated with SLE susceptibility included two transcription factors (EHF, and MED1), two components of the NFκB pathway (RASSF2 and RNF114), one gene involved in adhesion and endothelial migration (CNTN6), and two genes involved in antigen presentation (BIN1 and SEC61G). In addition, the strongly significant association of multiple single nucleotide polymorphisms (SNPs) in the HLA region was assigned to HLA alleles and serotypes and deconvoluted into four primary signals. The novel SLE-associated genes point to new directions for both the diagnosis and treatment of this debilitating autoimmune disease.
PMCID: PMC4156543  PMID: 24871463
Frontiers in Genetics  2015;5:450.
Genome wide association studies have identified variants in PXK that confer risk for humoral autoimmune diseases, including systemic lupus erythematosus (SLE or lupus), rheumatoid arthritis and more recently systemic sclerosis. While PXK is involved in trafficking of epidermal growth factor Receptor (EGFR) in COS-7 cells, mechanisms linking PXK to lupus pathophysiology have remained undefined. In an effort to uncover the mechanism at this locus that increases lupus-risk, we undertook a fine-mapping analysis in a large multi-ancestral study of lupus patients and controls. We define a large (257kb) common haplotype marking a single causal variant that confers lupus risk detected only in European ancestral populations and spans the promoter through the 3′ UTR of PXK. The strongest association was found at rs6445972 with P < 4.62 × 10−10, OR 0.81 (0.75–0.86). Using stepwise logistic regression analysis, we demonstrate that one signal drives the genetic association in the region. Bayesian analysis confirms our results, identifying a 95% credible set consisting of 172 variants spanning 202 kb. Functionally, we found that PXK operates on the B-cell antigen receptor (BCR); we confirmed that PXK influenced the rate of BCR internalization. Furthermore, we demonstrate that individuals carrying the risk haplotype exhibited a decreased rate of BCR internalization, a process known to impact B cell survival and cell fate. Taken together, these data define a new candidate mechanism for the genetic association of variants around PXK with lupus risk and highlight the regulation of intracellular trafficking as a genetically regulated pathway mediating human autoimmunity.
PMCID: PMC4288052  PMID: 25620976
lupus; PXK; fine-mapping; B cells; BCR
Annals of the Rheumatic Diseases  2012;71(7):1219-1226.
To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in systemic lupus erythematosus (SLE).
Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 single-nucleotide polymorphisms. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL's test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild type form were analysed for their effect on protein half-life using a protein stability assay, cycloheximide and western blot. CHiP-qPCR for detection of nuclear factor κ B (NFkB) binding.
Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2 Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR=2.31 (95% CI 1.38 to 3.86). The 71Thr decreased BLK protein half-life.
These results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently, lead to reduced levels of BLK protein.
PMCID: PMC3375585  PMID: 22696686
Genes and immunity  2008;10(3):10.1038/gene.2008.95.
The tumor necrosis factor ligand superfamily member 4 gene (TNFSF4) encodes the OX40 ligand (OX40L), a co-stimulatory molecule involved in T-cell activation. A recent study demonstrated the association ofTNFSF4 haplotypes located in the upstream region with risk for- or protection from Systemic Lupus Erythematosus (SLE) (Graham et al, 2008). In order to replicate this association, five single nucleotide polymorphisms (SNPs) tagging the previously associated haplotypes and passing the proper quality control filters were tested in 1312 cases and 1801 controls from Germany, Italy, Spain, and Argentina. The association of TNFSF4 with SLE was replicated in all the sets except Spain. There was a unique risk haplotype tagged by the minor alleles of the SNPs rs1234317 (pooled OR=1.39, p=0.0009) and rs12039904 (pooled OR=1.38, p=0.0012). We did not observe association to a single protective marker (rs844644) or haplotype as the first study reported; instead, we observed different protective haplotypes, all carrying the major alleles of both SNPs rs1234317 and rs12039904. Association analysis conditioning on the haplotypic background confirmed that these two SNPs explain the entire haplotype effect. This is the first replication study that confirms the association of genetic variation in the upstream region of TNFSF4 with susceptibility to SLE.
PMCID: PMC3867640  PMID: 19092840
Systemic lupus erythematosus; TNFSF4; OX40L; genetic association study
Arthritis and rheumatism  2012;64(11):3695-3705.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and altered type I interferon expression. Genetic surveys and genome-wide association studies have identified more than 30 SLE susceptibility genes. One of these genes, TNIP1, encodes the ABIN1 protein. ABIN1 functions in the immune system by restricting the NF-κB signaling. In order to better understand the genetic factors that influence association with SLE in genes that regulate the NF-κB pathway, we analyzed a dense set of genetic markers spanning TNIP1 and TAX1BP1, as well as the TNIP1 homolog, TNIP2, in case-control sets of diverse ethnic origins.
We fine-mapped TNIP1, TNIP2, and TAX1BP1 in a total of 8372 SLE cases and 7492 healthy controls from European-ancestry, African-American, Hispanic, East Asian, and African-American Gullah populations. Levels of TNIP1 mRNA and ABIN1 protein were analyzed using quantitative RT-PCR and Western blotting, respectively, in EBV-transformed human B cell lines.
We found significant associations between genetic variants within TNIP1 and SLE but not in TNIP2 or TAX1BP1. After resequencing and imputation, we identified two independent risk haplotypes within TNIP1 in individuals of European-ancestry that were also present in African-American and Hispanic populations. These risk haplotypes produced lower levels of TNIP1 mRNA and ABIN1 protein suggesting they harbor hypomorphic functional variants that influence susceptibility to SLE by restricting ABIN1 expression.
Our results confirmed the association signals between SLE and TNIP1 variants in multiple populations and provide new insight into the mechanism by which TNIP1 variants may contribute to SLE pathogenesis.
PMCID: PMC3485412  PMID: 22833143
PLoS ONE  2013;8(8):e69404.
Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a negative regulator of T-cell activation associated with several autoimmune diseases, including systemic lupus erythematosus (SLE). Missense rs2476601 is associated with SLE in individuals with European ancestry. Since the rs2476601 risk allele frequency differs dramatically across ethnicities, we assessed robustness of PTPN22 association with SLE and its clinical sub-phenotypes across four ethnically diverse populations. Ten SNPs were genotyped in 8220 SLE cases and 7369 controls from in European-Americans (EA), African-Americans (AA), Asians (AS), and Hispanics (HS). We performed imputation-based association followed by conditional analysis to identify independent associations. Significantly associated SNPs were tested for association with SLE clinical sub-phenotypes, including autoantibody profiles. Multiple testing was accounted for by using false discovery rate. We successfully imputed and tested allelic association for 107 SNPs within the PTPN22 region and detected evidence of ethnic-specific associations from EA and HS. In EA, the strongest association was at rs2476601 (P = 4.7×10−9, OR = 1.40 (95% CI = 1.25–1.56)). Independent association with rs1217414 was also observed in EA, and both SNPs are correlated with increased European ancestry. For HS imputed intronic SNP, rs3765598, predicted to be a cis-eQTL, was associated (P = 0.007, OR = 0.79 and 95% CI = 0.67–0.94). No significant associations were observed in AA or AS. Case-only analysis using lupus-related clinical criteria revealed differences between EA SLE patients positive for moderate to high titers of IgG anti-cardiolipin (aCL IgG >20) versus negative aCL IgG at rs2476601 (P = 0.012, OR = 1.65). Association was reinforced when these cases were compared to controls (P = 2.7×10−5, OR = 2.11). Our results validate that rs2476601 is the most significantly associated SNP in individuals with European ancestry. Additionally, rs1217414 and rs3765598 may be associated with SLE. Further studies are required to confirm the involvement of rs2476601 with aCL IgG.
PMCID: PMC3737240  PMID: 23950893

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